Patent Grant
7345150
Throughout this application various publications are referenced by numerals within parenthesis. Full citations for these publications may be found at the end of this application, preceding the claims. The disclosure of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
The present invention relates to the use of an albumin-based colloid composition, such as PEG-Alb, a polyethylene oxide (such as polyethylene glycol (PEG) modified albumin, for treatment of such diverse hypovolemic conditions as shock, sepsis, bleeding and surgery. In a preferred embodiment the composition has at least one protected thiol region. In another embodiment, the albumin is modified with an indicator reagent.
Massive resources have been expended on the development of potential therapies aimed at reversing the hypovolemia that is common to different manifestations of systemic inflammatory response syndrome (SIRS). Sepsis alone accounts for 750,000 cases per year in the United States, resulting in 200,000 deaths (1). This high mortality results from multi organ dysfunction (MODS), which is associated with organ edema secondary to capillary leak (CL). Patients with significant CL are typically managed by administering resuscitation fluids containing osmolytes (e.g., albumin, starches, or dextrans) in addition to vasopressors and other supportive measures.
Capillary leak, which is present in different conditions such as multiorgan dysfunction (MODS), sepsis, trauma, burn, hemorrhagic shock, post-cardiopulmonary bypass, pancreatitis and systemic capillary syndrome, causes morbidity and mortality among a large number of hospital patients. Capillary leak (CL) is a central component of MODS, secondary to severe sepsis and systemic inflammatory response syndrome (SIRS). It is characterized by increased capillary permeability resulting in interstitial edema and decreased tissue perfusion leading ultimately to organ failure and death. The leak aspect of capillary leak syndrome (CLS) is reflected in both the release of water into the interstitial space and high molecular weight components of serum which ordinarily would be retained within the capillaries.
Hypovolemic states often lead to hypoperfusion of vital organs, causing organ dysfunction and ultimately resulting in morbidity and death (2). Hypovolemia can occur either rapidly, as with hemorrhagic shock, or progressively due to an underlying disease, with both types involving a systemic inflammatory process. In hemorrhagic shock, hypovolemia occurs due to a rapid and sudden loss of intravascular volume. Upon resuscitation, an inflammatory process may be triggered in reperfused tissues (ischemic—reperfusion injury) causing endothelial cell (EC) injury and capillary leak (CL) leading to a secondary hypovolemic state. In sepsis and other diseases, systemic inflammation is triggered by the disease and in a similar sequence leads to EC injury, CL, and ultimately hypovolemic shock.
Resuscitation with plasma volume expanders remains a mainstay in treating hypovolemia, but with mixed results. The efficacy and safety of volume expanders, including both colloids (e.g., albumin and starches) and crystalloids, continue to be topics of intense research and controversy (3,4). The unpredictable effectiveness of albumin as a plasma expander may be linked to the severity of the underlying EC injury (5). Specifically, if the endothelial integrity is compromised such that albumin can readily extravasate, the leaking albumin may exacerbate the oncotic gradient favoring CL, as opposed to reversing it.
Though the biological mechanisms that induce CL syndrome are poorly understood, some evidence indicates the involvement of inflammatory cytokines. Fluid replacement with solutions of human albumin is only marginally effective since it does not stop the loss of albumin into the extravascular space. Albumin is important because it is responsible for plasma oncotic pressure as well as for retaining sodium ions in the blood.
Under normal conditions, albumin contributes to about 80% of the total blood colloid osmotic pressure (6) and is ideally sized such that it extravasates at a low physiologic rate (7). In CL patients, 5% to 20% albumin solutions are often administered to increase circulating blood volume and to augment intravascular osmotic properties. This method of retarding CL makes the tenuous assumption that albumin can maintain its normally low extravasation rate during shock. Clinical data, however, show that the efficacy of albumin is inconsistent at best (8,9). Some have even suggested that resuscitation with albumin may increase mortality in critically ill patients (10).
PEGylation has been used extensively (11,12). Modification of interferon beta-1a with polyethylene glycol prolongs its half-life, resulting in higher antiviral activity (13). There have been studies on the use of PEGylated hemoglobin (PEG-Hb) as a substitute for blood (14,15,16). Large amounts of PEG-Hb, constituting up to 80% vascular volume showed that PEG-Hb is effective in maintaining the hemodynamics and oxygen delivery in the rat (17). These studies suggest that PEG-Hb is safe even at very high doses.
Other colloids have been used to treat capillary leak conditions with varying degrees of efficacy. A variety of heterogeneous (Mr weighted average: 125,000-450,000 Da) starch colloids have been proposed or are in use as substitute for albumin (18). While these compounds are less expensive and more readily available than pooled human albumin, use of starch colloids has been restricted to low doses due to safety issues that severely limit their use. In addition, the high Mr (>1,000,000 Da) moieties within the heterogeneous starch colloids can alter blood rheological properties and cause coagulopathy (19). The relatively homogeneous Pentastarch (Mr=110,000) has been shown to attenuate lung injury in an aortic occlusion reperfusion injury model (20).
In a recent study, MAP and heart rate (HR) did not change favorably when hetastarch (HES) was given in a septic pre-treatment rat model (21). In contrast, favorable changes in MAP (increased) and HR (decreased) were observed in rats pre-treated with polymerized hemoglobin. This occurred despite the fact that, at the same molar concentrations, the colloid osmotic pressure of HES (27 mm/Hg) was higher than the polymerized hemoglobin (21 mm/Hg). Use of the latter as a routine plasma expander is however controversial and is complicated by potential side effects particularly in relation to the kidneys.
Finally, several studies have suggested that albumin has an endothelial anti-apoptotic effect by mediating regulation of cellular glutathione and nuclear Factor Kappa B activation (22,23,24). This may play a significant role in sepsis induced CL particularly in light of a recent report that linked CL in different systemic inflammatory response manifestations to endothelial cell apoptosis (25).
The available albumin today has a molecular weight of 69,000 with a very short half-life (4-6 hours) which can easily leak to the extravascular space in capillary leak conditions such as severe sepsis, pancreatitis, burn and trauma. This leaking can cause worsening edema and/or compartment syndrome. The use of pentastarch and hexastarch are of limited value since they are not for use in pediatric patients and can cause bleeding. Additionally, only 15 cc/kg can be used in patients. Further, the pentastarch and hexastarch have been shown to cause intractable pruritus (itching) after use and the effect lasted for years. In fact, some studies state that the use of albumin as a replacement or as a volume expander is counterproductive since it increases edema by drawing fluid out of the capillaries.
Therefore, there is a great need for a composition and a method to effectively prevent and/or treat hypovolemic conditions which does not have the above-described disadvantages.
In particular, it is to be noted that Hemorrhagic shock (HS) is a leading cause of death following trauma (1a-3a). Early management requires, in addition to controlling the hemorrhage, providing fluid therapy to restore tissue perfusion. The choice of initial fluid therapy can have a significant impact on the outcome. After hemorrhagic shock and resuscitation, nuclear factor-κB (NF-κB) is activated, triggering an inflammatory response, characterized by overproduction of cytokines such as TNF-α, chemokines and cell adhesion molecules which activate endothelial cells (EC), macrophages, neutrophils and other cells (4a). These activated cells (5a, 6a) generate oxidation products such as reactive oxygen species (ROS) which cause vascular damage and capillary leak (CL) (7a-10a). Oxidants and free radicals produced following reperfusion are potent inducers of apoptosis (11a), especially of the EC. Shrinkage of these cells worsens the widening of the inter-endothelial cell gaps and exacerbates the capillary leak (12a) leading to albumin loss. In this environment of oxidative stress with low levels of albumin, endothelial integrity is compromised (32a,34a,35a). Oxidation products, cytokines and vascular depletion, worsened by CL, contribute to vascular unresponsiveness to intrinsic and extrinsic pressors (10a, 13a, 14). These events are summarized in
In another area of note, recent studies indicate that the type of fluid used in hemorrhagic shock resuscitation affects the physiologic response, the immune response and the systemic inflammatory state.
Crystalloids—Lactated Ringer's (LR) and artificial (synthetic) colloids activate neutrophils and up-regulate cell adhesion molecules; these effects are not seen with albumin or fresh whole blood (10a,11a). Moreover, animals resuscitated with LR or artificial colloids developed significant apoptosis, especially in the lungs and spleen (15a, 16a). Aggressive high volume resuscitation, without controlling the bleeding, can exacerbate the hemorrhage by disrupting the early formed soft thrombi, and by diluting coagulation factors (17a). Conversely, small volume resuscitation using hypertonic saline (7.5%, HTS) alone or in combination with a synthetic colloid is superior to high volume resuscitation, especially in head trauma and in patients at increased risk for developing abdominal or extremity compartment syndrome. However, adverse effects have been reported with small volume HTS used alone or in combination with a synthetic colloid, including hyperchloremic acidosis (18a), and anaphylactoid reactions linked to the colloid component (19a). Other fluids in preclinical testing, such as lactate ethyl pyruvate and ketone based fluids, show less cellular injury and better survival in hemorrhaged animals compared to LR (20a, 21a).
Colloids—The efficacy and safety of colloid plasma expanders, including albumin, are controversial (22a, 23a). Artificial colloids, including starches (24a), have been substituted for albumin in treating capillary leak conditions with varying efficacy. While less expensive and more readily available than human albumin, starch colloids are restricted to low doses because the high Mr (>1,000,000) components alter blood rheological properties and cause coagulopathy (23a). In contrast to albumin, synthetic colloids activate inflammatory and apoptotic processes (25a). Albumin does not increase expression of neutrophil adhesion molecule CD-18, an important step in reperfusion injury, while artificial colloids do (26a). Albumin, which accounts for 80% of blood colloid osmotic pressure (27a), extravasates at a low physiologic rate (28a). In patients with CL, 5% or 25% albumin solutions are administered to increase blood volume and to maintain the oncotic gradient. The efficacy of albumin treatment is variable (29a) and some studies indicate that albumin resuscitation may actually increase mortality (30a). However, a recent randomized double blind controlled clinical study in New Zealand and Australia, involving more than 7000 trauma patients receiving normal saline or 4% albumin, showed no difference in 28 day mortality between the two groups (31a), (study presented by Dr S. Finfer at the 33 rd Congress of Society of Critical Care Medicine, February 2004, Orlando, Fla.).
Albumin as an anti-apoptotic and anti-inflammatory agent—In spite of the conflicting studies of the clinical efficacy of albumin resuscitation, a number of lines of evidence indicate that albumin maintains the integrity of the vascular endothelium (32a-34a) by filling hydrophilic pores of the endothelial surface layer, contributing to their stability (35a). Studies employing human tissue explants in rat skin (36a, 37a) indicate that albumin inhibits endothelial cell apoptosis. Albumin acts as a source of thiol groups (Cys-34); this effect has been demonstrated in septic patients with increases in overall thiol concentration of up to 50% following administration of 200 ml 20% albumin (38a). In vitro mechanistic studies showed that albumin exerts its endothelial anti-apoptotic effect by regulating cellular glutathione and NF-κB deactivation. Physiological concentrations of albumin inhibit TNFα induction by inhibiting NF-κB activation (39a). In a rodent model of HS, 25% albumin resuscitation diminished NF-κB translocation and cytokine-induced neutrophil chemoattractant messenger RNA concentrations (40a).
However, it is also to be noted that is albumin is ineffective in hemorrhagic shock. The ineffectiveness of unmodified albumin as a plasma expander in the previous studies (27a, 29a, 30a) may be linked to the severity of the underlying endothelial cell injury. If the endothelial integrity is compromised such that albumin can readily extravasate, the leaking albumin may exacerbate the oncotic gradient favoring capillary leak (41a).
One aspect of the present invention relates to a composition comprising an albumin-based colloid composition. In one aspect, the albumin-based colloid composition is modified such that its hydrodynamic radius is sufficiently large to preclude its leaking through the capillaries while retaining its oncotic properties and its ability to bind ligands such as sodium ions, fatty acids, drugs and bilirubin. While a number of proteins have been modified with polyethylene glycol, attached through the ε-amino group of lysine, without loss of biological activity and without significant toxicity, no one has before modified human albumin with a PEGylation product (including polyethylene oxide) at multiple sites on the albumin protein. The present invention contemplates the use of PEGylation products which expand the composition's hydrodynamic ratio to a degree such that, when administered to a patient in danger of, or suffering from a hypovolemic state, the albumin-based colloid composition reverses the hypovolemic condition.
The albumin-based colloid composition of the present invention is especially useful for volume expansion in states of shock such as severe sepsis, shock, pancreatitis, burn and trauma, thereby improving survival rates in those conditions.
The albumin-based colloid composition is also useful as a hyperosmotic agent driving, or causing, ultra filtration in peritoneal dialysis. Still other uses include, for example, use in head trauma, hyperviscosity states, patients with liver cirrhosis following parcenthesis, eukopheresis, nutritional albumin deficiency, nephrotic syndrome, liver failure, severe hypoalbuminemic patients, and severe burn patients.
In one aspect, the present invention comprises a composition of an albumin-based colloid composition having a preferred degree of hydration. The present invention further relates to two methods to produce the albumin-based colloid composition by modifying the albumin with polyethylene oxide: one is by using N-hydroxysuccinamide esters and the other is by using cyanuric chloride derivatives. The albumin-based colloid composition of the present invention is safe and has an extended useful half-life. The albumin-based colloid composition can be synthesized using recombinant albumin which decreases its immunogenicity.
The albumin-based colloid composition has a lessened tendency to extravascate because of its larger size, thereby avoiding worsening of the hypovolemic condition such as capillary leak syndrome and clinically, edema and compartment syndrome.
In another aspect, the volume-expanding properties of the albumin-based colloid (or example, albumin with covalently attached polyethylene glycol (PEG-Alb) is a large albumin-based colloid composition which has a greater degree of hydration and a larger hydrodynamic radius. The albumin-based colloid composition is less likely to enter the extra vascular space than normal albumin. Additionally, the albumin-based colloid composition retains the important physiologic functions of albumin, including roles as an osmolyte, as an antioxidant, and as a transporter of less soluble metabolites such as heme and bilirubin; the latter two features are not associated with other crystalloids and colloids.
In one aspect, the present invention relates to a composition comprising a large albumin-based colloid with a preferred degree of hydration. The composition is an albumin-based colloid and, in one embodiment, comprises a polyethylene glycol modified albumin having a hydrodynamic radius sufficiently large to preclude the molecule from leaking through a patient's capillaries. In certain embodiments, the albumin-based colloid composition has a molecular weight of at least about 80 to about 250 KD or greater. The composition can comprise human albumin, bovine serum albumin, lactalbumin, or ovalbumin.
The albumin-based colloid composition has an ability to bind ligands such as sodium ions, fatty acids, bilirubin and therapeutic drugs.
In another aspect, the present invention relates to an in vivo method of preventing or treating hypovolemic conditions comprising administering a therapeutic amount of the large albumin-based colloid composition to a patient in danger of developing such conditions.
In another aspect, the present invention relates to a method for the prevention of mammalian tissue injured or at risk of injury comprising the administration of a therapeutic amount to a mammal of a composition comprising an albumin-based colloid. The composition is incapable of leaking through the mammal's capillaries and is present in an amount of sufficient to protect the tissue from injury. The method is especially useful where the risk of injury is due to hypovolemia, sepsis, shock, burn, trauma, surgery, predisposition to capillary leak, hyperviscosity stress, hypoalbuminemia, and/or anoxia.
Yet another aspect of the present invention relates to a method for forming an albumin-based colloid composition which comprises modifying albumin with polyethylene oxide. The albumin is modified by using N-hydroxysuccinamide esters, or, alternatively, is modified by using cyanuric-chloride derivatives. In certain embodiments, the method includes dissolving albumin in potassium phosphate to form an albumin solution, activating methoxy polyethylene glycol with cyanuric chloride and dissolving in water to form a methoxy polyethylene glycol solution, adding the methoxy polyethylene glycol solution to the albumin solution to form a mixture, stirring the mixture for a suitable time at about room temperature, dialyzing the mixture against a phosphate buffered saline solution at about 4° C. for a suitable time, and collecting polyethylene glycol modified albumin. In certain embodiments, the ratio of a volume of the methyoxy glycol solution to a volume of the albumin solution is in the range of about 1 to about 3.
In a cecal ligation and puncture (CLP) and endoxtoxemic rat models, superior (2-3 hours) fluid resuscitation properties of albumin when conjugated at multiple sites with methoxy polyethylene (PEG-Alb) compared to either unmodified albumin or crystalloid. The larger PEG-Alb (about 16 times albumin size) and its enhanced colloid osmotic property lead to less extraveasation under sepsis—capillary leak (CL) conditions. Consequently, PEG-Alb treated rats showed improved blood pressure recovery and less CL-induced hemoconcentration. In addition in endotoxemic rats, there is evidence of less lung tissue injury with PEG-Alb. PEG-ylation of proteins increases their intravascular retention time (half-life) possibly by reducing physiologic turnover (e.g., protecting against proteolysis) and antigenicity. This invention describes and verifies a method that 1) allows for the simultaneous (i.e., in same subject) assessment of albumin and PEG-Alb intravascular retention times, and 2) provides visualization of extravascular (or leaked) albumin and PEG-Alb as a measure of vital organ injury. This method is based on a double chromophore technique where albumin and PEG-Alb tagged by spectroscopically distinct chromophores and their concentrations are repeated assess over time. The albumin is modified with an indicator reagent.
More specifically, the methods of this invention relates to the preparation of dye conjugated albumin and PEG-Alb. Human albumin (50 mg/ml) was incubated 1 hr in 50 mM potassium phosphate (pH 7.5), 150 mM NaCl, and 0.5 mM dithiothreitol. The dithiothreitol-treated albumin was incubated two hours with 4 mM 5-iodoacetamidofluorescein or 1.5 mM Texas Red maleimide (Molecular Probes). The dyemodified albumins were diluted five-fold and reconcentrated three times in a centrifugal concentrator (10,000 Mr cut off, Millipore) to remove most of the unincorporated dye, followed by dialysis for 48 hours against four changes of phosphate-buffered saline.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
According to one aspect of the present invention, unlike starches, the albumin-based colloid composition retains the important physiologic functions of albumin, including roles as an osmolyte, as an antioxidant (26), and as a transporter of less soluble metabolites such as heme and bilirubin (27); the latter two features are not associated with other crystalloids and colloids. Protein unfolding studies performed on PEG-Alb indicated that albumin functionality is highly preserved).
According to the present invention, the colloid oncotic properties of the albumin-based colloid composition are superior to those of unmodified albumin with regard to plasma volume expansion during treatment of hypovolemic. The albumin-base colloid composition reduces the likelihood of end organ injury, and hence morbidity and mortality, in critically ill patients. The present invention also relates to a method for the pretreatment of septic patients to prevent or ameliorate ARDS and maintain blood pressure. The albumin-based colloid composition of the present invention, with its larger molecular weight (preferably about 80 KD or greater) and augmented colloid osmotic function, is vastly superior to saline or albumin with regard to improving the physiological and histologic manifestations of endotoxin-induced shock.
The albumin-based colloid composition is kept in the intravascular compartment in patients, even in sepsis conditions where capillary leak occurs. In the lipopolysaccharide (LPS) induced model of sepsis in rats, there was no difference in hematocrit (HCT) pre-experiment, however after inducing sepsis, the hematocrit of the saline and albumin treated groups increased while that of the PEG-Alb group decreased.
The maintenance of blood pressures in sepsis is also important. The efficacy of PEG-Alb, saline and albumin treatments for prevention of sepsis induced hypotension are shown in
The histopathologic findings clearly show that the PEG-Alb treated group exhibits less alveolar damage than the albumin group. (
Lanes 1 and 4 contain standard markers which are from top to bottom: 1) Myosin (MW 205 KD); 2) Phosphorylase (97 KD); and 3) Bovine serum albumin (66 KD). Lanes 2 contains human serum albumin after pegylation and its molecular weight over 200 KD. Lane 3 contains human serum albumin before pegylation.
According to the present invention, pretreatment of rats with PEG-Alb prior to induction of sepsis with LPS dramatically reduces the manifestations of LPS-induced shock when compared to pretreatment of animals with saline or unmodified albumin. High dose of LPS was given because rodents are relatively resistant to LPS, and sustained hypotension is needed to simulate the severe human sepsis with MODS. PEG-Alb gives a more rapid recovery in blood pressure, a lower hematocrit—suggesting hemodilution as opposed to the hemoconcentration that characterizes CL—and significantly reduced lung injury. The larger effective size of the PEG-Alb molecule renders it less likely to extravasate in the presence of cell injury and during a loss of endothelial integrity.
The shock that follows administration of an endotoxin is characterized by a biphasic blood pressure response. In the first phase, a drop in blood pressure occurs 10-15 minutes after LPS is injected. This was evident in all of the LPS-injected animals, suggesting that PEG-Alb does not act by neutralizing the endotoxin itself. The second phase of hypotension is caused predominantly by the action of inducible nitric oxide (iNOS), which substantially reduces plasma volume (28). It is during this second phase that PEG-Alb has a superior effect when compared with albumin or saline. Although iNOS m RNA or peptide was not measured, it is very likely under these conditions employed here; i.e., intravenous administration of 20 mg/Kg LPS that iNOS was induced. While inherent limitations exist with any pretreatment model, the data show that administering PEG-Alb prior to LPS protects rats from developing ARDS.
The hematocrit, mean arterial pressure, and histology all indicate that PEG-Alb is a beneficial treatment for the LPS-induced hypovolemia. Both the hemodilution and the unchanged MAP achieved with the PEG-Alb treatments are indicative of plasma volume expansion (or at least maintenance), while the opposite effects were observed with both albumin and saline. Maintenance of intravascular volume with PEG-Alb is consistent with reduced capillary leak. Histopathologic findings (
The improved colloidal properties of PEG-Alb result from increased hydrophilic properties, which are shown by its very large hydrodynamic radius—as reflected in its behavior on a gel filtration column and its larger molecular radius of gyration (RG) and excluded volume (Λ) as inferred from its nonideal osmotic properties. This was also demonstrated using size exclusion chromatography where the elution ratio of PEG-Alb/albumin agreed with the excluded volume of PEG-Alb/albumin (
The colloid oncotic properties of PEG-Alb are superior to those of unmodified albumin with regard to plasma volume expansion during treatment of hypovolemia associated with CL. PEG-Alb is useful to reduce the likelihood of end organ injury, and hence morbidity and mortality, in critically ill patients. The present invention is useful in the pretreatment of patients to prevent or ameliorate ARDS and maintain blood pressure. PEG-Alb, with its larger molecular weight and augmented colloid osmotic function, is vastly superior to saline or albumin with regard to improving the physiological and histologic manifestations of endotoxin-induced shock.
The following examples are provided merely to further illustrate the present invention. The scope of the invention shall not be construed as merely consisting of the following examples.
Materials and Methods: Preparation of PEG-Alb. 2 gms of human albumin (Sigma, St. Louis, Mo.) was dissolved in 45 ml. of 50 MM of potassium phosphate (mixture of mono and dibasic), pH 7.4. 500 mg of methoxy polyethylene glycol (Sigma, St. Louis, Mo.) was activated with cyanuric chloride and dissolved in 4 ml. of water. 1.4 ml of methoxy polyethylene glycol solution was added to 45 ml of the human albumin solution and the mixture was stirred for two hours at room temperature. The mixture was transferred to a dialysing tube (molecular weight cut off—12500) and dialysed against 3000 ml of phosphate buffered saline at 4° C. for 72 hours. The polyethylene gylcol modified albumin (PEGA) was collected and then frozen at −20° C. until its use.
Animals. Adult male Sprague-Dawley rats (Charles River Laboratories, Portage, Mich.) weighing 400-480 grams were used. Animals were housed in an American Association for Accreditation of Laboratory Animal Care, International (AAALACI) approved facility. They were provided standard rat chow and water ad libitum. All protocols were approved by the Institutional Animal Care and Use Committee and the ABC (Hazard) Committee.
Methods
The animals were fasted overnight, but given water ad libitum. Animals were anesthesized using Sodium pentobaribital (50 mg/kg) intraperitoneally and given additional doses as needed during the course of the experiment. An arterial cathether (Intramedic PE-50, Clay Adams) was placed on the carotid artery and hooked to the transducer/amplifier for continuous blood pressure monitoring (TestPoint, Capital Equipment Corporation, Billerica, Mass.). An intravenous line was placed on the opposite internal jugular vein using G24 cathether. A blood sample was taken from the carotid line for baseline hematocrit and albumin and replacement fluid (1 ml 0.9% saline) was infused via the intravenous line. Normal saline 5 ml was infused in group 1. Albumin 0.6 gms/kg body weight (BW) was given to group 2 and PEGA 0.6 gms/kg BW was given to group 3. After 30 minutes, endotoxin (LPS) (Sigma Chemicals, St. Louis, Mo.) was given to the three groups at varying doses. The rats were divided into 3 groups based on the received resuscitation fluid: Group 1 (n=9) received unmodified albumin in normal saline solution at a 0.6 gm/kg dosage; the injection concentration of albumin was 40 mg/ml, yielding an injection volume of 1.5 ml/100 g body weight (BW). Instead of albumin, Group 2 (n=12) received PEG-Alb at the same dosage, protein concentration, and injection volume as at Group 1. Group 3 (n=6) received 1.5 ml/100 gm BW of normal saline. Blood pressure monitoring was done for three hours after endotoxin infusion after which the rats were euthanized.
Post-experiment blood samples for hematocrit and albumin were taken. The right lung was put in formalin and set to pathology for hematoylin-eosin staining.
PEG-modified albumin (PEG-Alb) was examined as a potential plasma volume expander. Albumin modified at multiple sites, exhibited a larger effective molar volume and exerted greater osmotic pressure than unmodified albumin. Solutions of PEG-Alb, albumin, and saline were tested in a rat endotoxin-induced model of shock. Pretreatment with polyethylene glycol modified-human albumin (PEG-Alb) maintained mean arterial pressure (p=0.023), retained volume as evidenced by hemodilution (p=0.001) and attenuated the histologic manifestations of acute respiratory distress syndrome (ARDS) (p=0.002). Rats were pretreated with fluorescence labeled PEG-Alb and rhodamine labeled albumin, separately and in combination, followed by treatment with LPS. Fluorescence microscopy of lung sections indicated that fluorescence-labeled PEG-Alb was retained within the blood vessels rhodamine-labeled albumin was not. Compared with the use of saline or unmodified human albumin, PEG-Alb is a useful alternative plasma volume expander that may be of use in hypovolemic states.
Preparation of Albumin and PEGA (PEG-Alb)
Methoxypolyethylene glycol cyanuric chloride (average Mr 5000) was added to human albumin (type V, Sigma Chemical Co.) dissolved in 50 mM KPI (pH 7.5) at 50 to 60 mg/ml with gentle stirring four times (0.2 mg per mg of albumin per addition) at 10-minute intervals at 22° C. The reaction was allowed to stir 40 minutes after the last addition of the reagent. Modification was rapid, being complete in less than 15 minutes at room temperature with the extent of modification depending primarily on the amount of reagent added. Prior to infusion into animals, both albumin and PEG-Alb were dialyzed against phosphate-buffered saline for 48 hours with three changes of buffer using high-molecular-weight-cutoff dialysis tubing (50 kDa molecular mass cutoff).
FITC-Albumin and FITC-PEG-Alb
Human albumin (50 mg/ml) was incubated 1 hr in 50 mM KPi (pH 7.5), 150 mM NaCl, and 0.5 mM dithiothreitol. The dithiothreitol-treated albumin was incubated two hours with 4 mM 5-iodoacetamido fluorescein or 1.5 mM tetramethylrhodamine-5-iodoacetamide. The flourescein-modified albumin was dialyzed 48 hours against four changes of phosphate-buffered saline to remove free flourescein. Rhodamine-labeled albumin was chromatographed on Sephadex 50 followed by extensive dialysis against phosphate-buffered saline.
Some of the flourescein-labeled albumin was modified with methoxypolyethylene glycol cyanuric chloride and purified by gel filtration on Sephacryl S200. Fractions from Sephacryl S200 eluting with apparent molecular weights in excess of 200,000 were pooled and concentrated using an Amicon ultrafiltration cell with a PM10 membrane. Analysis of the flourescein and rhodamine-labeled albumins by gel electrophoresis revealed that the fluorescence was associated with the protein; no fluorescence was detected at the positions of free flourescein or rhodamine.
Physiological Studies
Experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) and the Academic Chemical Hazardous Committee (ACHC) at the Medical College of Ohio. Adult male Sprague-Dawley rats (Charles River Laboratories, Portage, Mich.) weighing 400-480 grams were used. Animals were housed in an American Association for Accreditation of Laboratory Animal Care, International (AAALACI) approved facility. They were provided standard rat chow and water ad libitum. Prior to the experiment, the animals were fasted overnight, but given water ad libitum.
All rats were anesthetized using sodium pentobarbital (50 mg/kg body weight) intraperitoneally followed with additional intravenous maintenance doses at 1 hour intervals. Mean arterial pressure (MAP) was continuously measured via a catheter (Intramedic PE-50, Clay Adams) placed in the right carotid artery and attached to a blood pressure transducer and amplifier (BLPR and TBM4, World Precision Instruments, Sarasota, Fla.) and collected on a computer (TestPoint, Capital Equipment, Billerica, Mass.). An intravenous line for infusion was inserted in the left jugular vein (G24 Protectiv*Plus, Johnson and Johnson/Ethicon, Arlington, Tex.).
The rats were divided into 3 groups based on the received resuscitation fluid: Group 1 (n=9) received unmodified albumin in normal saline solution at a 0.6 gm/kg dosage; the injection concentration of albumin was 40 mg/ml, yielding an injection volume of 1.5 ml/100 g body weight (BW). Instead of albumin, Group 2 (n=12) received PEG-Alb at the same dosage, protein concentration, and injection volume as at Group 1. Group 3 (n=6) received 1.5 ml/100 gm BW of normal saline. A 1 ml baseline blood sample was taken for baseline hematocrit (Hct) measurement from the carotid line and replaced with the same volume of 0.9% saline. MAP monitoring was initiated at the start of the fluid infusion. After 30 minutes, 20 mg/kg BW of Endotoxin (E. Coli lipopoly-saccharide [LPS] from serotype 055: B45, Sigma Chemicals, St. Louis Mo.) dissolved in 1 ml of saline was administered, and the rats were monitored for 3 hours thereafter. A blood sample was then taken for post sepsis Hct assessment, and then rats were euthanized with 150 mg/kg/BW of Pentobarbital IP and exsanguinated. Finally, one kidney and the lungs were harvested and immediately fixed in 10% formalin for subsequent histologic examination.
Histologic Studies
The lung and kidney tissues were removed from formalin solution and subjected to standard processing, including a hematoxylin and eosin stain. These coded preparations were examined with a light microscope by a blinded pathologist, who scored the inflammatory histopathologic features using the following five-point system: 0=no significant histopathologic changes; 1=minimal interstitial inflammatory infiltrates; 2=mild interstitial inflammatory infiltrates with mild hyalinization; 3=moderate interstitial inflammatory infiltrates with moderate hyalinization; 4=severe interstitial inflammatory infiltrates with severe hyalinization. In order to ensure consistency, the same pathologist examined samples on two separate occasions, and the averaged score was used.
Molecular/Biophysical Studies
SDS Gel Electrophoresis. Samples of unmodified albumin and PEGA were prepared for electrophoresis by adding SDS (1%, W/V) and beta mercaptoethanol (5%, V/V) and heating in a boiling water bath for 1 minute. Samples were subjected to electrophoresis on 7.5% or 10% acrylamide gels (30).
Size Exclusion Chromatography
Albumin and PEGA were analyzed by size-exclusion chromatography on a 24 ml bed volume Superose 6 column (Pharmacia). Samples or a mixture of standards (in 0.5 ml) were applied to the column and eluted with 10 mM potassium phosphate (pH 7.5) and 150 mM NaCl at 0.5 ml min−1. Absorbance at 280 nm was monitored continuously.
SELDI-TOF Protein Analysis
Surface-enhanced laser desorption/ionization-time of flight (SELDI-TOF) mass spectrometry was used to characterize the PEG-albumin and albumin samples. One microliter of sample (at 1 to 5 mg ml−1) was deposited and allowed to air dry directly onto a 2 mm spot of an alaphatically coated aluminum ProteinChip array (H4 ProteinChip, Ciphergen Biosystems, Palo Alto, Calif.). Twice, one half microliter of energy absorbing matrix (EAM, a saturated solution of 3,5-Dimethoxy-4-hydroxycinnamic acid in aqueous 50% acetonitrile and 0.5% triflouroacetic acid) was applied to the sample and allowed to air dry.
The ProteinChip array was transferred to a ProteinChip reader and a laser (N2 320 nm-UV) was focused on the sample in a vacuum chamber. After 2 warming laser shots, proteins absorbed to the matrix were ionized and desorbed from the array surface. Ionized proteins were detected and molecular masses were determined using TOF analysis. The TOF mass spectra were collected in the positive ion mode with a ProteinChip System (PBSII series, Ciphergen) using Ciphergen Peaks (version 2.1b) software. Real-time signal averages of 65 laser shots were averaged to generate each spectrum.
Colloid Osmotic Pressure (COP)
Both PEGA-Alb and albumin were prepared for COP measurements in similar fashion. Briefly, samples were dissolved in 10 mM potassium phosphate (pH 7.5), 150 mM NaCl at 50 mg ml−1, treated with dithiothreitol (0.5 mM dithiothreitol) for 1 hour at 30° C., and then incubated with iodoacetamide (5 mM iodoacetamide) for 1 hour at 30° C. The acetamidated albumin (5 ml at 50 mg ml−1) was then subjected to chromatography on Sephacryl S300 (2.8 cm×40 cm) equilibrated in 10 mM potassium phosphate (pH 7.5) and 150 mM NaCl to reduce albumin dimer and other low and high molecular weight contaminants that otherwise interfere with determination of osmotic pressure. Finally, both albumin and PEGA were dialyzed against several changes of 0.9% NaCl.
COP measurements with each colloid were repeated over a wide range of concentrations using the Wescor Model 4420 colloid osmometer (Logan, Utah). The instrument was blanked with 0.9% saline and calibrated with a 20.2 mOsm albumin standard solution. Note, the concentration of unmodified albumin was determined from absorbance at 280 nm (ε280 nm, 1%=5.31) (31) and were confirmed by dry weight measurements. PEG-Alb concentrations were estimated from dry weight determination.
COP [π] in terms of concentration [c] were analyzed via a nonlinear least squares fit of the equation to estimate 1) estimate the weighted molecular Mass [Mr] reflected from the ideal component of the π-c relation (32) and 2) the non-ideal contributions of all other virial coefficients via the two parameters B and α:
This form of the equation is a slight modification yet more flexible form of the traditionally employed equation [π=RT(c/Mr+Bc2+Cc3 . . . )] that avoids a priori assumptions of number of virial coefficients; R=63.364 mm Hg M−, c is concentration (g per dl), and T is temperature (295° K).
Statistical Analysis
The difference between pre and post-LPS hematocrits among these three treatment groups was compared by ANOVA, whereas two-way repeated measures of ANOVA were used to compare mean arterial pressure (MAP) before LPS and at multiple time points after LPS. Individual differences between groups were assessed using a Tukey multiple-comparison test. A p<0.05 was used to indicate statistical significance.
Physiological Studies
Vascular volume contraction/expansion following LPS—induced sepsis was inferred from the changes in MAP and Hct. Both of these measures varied significantly for rats pre-treated with PEG-Alb, albumin or saline. Initially, within 15-25 minutes post LPS bolus infusion, all three groups showed a similar drop of ˜40% in MAP (Saline: 135±11 down to 81±30 mmHg; Albumin: 134±14 down to 85±20 mmHg; PEG-Alb: 125±12 down to 79±19 mmHg) (
Pre-LPS hematocrit was similar in all study groups [44±2 (saline), 42±3 (albumin) and 45±2 (PEG-Alb)]. At 3 hours after LPS, hematocrit (post) was elevated relative to baseline (pre) levels for both the albumin (Hct Ratio (post/pre)=1.09±0.11) and saline (Hct Ratio=1.19±0.09) treated rats indicating a relative decrease in intravascular fluid volume or hemoconcentration (
Histologic Studies
Microscopic examination of lung tissue sections taken from PEG-Alb-treated and control (no-sepsis) rats did not reveal significant histopathological changes (
Results from example normal (
Biophysical Properties of PEG-Alb
Molecular size—The results of SDS gel electrophoresis of albumin and PEG-Alb are contrasted in
To examine the extent of PEG modification by a different technique, albumin and PEGA were analyzed by SELDI-TOF mass spectrometry. Both spectra showed multiple peaks that resulted from the a) presence of monomers and multimers and, more relevantly, b) the detection of singly charged (z=1) as well as multi-charged (z≧2) species. Accounting for these effects, the dominant single-charged albumin monomer spectral peak was centered around a molecular mass of 66,880±2,800 Da (
Colloid osmotic pressure (π). To evaluate the properties of PEG-Alb as an osmolyte compared to albumin, we examined their osmotic pressure (π) over a wide range of concentrations (g/dL). Both albumin and PEG-Alb, albeit differently, showed nonlinear dependence of osmotic pressure with respect to protein concentration (
The two methods for estimating molecular weight (SELDI and colloid osmometry) provided similar estimates for albumin but not PEG-Alb. For the latter, the π-based estimate was greater than expected at 128,000 Da. Since the osmotic pressure derivation provides a number averaged molecular for all species in the solution, then an overestimate of molecular weight by this method is consistent with the presence of multimers. While not wishing to be bound by theory, it is believed this is a likely explanation of these apparent differences since the SELDI data does indeed suggest the presence of PEG-Alb multimers (
Compared to saline and albumin, pre-treatment of rats with PEG-Alb prior to LPS-induced septic shock resulted in: 1) a more complete recovery in blood pressure, 2) unchanged or slightly lowered hematocrit, suggesting hemodilution as opposed to hemoconcentration that usually characterizes CL, and 3) significantly reduced lung injury.
Since rodents are fairly resistant, a relatively high dose of LPS was used in the experiments to ensure significant and sustained hypotension as a way of simulating severe human sepsis with MODS (35). The hypotension that follows LPS is characterized by a biphasic response. In the first phase, a sharp rapid drop in arterial pressure occurs within 15-25 minutes of LPS bolus infusion. This phase did not differ among the treatment groups indicating that albumin and PEG-Alb did not alter the initial effects of endotoxin relative to saline. The second phase of hypotension is caused predominantly by the action of inducible nitric oxide (iNOS), which substantially reduces plasma volume via CL (36). While iNOS mRNA or peptide was not measured, it is highly likely that iNOS was induced by the administration of a high LPS dose (20 mg/Kg)(37).
The superior effects of PEG-Alb compared to albumin or saline were manifested in this second hypotension phase of endotoxin shock. Evidence of this included the more complete blood pressure recovery and relative hemodilution. Also, minimal interstitial infiltrates and hyalinization in the lung tissues of PEG-Alb-treated rats were evident from lung histopathology while immunflourescence studies in lung tissues showed greater retention of PEG-Alb intravascularly compared to apparent albumin extravasation in the presence of CL. All these are consistent with less capillary leak and greater plasma expanding properties.
The in vitro measurements show that the substantially larger effective size and greater colloid osmotic pressures of the PEG-Alb molecule, relative to albumin renders, is less likely to extravasate in the presence of cell injury and loss of endothelial integrity. Indeed, SEC and colloid osmometry indicated a 13-16 fold increase in the extended molecular structure/excluded volume after pegylation. The improved colloidal properties of PEG-Alb resulted from increased hydrophilic properties, which are reflected by the larger hydrodynamic/gyration radius (RG) and excluded volumes (Λ). In a canine model of endotoxic shock, the severity of capillary permeability was inferred by the measurment of different proteins molecular weights by electropheresis (38). The larger molecular weights corresponded to MW of 900,000 Da and the smallest being the albumin (60,000 Da). The albumin corresponded to a radius of gyration 3.4 nm and Apopferritin dimer, the largest protein to 12.1 nm, knowing that the larger gaps are far less represented at the endothelium compared to the medium gaps (60,000-500,000 Da) (39), PEG-Alb with its 10. nm size should be retained in the vascular space in moderate to severe leak.
Human albumin (Sigma Chemical Co. type V) at 50 mg ml-1 in 10 mM potassium phosphate (pH7.5), 150 mM NaCl, and 0.5 mM dithiothreitol was incubated for 1 hour at 30° C. Maleimide-methoxypolyethylene glycol 20,000 Mr (Shearwater Inc. cat. Number 2D2MOP01) or maleimide-methoxypolyethylene glycol 40,000 Mr (Shearwater Inc. cat number 2D2MOP01) was added to 1 mM and the reactions were incubated for 1 hour at 30° C. PEG-modified albumins were purified by ion exchange chromatography on Q-Sepharose) Pharmacia).
In another aspect, the present invention relates to a polyethylene glycol-modified albumin (PEG-Alb) developed by the inventors herein that is 16 times larger than albumin (42a); a representation of PEG-Alb is shown in
Unlike synthetic colloids, PEG-Alb retains important physiologic functions of albumin, including roles as an osmolyte, as an antioxidant (38a) and as a transporter of less soluble metabolites such as heme and bilirubin, features that are not associated with other crystalloids and colloids. Studies involving a variety of PEG-modified proteins demonstrate no significant toxicity (48a). The first generation (PEG-Alb1) developed was more effective than albumin or saline in cecal ligation and puncture (CLP) and lipopolysaccharide (LPS) models of severe sepsis. Animals treated with PEG-Alb1 exhibited more intravascular retention of the colloid, better hemodynamics, less capillary leak, and less lung injury. The increased hydrodynamic radius of PEG-Alb1 reduced its extravasation and reduced end organ injury while maintaining blood pressure and organ perfusion. In addition, the biophysical characteristics of PEG-Alb1 such as high colloid osmotic pressure (COP) and high viscosity allows for lowering the “transfusion trigger” point, which is defined as the hemoglobin (Hb) level below which peripheral tissues suffer from inadequate perfusion (49a).
The extravasation of albumin during capillary leak (ischemia/reperfusion) in hemorrhagic shock is critical. Specifically, this loss of albumin from the intravascular space is injurious in two major ways. First, the oncotic force of the albumin is lost, allowing for tissue edema contributing to the development of multi-organ dysfunction. Second, the antioxidant effect offered by albumin is significantly diminished, allowing for oxidant stress to continue to cause vascular injury and perpetuate the capillary leak and extravasation of more albumin. While not wishing to be held to theory, the inventors herein believe that administration of a larger (larger hydrodynamic radius) and functionally preserved (Cys-34 preserved as a thiol for its antioxidant function) albumin (PEG-AlbCys-34) improves outcomes in experimental hemorrhagic shock.
In one aspect, the present invention relates to PEG-AlbCys-34 as a resuscitation fluid for treatment of hemorrhagic shock. PEG-AlbCys-34, with a large effective hydrodynamic radius, will not leak from the intravascular space as is seen with unmodified albumin in capillary leak accompanying ischemia-reperfusion injury (I/R) and shock states. Retention of PEG-AlbCys-34 in blood vessels makes of PEG-AlbCys-34 more effective than unmodified albumin and other resuscitation agents, while retaining the ligand binding, antioxidant, anti-inflammatory and anti-apoptotic functions of albumin.
In another aspect, the present invention is especially useful in military applications. First, PEG-Alb maintains vascular volume as evidenced by better blood pressure recovery after resuscitation in LPS and CLP models of shock. The data also indicate that PEG-Alb is also effective in hemorrhagic shock. Second, because of its biophysical characteristics (high COP, high viscosity), PEG-Alb can lower the transfusion trigger to levels below 7 g/dl. This means that oxygen delivery to peripheral tissues is maintained at lower hemoglobin level for a longer time prior to blood transfusion. Third, PEG-Alb can be lyophilized and rehydrated so that it can be stored and reconstituted under adverse conditions.
Physiological Studies
PEG-Alb1 was examined in three different models of shock, two that mimic septic shock (CLP and LPS) and one that mimics hemorrhagic shock (HS). These studies show that PEG-AlbCys-34 is a more effective resuscitation agent than PEG-Alb1, starches and HTS.
Animal Models
CLP model—Albumin modified at multiple sites with methoxy polyethylene glycol was evaluated. This material is more effective than albumin or saline in maintaining MAP. PEG-Alb1 was also more effective in maintaining serum colloid osmotic pressure. A mixture of mPEG5000 and albumin was no more effective than albumin alone or saline in maintaining blood pressure, indicating that the effectiveness of PEG-Alb requires that the PEG be covalently attached to the protein. As shown in
Endotoxin model—PEG-Alb1 in a rat LPS model of shock was also examined. Consistent with the result in the CLP model, PEG-Alb1 was more effective at maintaining MAP compared to unmodified albumin or saline. In addition, administration of PEG-Alb1 before LPS treatment significantly reduced lung injury compared to saline or albumin treatment. Inflammatory histopathologic changes consistent with severe acute lung injury, including hyalinization and interstitial lymphocyte infiltrates, were detected in most rats treated with saline or albumin while these changes were less evident in rats pretreated with PEG-Alb1; representative H&E sections are shown in
Hemorrhagic shock model (HS)—The effectiveness of PEG-Alb1 to unmodified albumin and saline in a rat volume controlled HS model was compared. Blood (2.6 ml/100 g b.w.) was drawn over 10 minutes simulating hemorrhage; after 90 minutes, resuscitation was initiated with saline, albumin or PEG-Alb1. As shown in
Experimental Model of Hemorrhagic Shock
PEG-Albs, including albumin in which cys 34 is retained as a thiol (PEG-AlbCys-34) are compared with other resuscitation agents in a well characterized rat HS model (51a-53a). a number of physiological parameters are examined that reflect the severity of different aspects of shock, including those related to lung injury, tissue perfusion (base excess, lactic acid), arterial blood gases (ABG), mean arterial pressure (MAP), heart rate (HR) and indices kidney function (creatinine). This information is used to compare PEG-Albs to established agents such as unmodified albumin, starch and hypertonic saline. PEG-Albs is also compared with different extents of PEG modification, with different size PEG, and with different protein-PEG linkages in order to optimize the performance of the PEG-Albs. The experimental model mimics circumstances that occur in real life. Phase I (pre-hospital) corresponds to the initial trauma and the time required to transport an individual to location where resuscitation can be given in the field. This could correspond in practice to resuscitation given by an EMT arriving in ambulance or to resuscitation provided by a medic in a combat zone. Phase II (hospital) corresponds to treatment that would be provided after an individual has been transported to a hospital and where blood transfusion can be administered. Phase III (observation phase) is meant to correspond to the time after treatment in a hospital or a rehabilitation center. The following protocol is used:
Phase I (Pre-hospital)—HS is initiated by volume-controlled hemorrhage (2.6 ml/100 g b.w. over 20 min (H20). Shed blood is retained for reinfusion. At 20 min, MAP is controlled between 40-45 mm/Hg by fluid resuscitation with LR or by blood withdrawal until 80 minutes. At 80 minutes rats are randomized to treatment groups. Treatment is infused over 30 minutes until 110 minutes to simulate resuscitation that would be given in the field.
Phase II (Hospital phase)—At 110 minutes, the shed blood is infused over 10 minutes to simulate transfusion. In previous studies, using this model, some rats died early in phase II with severe hypoglycemia and metabolic acidosis; bicarbonate solution and glucose will be infused to restore MAP to >70-80 mm/Hg and glucose >150 mg/dl until H 270 minutes (53a).
Phase III (Observation phase)—Catheters are removed; anesthesia is discontinued, rats are returned to their cages with access to food and water, and observed until 72 hours. Survivors are evaluated every 24 hours using the rat overall performance score (54, 55) 1=normal, 2=moderate disability, 3=severe disability, 4=coma 5=death). Necropsies are performed on rats that die before 72 hours. Survivors are euthanized. In phases 1, and II rats are anesthetized with pentobarbital (50 mg/Kg i.p) with extra doses (12.5 mg/Kg) given as needed for agitation. Incisions are treated with Bupovacaine (Marcaine 0.025%). The protocol is shown schematically in
Results: Data indicates that the first generation PEG-Alb (PEG-Alb1) is more effective than saline or albumin
The comparison is extended to other standard resuscitation agents. PEG-Alb with protected thiol (PEG-AlbCys-34) is tested. Albumin at 25% has proven to be effective in hemorrhagic shock while 5% albumin has not (40a, 56a); it is important to point out that the volume of resuscitation agent per se is significant (the same amount of albumin is given but in a more concentrated form). The reason the concentrated form is superior may be explained by the fact that threshold concentration of albumin being required to exert the antioxidant effect. Alternatively hyperosmolarity associated with the use of 25% albumin might contribute to the anti-inflammatory effect (40a). Albumin and PEG-Alb at 5% and 25%, are compared based on albumin content. Hetastrarch 6% (Hexstend®) is also used in resuscitation and is compared to PEG-Albs Hypertonic saline (7.5%) is a third resuscitation agent that is compared to PEG-Albs.
Capillary Leak Studies
While not wishing to be held to theory, the inventors here believe that PEG-Albs will be retained within blood vessels during capillary leak conditions and thus maintain the colloid-osmotic pressure of blood. We have shown that PEG-Alb1, which is 16 times larger than albumin, extravasates less in capillary leak conditions associated with CLP and LPS models (42a). We determined this is also the case in hemorrhagic shock model. Using a method we developed, fluorescently labeled albumin and PEG-Alb (Texas Red, TR) and PEG-Alb (fluorescein, F) are injected into rats and a small volume of blood is taken through the tail vein for analysis at different times after injection. Preparation of the labeled albumins is described in the section of this proposal dealing with the biophysical characterization of the PEG-Alb. If albumin is lost to the extravascular space and PEG-Alb to be retained, the ratio of fluorescein to Texas Red (F/TR) will increase with time, consistent with loss of albumin and preferential retention of PEG-Alb. The excitation and emission spectra of Texas Red and fluorescein are sufficiently different that mixtures of the two dyes can be examined quantitatively in serum samples. The distribution of albumin and PEG-Alb is also examined qualitatively in frozen tissue sections of lung by fluorescence microscopy to determine if the PEG-Alb is retained within blood vessels, as observed in the models of septic shock (
The labels (fluorescein albumin and Texas Red PEG-Alb) are switched to verify that the fluorophor does not alter the distribution of the protein. The bronchoalveolar lavage (BAL) is examined for the fluorescent albumin and PEG-Alb; if TR albumin leaks preferentially, one would expect the ratio of F/TR in BAL to decrease. This method is used to compare PEG-modified albumins that we have produced to determine if one is more effectively retained than others.
In an other aspect, an alternate approach is useful to study lung permeability and employs Evans Blue Dye (EBD), which is not permeable to blood vessels (57a). Rats are injected with 1% EBD solution through an internal jugular vein catheter twenty minutes before euthanasia. After allowing for complete circulation of the dye (5 minutes), blood is drawn and EBD concentration is determined in plasma. Rats are euthanized and the lungs, livers are harvested. BAL is performed on the excised lungs by instilling five milliliters of normal saline three times. The left lung lobe is tied off to prevent influx of saline to preserve this lung for the wet-to-dry weights. The lung that was not infused with saline is taken for weighing and is put in a vacuum oven for drying and subsequently measure the wet/dry as a surrogate for extravascular fluid leak. The combined BAL fluid is centrifuged to remove cells, and the supernatant is assayed for EBD. The concentration of EBD in the BAL fluid is expressed as the percentage of that present in the plasma. That is, BAL/Plasma EBD is compared between the treatment groups along with the wet/dry of the lung tissue.
Hemodynamics
A feature of PEG-Alb1 compared to saline and albumin in the septic shock models is its capacity to maintain blood pressure and prevent hemoconcentration. An important issue in the hemorrhagic shock model is how well PEG-AlbCys-34 performs compared to standard resuscitation agents. Rats are anesthetized with sodium pentobarbital followed by maintenance sedation as needed. An arterial catheter (Intramedic PE-50, Clay Adams) is inserted into the right carotid artery, connected to a pressure transducer, amplified and continuously monitored (sampling rate 100 Hz; MP 100, BioPac Systems Inc., Santa Barbara, Calif.) and collected on a computer. An intravenous line (G24 Protective*Plus, Johnson and Johnson/Ethicon, Arlington, Tex.) is inserted in the left jugular vein for infusion of fluids. MAP and HR in animals given various fluid resuscitation agents is monitored. In the pre-hospital phase, all the rats are subjected to similar levels of ischemia for a minimum of 60 minutes, after which they are randomized to treatment groups. The crystalloid group receives three times the volume of the colloid groups, and eight times the volume of HTS group. PEG-Albs shows superior performance in MAP starting in the initial phase (pre-hospital) based on the fact that CL can occur as early as 20 minutes after hemorrhagic shock (58a). In the Hospital phase, PEG-AlbCys-34 group performance is superior to the other treatment groups for the following reasons: 1) Following treatment (reperfusion), capillary leak becomes even more severe and here PEG-Alb is more retained in the vascular space; 2) In contrast to crystalloids and synthetic colloids. PEG-AlbCys-34 improves the sensitivity of the blood vessels to the endogenous pressors by decreasing the oxidation products (10a).
Perfusion Studies
Hypoperfusion of vital organs during hemorrhagic shock is a primary cause of organ dysfunction. A number of physiological parameters indicative of reduced blood flow are examined in order to compare PEG-Alb to other resuscitative agents.
a. Lactic Acid—Lactic acid levels correlate with subsequent organ failure in hemorrhagic shock (59a). Increased levels of epinephrine (secondary to shock) decreases ATP by stimulating the activity of Na+-K+ ATPase (60a), as a result lactate production increases due to mitochondrial dysfunction and anaerobic glycolysis. Improving the perfusion in PEG-Alb treated groups is expected secondary to the following factors: 1) better maintenance of systemic blood pressure and 2) better perfusion at the microcirculation level enhanced by the biophysical characteristics of PEG-Alb (increased COP and viscosity). This increases the shear stress at the capillary level stimulating the increase in nitric oxide, which results in vasodilation and improved perfusion (49a).
b. Base Excess (BE) Fencl-Stewart (61a, 62a) method. Base deficit is defined as the amount of base required to titrate one liter of whole blood to a normal pH at normal physiologic values of PaO2, PaCO2, and temperature (63a). BE is obtained by multiplying the deviation in standard bicarbonate from a mean of 22.9 by a factor of 1.2 (64a). Calculation of BE assumes normal water content, electrolytes, and albumin. This is relevant since significant change in plasma albumin concentration is expected. A decrease in albumin by 1 g/dl decreases the BE by 3.7 mEq/L (61a). BE corrected for changes in sodium, chloride, and albumin in a cohort of pediatric ICU patients showed a better correlation with mortality than calculated BE, anion gap and lactate (62a). Any value ≧−5 mEq/L is significant. Base excess corrected for unmeasured anions (Beua) is defined by:
Beua=BE−(Befw+Becl+Bealb)
c. Viscosity—During treatment of hemorrhagic shock, resuscitation using large volumes of crystalloids and colloids lowers hematocrit and blood viscosity. Historically, the use of colloids and crystalloids in the correction of blood loss was considered safe up to a level called the transfusion trigger (50% Hb lost or Hb of 7 g/dl) (65a). When the hematocrit drops below 50% of baseline, the shear stress at the capillary level will be lowered, resulting in vasoconstriction and decreased oxygen delivery to the tissues. Studies bt Tsai's group and others (49a, 65a, 66a) indicate that increased viscosity helps maintain oxygen delivery to tissues prior to blood transfusion or other agents for delivery of oxygen to tissues. PEG-Alb should increase viscosity as other polymerized proteins do (67a). When PEG-Alb was given at 3 g/dl to CLP rats, serum viscosity (measured with a Cone-Plate Viscometer) was at 3 cP, a level considered necessary to maintain the oxygen delivery at that degree of hemodilution (66a). As shown in
In vivo studies show that “maintaining PEG-AlbCys-34 in the vascular space following ischemia/reperfusion injury where the oxidative stress is intense and the native albumin is leaking” results in augmenting the antioxidant capacity in the vascular space, decreasing apoptosis and controlling of inflammation.
Inflammation Studies
NF-κB is activated following hemorrhagic shock, leading to overexpression and production of cytokines such as TNF-α (68a). The activation of NF-κB during ischemia (69a) or during resuscitation (70a) is considered an important step in initiating and maintaining the exaggerated inflammatory response. Importantly, the volume in which albumin is administered appears to play a significant role in inflammation. 25% Albumin, but not 5% or R/L, decreased neutrophil sequestration in the lung and prevented lung injury following shock/resuscitation (40a). This is the basis for testing albumin preparations using the two concentrations.
a. Histology—Acute lung injury (ALI) and diffuse alveolar damage (DAD) are frequent complications after hemorrhagic shock and are frequently associated with severe inflammatory response (71a). Formalin fixed lung tissues are subjected to standard hematoxylin and eosin stain processing. Coded specimens are examined by light microscopy by a blinded pathologist, who score the acute inflammatory lung injury using a five-point system: 0, no significant histopathologic changes; 1, minimal interstitial inflammatory infiltrates; 2, mild interstitial inflammatory infiltrates with mild hyalinization; 3, moderate interstitial inflammatory infiltrates with moderate hyalinization; 4, severe interstitial inflammatory infiltrates with severe hyalinization. To ensure consistency, samples are examined twice, and the scores are averaged.
b. Myeloperoxidase in lungs—The interaction between neutrophils and different cells, especially endothelial cells, plays a critical role in organ injury after resuscitation. Myeloperoxidase activity in lung extracts is measured as a measure of neutrophil sequestration, which is related to the severity of inflammation (72a).
c. Cytokines—Following reperfusion, the local inflammatory reaction involves cytokines such as TNF-α (73a, 74a) in addition to neutrophil recruitment. In the same HS rat model, plasma levels of TNF-α and TNF-α mRNA in liver increased significantly 20 minutes after the end of bleeding (4a). It has been shown that high concentrations of albumin decreased the production of proinflammatory cytokines such as TNF-α and IL-6 (39a, 75a). TNF-α and IL-6 is measured in lung and liver tissue during phases II and III. Standard cytokine assays is performed also in sera at baseline and following the end of phases I, II and III according to the manufacturer's protocol (Pharmingen, San Diego, Calif.).
d. NF-κB activation−NF-κB activation occurring in the ischemic phase or following resuscitation is tied to the dysfunctional inflammatory response in hemorrhagic shock and resuscitation. Liver NF-κB binding activity measured by electrophoretic mobility shift assays increased in the nuclear extracts 10 minutes after the end of bleeding. Western blot studies showed that the levels of inhibitory protein IκBa in cytoplasmic extracts decreased at 5 minutes after the end of bleeding (4a). Proinflammatory cytokines contain NF-κB binding sites (76a); increased NF-κB binding to their sites results in increased cytokine expression leading to increased inflammation and tissue injury. This means that down regulation of NF-κB is expected to reduce inflammation. It had been shown in cell culture systems that albumin increased intracellular glutathione sufficiently to prevent TNFα-induced NF-κB translocation (77a). NF-κB is measured in lung and liver following phases II and III. Reduction in NF-κB is used as an indicator of a positive resuscitation effect. Electrophoretic mobility shift assays are used to measure NF-κB and Western blot analysis to measure IκBα (4).
Ischemia-reperfusion results in disrupting endothelial integrity (78a, 79a). When pulmonary artery endothelial cells (EC) were exposed to ischemic human plasma, ten minutes later they became rounded, formed gaps and then blebbed (80a, 81a). The same morphologic changes occurred in microdermal EC culture after exposure to sera from capillary leak syndrome patients (12a). Apoptosis of EC was evidenced by morphologic criteria, plasma phosphatidylserine exposure (Annexin staining), and DNA fragmentation. Increased Bax/Bcl2 in endothelial cells was detected by immunohistochemistry. The mechanism of these effects was explored by measuring intracellular reactive oxygen species (ROS) and the results suggested that oxidative injury played a role in the mechanism of EC apoptosis (12a). Oxidative stress is a well known inducer of apoptosis (11a). In addition increased apoptosis occurs after trauma and hemorrhage (15a, 78a, 79a, 82a). Inhibition of apoptosis by caspase inhibitors attenuated I/R induced inflammation (36a, 83a, 84a). In tissues exposed to ischemia-reperfusion, antioxidants minimized the damage from this injury. Albumin is the major extracellular antioxidant in plasma. It exerts this function through the enzyme gamma glutamylcysteine dipeptide, where albumin plays a significant role in glutathione synthesis (38a). Glutathione is the main low molecular weight soluble thiol present in mammalian cells, (85a) its depletion plays a role in the induction of apoptosis (86, 87). In another study looking at how albumin exerts its antioxidant activity (40), modification of the single free thiol (cys 34) was accompanied by a 45% decrease in antioxidant activity (88a). Albumin is protected against oxidation by its capacity to increase glutathione (GSH). Conversely, reduction in GSH led to a) activation of caspase 3 and poly ADP ribose polymerase (PARP) fragmentation (89a), and b) the decrease in Bcl-2/Bax ratio. The latter ratio is a strong indicator of cell survival, particularly in defense against oxidative injury (90a, 91a). As a result, albumin, through its function as antioxidant, contributes significantly to, the protective effect against apoptosis. In reference to the endothelium, albumin reduced microvascular permeability (33a, 92a, 93a) and played an essential role in preventing apoptosis of endothelial cells (36a, 84a).
a. TUNEL assay—This method uses terminal deoxynucleotidyl transferase to label DNA strand breaks with fluorescein-conjugated nucleotides (94a). Apoptosis detection kit (Boehringer, Indianapolis, Ind.) will be used. Tissue samples are examined by a blinded pathologist. B. Western blot analysis of apoptosis markers—Tissue samples are quick-frozen and stored at −80° C. until extracted for Western blot analysis. Apoptosis is detected by examining a number of proteins whose presence or modification is associated with apoptosis. Rhe expression of proapoptotic protein bax and the antiapoptotic protein bcl-2 using western blot analysis are examined. Tissue extracts for cleavage products of poly ADP-ribose polymerase (PARP) are analyzed. PARP is a substrate for caspases 3 and 7 and an accepted marker for apoptosis. Full length PARP (115 Kda) is cleaved into fragments of 85 to 90 Kda and 23 to 24 Kda resulting in inactivation of its enzymatic activity (11, 95).
b. Immunohistochemical staining for bax and caspase-3—Tissues are embedded in paraffin and cut into 5-micron thick sections for immunostaining. Sections are prepared from HS animals and control animals. A polyclonal rabbit antibody specific for active caspase-3 is used. Distribution of caspase 3 in thin sections of tissue are determined by immunostaining using a fluorescent secondary antibody. For co-localizing the endothelium, CD34 and factor VIII stains are used. Negative control sections receive identical treatment except for the primary antibody. Immunostained slides from control and treated animals are coded and read at 40× magnification by blinded readers. Two separate readings are obtained for each slide and expressed as the percentage of positive cells/mm2 tissue.
c. Measurement of glutathione—Oxidative stress accompanying HS is reflected in the ratio of reduced to oxidized glutathione GSH/GSSG. Accordingly, reduced and oxidized glutathione in the lung according are measured according to the procedure described by Hissin and Hilf (96). Frozen tissues are extracted with TCA, neutralized and GSH and GSSG content in the extract are determined by reaction with o-phtaldialdehyde (OPT) and the resulting fluorescence is monitored using authentic GSH and GSSG as standards. The GSH/GSSG increases following treatment with PEG-AlbCys-34 compared to the other groups including PEG-Alb1. This correlates with less apoptotic activity as evidenced by less PARP and decreased bax/bcl-2.
d. Measurement of malondialdehyde—Malondialdehyde (MDA) in tissue extracts is also used as a marker for oxidative stress associated with HS. Malondialdehyde (MDA) (97a) and Total Antioxidant Capacity (TAOC) (98a, 99a) in the lung and liver harvested after sacrifice is determined. MDA is assayed employing an HPLC method (94a). MDA an early marker of lipid peroxidation and, along with the TAOC, increases while the GSH/GSSG ratio is expected to decrease.
In parallel with the examples of the in vivo efficacy of the PEG-albumin, physical studies on the modified albumins are performed to identify properties that correlate with its in vivo effectiveness in treating shock.
Methods of synthesis, product size distribution, effects of modification on protein secondary structure and conformation, the effect of PEG modification on oncotic properties of albumin and effects on the binding of physiologically relevant ligands are evaluated. The example IV-5a describes preliminary studies on the preparation and properties of PEG-Albs and the example IV-5b describes the proposed studies.
1. Preparation and Size Analysis of PEG-Albs
Because the mode and extent of modification and the size of mPEG (methoxypolyethylene glycol) attached to albumin may alter its biophysical properties and in vivo properties, we have examined different methods for linking PEG to albumin and have characterized the modified proteins with respect to size, stability and osmotic properties. We examined N-hydroxysuccinimide esters (mPEG5000), cyanuric chloride (mPEG5000), and thiol selective maleimide derivatives (mPEG20000 and mPEG40000).
The cyanuric chloride (mPEG5000) derivatives have been tested in animals. These modes of modification are simple, rapid and most of the albumin is modified. Excess reagent and any unmodified albumin are removed by gel filtration or ion exchange chromatography. NHS esters and cyanuric chlorides (both selective for lysyl ε-amino groups) and maleimides (selective for cysteinyl thiols) are commercially available and react readily under mild conditions.
Because human albumin's single thiol (100-102) has an unusually low pKa (approximately 5.5), it is modifiable with thiol selective reagents without perturbing the disulfide structure of the protein. Acccording to one aspect of the present invention we have attached mPEGs of different sizes (a 20,000 Mr derivative and a branched 40,000 Mr derivative). Albumin is incubated with dithiothreitol and low molecular weight products linked to the albumin through cys 34 are removed by Sephadex G50 chromatography followed by modification with maleimide mPEG40000.
2. Thermodynamic Stability and Conformation of the PEG-Albs
An issue in the analysis of these PEG-Albs is whether the modification alters native structure and potentially the ligand binding properties and stability of the albumin. We examined the stability of PEG-Albs by analyzing urea induced unfolding; this is a standard method for studying the thermodynamic stability of proteins which gives the free energy of unfolding and can reveal whether the protein assumes unfolded intermediates (103a-105a). The protein is incubated with increasing concentrations of denaturant and a spectroscopic signal characteristic of the native and unfolded states is examined. We used the shift in the fluorescence emission wavelength (intensity averaged emission wavelength <λ>) of the tryptophan (trp 214) as a signal since there is a significant red shift when the protein unfolds (106a). Examples of results of such studies comparing unmodified albumin (panel A), albumin modified with mPEG20000 (panel B) and albumin modified with mPEG40000 (panel C) are shown in
We also compared one of the PEG-Albs (PEG40-Alb) to unmodified albumin by differential scanning calorimetry (DSC). This approach gives information on the thermodynamic stability and can be employed to study the effects of ligands on conformation and stability.
To extend the studies of stability, we examined the fluorescence of the single typtophanyl residue to quenching by different agents. The tryptophan fluorescence can be used as an indicator of native structure, since subtle changes in protein conformation can alter the emission intensity and the shape of the emission spectrum (112a, 113a). Modification of albumin with mPEG5000 contributes to absorbance in the ultraviolet (between 240 nm and 280 nm), while the absorption spectra and the fluorescence emission spectra of the PEG20000 and PEG40000 modified albumins were virtually indistinguishable from unmodified albumin. Fluorescence emission spectra for the mPEG 5000, PEG20000 and PEG40000 derivatives were similar to unmodified albumin indicating that the environment of the single tryptophanyl residue has not been altered significantly.
We examined the accessibility of tryptophan to the solvent by determining how readily its fluorescence could be quenched by iodide or acrylamide.
3. Osmotic Properties of PEG20-Alb and PEG40-Alb
Because the osmotic properties of the modified albumins are essential for function in vivo, we examined the dependence of colloid osmotic pressure on the concentration (114a, 115a) of mPEG20000, mPEG40000, multiply modified mPEG5000 albumins and unmodified albumin as shown in
4. Albumins with Fluorescent Labels
We have prepared unmodified albumin and mPEG-Albs with fluorescein or Texas Red linked through cys34. These fluorescent albumin derivatives are used to examine how effectively the albumin is retained in the circulation in animals with capillary leak; disposition of these albumins can be monitored fluorometrically in body fluids or by fluorescence microscopy of tissue sections. The two fluorophors have well separated excitation and emission spectra, so samples containing a mixture of two albumins (e.g., unmodified albumin with Texas Red and PEG-albumin with fluorescein) can be examined in the same animal. When PEG is linked through cys34, we couple amine reactive versions of fluorescein or Texas Red through a lysyl ε-amino group. Having albumin with two different fluorophors allows for the determination of how efficiently the PEG albumin with fluorescein is retained in the circulation compared to the unmodified albumin with Texas Red. These fluorescent albumins are only employed analytically for monitoring retention of unmodified versus PEG-albumin in models of shock or to monitor the in vivo half-life. We have readily detected the fluorescence of fluorescein-albumin in dilutions of serum well above the background of other fluorescent material. As necessary, measurements of intensity is corrected for the inner filter effect (112a, 113a) arising from other chromophors in serum samples; however, our studies with the fluorescent albumin indicates that any interference is negligible due to the large dilution of the serum that is required (1:1000 to 1:2000).
1. Preparation of PEG-Albumin
We have examined a number of reagents for linking PEG to albumin and the mPEG5000-Alb in vivo. Because the size of the PEG attached to albumin, its location and the nature of the covalent linkage results in products with significantly different stabilities, biological half-lives and ligand binding, various modes of attachment and types of mPEG (46a, 116a-120a) are examined. For the amine selective reagents that tend to modify multiple lysyl residues, specific methods are used for preparing material with a more defined size distribution so that the dependence of efficacy on size is examined. Controlling the size distribution is achieved, in part, by limiting the extent of modification in the initial reaction, by purifying the product by ion exchange or gel filtration chromatography, and by the selective modification of specific residues, as we have done with the maleimide-PEGs.
Modes of linkage—While the modes of linking the reagent to albumin that used thus far have produced a product with the desired in vivo effect, it is also within the contemplated scope of the present invention that other modes of attachment are useful to generate products with differences in stability or binding of relevant ligands. PEGs of various sizes, with different reactive groups (primarily amine and thiol selective) are available (Shearwater Corp., Huntsville Ala.); this supplier develops reagents specifically for PEGylation of biological materials. Others have emphasized the importance of attention to the quality of the mPEG reagents and biological optimization (119a). The present invention also contemplates the use of such additional method steps of modifying conditions (e.g., pH, ionic strength, temperature) and maintaining of native structure; for example, the disulfide bonding and structure of albumin may be disrupted at high pH due to protein thiol-disulfide exchange.
1. Amine selective reagents—The most abundant class of nucleophiles available for modification are surface lysyl residues that are readily modified to give a highly substituted product. While mPEG-succinimidyl-succinate generates a product with an ester linkage that might be a substrate for serum esterases, other reagents such as mPEG-succinimidyl-propionate (1 in
2. Thiol selective reagents—Modification through a thiol is a useful approach for human serum albumin since it has a single thiol (cys34) (100a, 101a, 125a). Human serum albumin is a mixture of protein with cys34 as a free thiol and a substantial fraction with the thiol modified with glutathione or as a disulfide dimer of two albumins. Under mild conditions, Cys34 disulfides can be reduced such that all of the cys34 is available as a free thiol without reduction of the less accessible disulfides. Cys34 is reactive with thiol selective reagents, including N-ethylmaleimide and iodoacetamide (100a, 101a, 125a). In one embodiment, albumin is modified with mPEG-maleimide derivatives (4 in
PEG Derivatives of Different Sizes and Geometries
Albumins modified with different size PEGs and PEGs with branched structures are examined. Sizes available include 3,400 Mr, 5,000 Mr, 20,000 Mr, and 40,000 Mr. There are branched (3 in
Preservation of Cys 34—The activity of albumin in inhibiting apoptosis and other biological properties depend on thiols (presumably cys34). mPEG-Albs that retain cys 34 as a thiol are prepared. Albumin is treated with a slight excess of dithiothreitol followed by modification of cys 34 with 5,5′-dithiobis-2-nitrobenzoic acid. Low molecular weight products are removed by gel filtration and the protein is modified with an amine selective PEG reagent. The free thiol is regenerated by treating the protein with dithiothreitol to release the thionitrobenzoic acid (monitored spectrally at 412 nm). The mPEG albumin is purified to remove unmodified protein, excess reagent and reaction byproducts. The mPEG-albumins produced using this approach are modified at multiple sites since the reagents modify lysyl residues. However, it is also within the contemplated scope that the method can include using larger PEG reagents (e.g., PEG20000 and PEG40000) the number of residues modified can be minimized by varying reagent concentration and reaction conditions.
Size selection and analysis of PEG-albumin—The size distribution of the product is important both because the PEG-albumin must be large enough to be retained within blood vessels during capillary leak and because a product that is too extensively modified might have undesirable attributes, such as loss of ligand binding properties or toxicity. Controlling the size distribution is achieved, in part, by limiting the extent of the reaction or, in the case of modification of cys34, modification of a single residue. The modified product is purified by gel filtration or ion exchange chromatography to select for PEG-albumin of a relatively narrow size distribution. The size distribution of the preparation is determined by gel filtration using proteins of defined molecular dimensions and Mr as standards and by mass spectrometry. One cannot really determine a molecular weight of the modified albumin by gel electrophoresis (127a, 128a) or by gel filtration since the PEG has an extended structure, and likely does not bind SDS the way proteins do. A more appropriate parameter is the equivalent or Stokes radius. The number average molecular weight and the effective molar volume can be obtained from the concentration dependence of colloid osmotic pressure (114a, 115a). Although the exact physical meaning of these measurements is subject to interpretation, they do provide a basis for comparing different preparations and parameters that can be correlated with in vivo effectiveness. These analyses define the extent of modification that is required for retention of PEG-albumin within blood vessels in models of shock and determine the merits of different extents of modification.
2. Effect of PEG Modification of Albumin on Protein Structure and Stability
The structure and stability of albumin are important for its physiological functions. Spectroscopic techniques are used to examine conformation and secondary structure to determine the extent to which modification of albumin with PEG alters the protein's structure and stability. Circular dichroic (CD) spectra in the near and far ultraviolet are obtained on unmodified albumin and on albumin modified with PEG. Analysis of the near ultraviolet spectra (250 to 320 nm) gives information on the extent to which modification has perturbed the microenvironment of tyrosyl and tryptophanyl residues (129a, 130a). The far ultraviolet CD spectra (180 to 250 nm) gives information on the extent to which secondary structure has been perturbed (129a, 130a). Human serum albumin is dominated by α-helix (67%) (100a-102a), and both spectra reflect this type of secondary structure. Environment of tryptophanyl residues is examined by iodide and acrylamide quenching of intrinsic tryptophan fluorescence (112a, 113a); examples of such experiments are shown in the results section. Tryptophan fluorescence, and its susceptibility to quenchers, is a sensitive probe of protein conformation. We have examined unmodified albumin and PEG modified albumin by iodide quenching and the single tryptophan is relatively inaccessible to this quencher with both proteins, consistent with PEG modification not altering its environment. In addition, the emission spectra of tryptophan for the two native proteins are essentially identical. These examples identify conditions for modification that result in PEG-albumin with minimal alterations in protein conformation and secondary structure.
The effect of PEG modification on the stability of albumin is evaluated by examining spectroscopic signals (intrinsic tryptophan fluorescence and CD) characteristic of native structure in the presence of increasing concentrations of chaotropic solutes (guanidine-HCl or urea). Analysis of such experimental data gives the free energy of unfolding in the absence of denaturant (ΔG0H2O) (103a, 105a), reflecting the thermodynamic stability of the protein.
In another aspect, the present invention provides a method to identify conditions for modification that result in a product with the desired biological activity without altering stability. Stability of the PEG-Albs is also be examined by differential scanning calorimetry (DSC) (133a-135a). In this approach one heats a protein solution slowly and measures the excess heat capacity associated with unfolding; this approach is useful to study the effects of fatty acids and tryptophan on the stability of albumin (108a-111a). To compare both the ligand-free proteins and the ligated species, fatty acids, tryptophan and other hydrophobic ligands are removed by charcoal treatment (108a) and the effect of adding various ligands including fatty acids, heme, N-acetyltryptophan is examined. This analysis gives information about protein stability (including the enthalpy of unfolding) and the number of states involved in the unfolding process and can be used to assess the integrity of the ligand binding sites.
1. Analysis of the oncotic properties of PEG-Albumin—The concentration dependence of colloid osmotic pressure of PEG-albumins is examined to see how this property relates to in vivo effectiveness. Unmodified albumin, PEG-Albs and comparable concentrations of the corresponding unconjugated mPEGs are examined. In the simplest case, the osmotic activity of PEG-albumin is the sum of the osmotic activities of a comparable concentration of unmodified albumin and the free PEG. However, interaction of solvent and solute with proteins is not necessarily simple and results may not be a simple arithmetic sum. The present invention provides a PEG-albumin preparation with a high osmotic activity that retains overall native structure.
2. Analysis of the ligand binding properties of PEG-albumin—Albumin binds a number of important ligands, including sodium ions, bilirubin, magnesium ions, fatty acids and many drugs. Ligands bind at multiple, distinct sites on the three major domains of albumin (131a, 132a). We examined whether modification of albumin with PEG alter binding of important ligands. Representative ligands that bind to the various sites including bilirubin (137a), fatty acids (138a), heme (139a) and various drugs (125a) are examined. While binding of these ligands can be measured by spectroscopic assays (131a), the most informative and thermodynamically rigorous approach is titration calorimetry (ITC) (140a-142a). A solution of ligand is titrated into a protein solution and the heat released or absorbed during binding is measured. This approach requires no chromophor and is applicable to any ligand and acceptor. ITC experiments give the association constant, binding enthalpy, binding entropy and the stoichiometry. The only significant limitations relate to analysis of tight binding and weak ligands and ligands of limited solubility. The extent to which modification alters ligand binding is determined by examining binding isotherms for the ligand to determine the binding constant(s) and the number of binding sites. The present invention also provide examples of ligands that are useful to evaluate the functional integrity of the three binding sites in the modified albumins compared to unmodified albumin.
1. Determination of the Half-Life of PEG-Albumin—The half-life of PEG-albumin is a consideration both in its efficacy and possible side effects. PEG modification of proteins in general (116a, 119a) and albumin specifically increases the half-life, reduces antigenicity, and reduces their susceptibility to proteolysis. PEG modification has a profound effect on the half-life of interferon α (from 6 hrs to 75 h) and its therapeutic effectiveness in treating hepatitis c (143a, 144a); with bovine albumin the change in half-life in rabbits is modest (143a). The latter result with albumin is not unexpected as it is a relatively long-lived protein (20 days in humans) even without PEG modification. As such, PEG albumin and normal albumin modified with fluorescein or Texas Red is administered; these fluorophors provide a signal for monitoring clearance from the circulation. Use of the two chromophors, one on unmodified albumin and the other on the PEG modified albumin allows for the two types of albumin to be monitored in the same animal so that the extent of preferentially retention in the circulation can be assessed. We have prepared both of these dye-albumin conjugates. The dye-albumin conjugates are administered to animals essentially as tracers and small blood samples (˜100 to 200 μl) are taken through the tail vein over one to two weeks for analysis. Clearance if followed by qualitatively by examining the protein by Western blot analysis using a commercially available monoclonal antibody specific for human albumin; this approach avoids any effects that addition of a fluorophor to the protein might have. PEG-albs have dramatically different migrations on SDS gels compared to unmodified albumin and the monoclonal antibody discriminates between human and rat albumin. Using antibody specific for the human albumin, clearance is monitored quantitatively using an enzyme-linked immunoassay (ELISA); use of an antibody requires verification that that it still binds to albumin after PEG modification.
2. Analysis of the Toxicological Properties of PEG-Albumin—For PEG modified albumin to be effective in treating capillary leak syndrome it must be administered at relatively high doses compared to other PEG modified proteins that have been used therapeutically, such as interferon. An obvious difference is that a gram or more of PEG modified albumin must be given compared to micrograms of interferon. It is essential that PEG albumin not be significantly toxic at these doses. Relatively large doses of higher molecular weight PEGs (4000 to 6000 Mr) show little toxicity in a number of animals (rats, rabbits and dogs)(45a, 143a, 145a-147a) while some evidence suggests that the lower molecular weight PEGs (e.g., 400 Mr) exhibit toxicity (45a, 148a, 149a). A large fraction of blood volume of dogs (30 to 50%) can be replaced with PEGylated hemoglobin without significant toxicity over two weeks (149a). In some studies where large amounts were administered, inclusions in cells of the liver and kidney were observed, indicative of phagacytosis. In most studies of toxicity, free PEG was examined and not PEG coupled to a relatively long-lived protein. Daily intravenous doses of PEG 4000 administered to dogs at up to 90 mg/Kg for one year elicited no toxic effects (150a); there were no gross anatomical, microscopic or biochemical abnormalities. In experiments that would probably not be approved by an IRB committee if they were proposed today, PEG 6000 was administered intravenously to six human volunteers with no apparent ill effect (146a); 94 to 99% of the PEG 6000 was excreted in the urine within 12 hours. PEGs in the 1000 to 10,000 Mr range are toxic in rats (147a) (LD50 10 to 20 gm/Kg), but only at doses that are approximately 50 to 100-fold higher than those given in the studies involving humans and dogs; the equivalent dose for a 75 Kg human extrapolated from these studies would be 0.75 to 1.5 Kg. We have seen no overt toxicity in the studies we have performed, but since all of our work has examined short term effects that are evident in less than 4 hours, toxicity arising from catabolism of PEG-albumin and release of PEG-peptides would not be observed.
Toxicological Evaluation
The most promising PEG-albumin conjugates are evaluated for toxicity by administering them at doses in a range that starts with an anticipated therapeutic dose and going to much higher doses; animals are monitored over periods of up to four weeks. Both single doses and multiple doses are tested. Data collected prior to sacrifice of the animals includes body weight, food consumption, water consumption, production of feces and urine production. Also, the animals are observed for signs of behavioral changes. Small amounts of blood are withdrawn periodically and enzyme assays are performed on serum for markers characteristic of hepatotoxicity. At the end of the experiment, the animals are sacrificed and tissues and organs are examined for macroscopic evidence of damage. A number of tissues are examined microscopically for evidence of toxicity, including liver, kidney, lung, brain, heart and skeletal muscle. Control animals that are given the vehicle are also examined in the same fashion.
In the following Example V, the indicator reagent may be a dye or a combination of dyes. The two dyes may be red, green or the same color. Their emission and exitation wavelength has to be widely and significantly distant. The preferred method is on a double chromophore technique. However, it can be multiple chromophores.
A preferred dye is a red maleimide dye, Texas Red. Indocyanine Green is an excellent fluorescent material. It can be used to replace Texas Red in the mixture of Texas Red and Fluorescine. Indocyanine green emission is in the near infrared (˜840 nm) and is an excellent tracer with distant emission from fluoroscein.
The preferred use of this technique is the assay to be used as a marker to measure and quantify the vascular leak which is a surrogate for multiple organ failure. The implications is that of predicting patients in danger of developing the organ failure. This allows the assay to be used to tailor certain therapies for such patients. Also this is a novel technique to study the half life of proteins.
In one embodiment, this invention is a technique of predicting the development of multiorgan dysfunction before it happens or earlier. The process is based on administering two or more proteins. For example, albumin and PEG-albumin. The proteins have significantly different molecular weights and are tagged with chromophores with distant emission and excitation wavelengths. Predicting occurs by assessing the concentrations of the chromophores over time. Besides the albumins, we can use for example another protein with known molecular weight. For example, immunoglobulin G molecular weight 150.000 or other proteins such as VonWillebrand factor MW 300.000.
The methods for the preparation of dye conjugated albumin an PEG-Alb were as follows. Human albumin (50 mg/ml) was incubated 1 hr in 50 mM potassium phosphate (pH 7.5), 150 mM NaCl, and 0.5 mM dithiothreitol. The dithiothreitol-treated albumin was incubated two hours with 4 mM 5-iodoacetamidofluorescein or 1.5 mM Texas Red maleimide (Molecular Probes). The dye-modified albumins were diluted five-fold and reconcentrated three times in a centrifugal concentrator (10,000 Mr cut off, Millipore) to remove most of the unincorporated dye, followed by dialysis for 48 hours against four changes of phosphate-buffered saline.
The fluorescein-labeled albumin was modified with methoxypolyethylene glycol cyanuric chloride and purified by gel filtration on Sephacryl S200. Fractions from Sephacryl S200 eluting with apparent molecular weights in excess of 200,00 were pooled and concentrated by ultrafiltration employing a PM 10 membrane (Millipore) followed by dialysis against several changes of 0.9% saline. Analysis of the fluorescein- and Texas Red-labeled albumins by gel electrophoresis revealed fluorescence was associated with the protein. No fluorescence was detected at the positions of free dye. Steady state fluorescence meansurements were made on a QM4SE fluorometer (Photon Technology International, Monmouth Junction, N.J.).
Next unmodified albumin and mPEG-Albs with fluorescein or Texas Red linked through cys34 was prepared. These fluorescent albumin derivatives were used to examine how effectively the albumin is retained in the circulation in animals with capillary leaks. Disposition of these albumins can be monitored fluorometrically in body fluids or by fluorescence microscopy of tissue sections. The two fluorometrically in body fluids or by fluorescence microscopy of tissue sections. The two fluorophores have well separated excitation and emission spectra, so samples containing a mixture of two albumins (e.g., unmodified albumin with Texas Red and PEG-albumin with fluorescein) can be examined in the same animal. When PEG is linked through cys34, an amine reactive versions of fluoroescein or Texas Red was coupled through a lysyl ε-amino group. Having albumin with two different fluorophores allows for the determination of how efficiently the PEG albumin with fluorescein is retained in the circulation compared to the unmodified albumin with two different fluorophores allows for the determination of how efficiently the PEG albumin with fluorescein is retained in the circulation compared to the unmodified albumin with Texas Red. These fluorescent albumins only were employed analytically for monitoring retention of unmodified versus PEG-albumin in models of shock or to monitor the in vivo half-life. The fluorescence of fluorescein-albumin in dilutions of serum was detected well above the background of other fluorescent material. As necessary, measurements of intensity were corrected for the inner filter effect arising from other chromophores in serum samples. However, studies with the fluorescent albumin indicate that any interference is negligible due to the large dilution of the serum that is required (1:1000 to 1:2000).
Animals/Measurement Protocol
Measurements were done in normal healthy rats (n=4) and CLP rats (n=11). For CLP, 6 rats were injected with (PEG-ALB-FL+Albumin-TR) and 5 were injected with the chromophores reversed. The Institutional Animal Care and Use Committee and the Academic Chemical Hazards Committee at the Medical College of Ohio approved experimental protocols. Animals were housed in an American Association for Accreditation of Laboratory Animal Care; International (AAALACI) approved facility. Adult male Sprague-Dawley rats (Charles River Laboratories, Portage, Mich.) weighing 400-480 grams were used. They were provided standard rat chow and water ad libitum. Prior to experiments, animals were fasted overnight, but given water ad libitum. We injected the fluorophores of PEG-Alb and that of albumin using a cecal ligation and puncture (CLP) induced sepsis rat model and a sham model. Rats were anesthetized with sodium pentobarbital (50 mg/kg BW, i.p.) followed by pentobarbital as needed. A laparotomy was perfoemd through a midline abdominal incision. The cecum was ligated just below the ileocecal valve with 3-0 silk ligatures such that intestinal continuity was maintained. The cecum was perforated with a 16-gauge needle in two locations and gently compressed until feces were extruded. The bowel was returned to the abdomen, and the incision was closed with a layer of proline sutures for the muscles and 3-0 silk for the skin. Sham rats underwent the same protocol; the cecum was manipulated but not punctured before the bowel was returned to the abdomen. Three ml of sterile 0.9 percent sodium chloride solution per 100 grams of body weight were administered subcutaneously on the back for resuscitation. The rats were deprived of food, but had free access to water after surgery.
Twenty hours after surgery, animals were anesthetized and instrumented to cannulate the internal jugular vein. Blood samples, each 100-150 μl, were taken at 40 minutes after injection (allowing for mixing of the chromophores and distribution in compartments), that is the time 0, then at 30 minutes, 1 h, 3 h, 5 h, 8 h. After 8 h, the rats will be allowed to recover for 2 hours after discontinuation of the internal jugular line. More blood samples now will be taken from the tail vein at 22, 28, 45, 52, 70, 96, 102, 148, 160, 171 hour or until the rat dies.
Histology/Fluorescence Microscopy
Formalin fixed lung and kidney tissues were subjected to standard processing, including a hematoxylin and eosin stain. For the immunofluorescence studies, lung sections were examined with a Nikon Eclipse E800 fluorescence microscope and pictures recorded using Image Pro Plus Version 4.0 (Media Cybernetics, L.P.) using 20× and 40×objectives.
Statistic Methods
Values are presented as mean±SD unless otherwise indicated. Within a treatment group, data analyzed at repeated time points (concentration of fluorescence, time in hours) were evaluated by repeated measures analysis of variance using a post-hoc paired t-test employing correction for multiple comparisons. Differences among the treatment groups at comparable time periods were evaluated with analyses of variance. Statistical significance is repreted at the p<0.05 and p<0.01 levels.
Results
The dye-albumin conjugates was administered to animals essentially as tracers and small blood samples (˜100 to 200 microliters.) were taken through the Jugular vein (the first 8 hours) and through the tail vein thereafter. The disposition of these albumins was monitored by measuring the fluorescence in the blood and by fluorescence microscopy through examining lung tissue sections. Using a method we developed, fluorescently labeled albumin and (Texas Red, TR) and PEG-Alb (fluorescein, F) were injected into rats and a small volume of blood is taken through the tail vein for analysis at times after injection. If albumin is lost to the extravascular space and PEG-Alb is retained, the ratio of fluorescein to Texas Red (F/TR) will increase with time, consistent with loss of albumin and preferential retention of PEG-Alb. The excitation and emission spectra of Texas Red and fluorescein are sufficiently different that mixtures of the two dyes can be examined quantitatively in serum samples. Switching the labels (fluorescein albumin and Texas Red PEG-Alb) give exactly complementary results verifying that clearance of the protein is not a property of the fluorophores. The clearance exhibits a fast phase, consistent with redistribution of the material into another compartment and a slow phase reflecting clearance. The PEG-albumin is cleared ˜3 times less rapidly compared to albumin. The distribution of albumin and PEG-Alb was also examined qualitatively in frozen tissue sections of lung by fluorescence microscopy to determine if the PEG-Alb is retained within blood vessels, as we observed in septic shock.
To demonstrate that PEG-Alb is retained within vessels while normal albumin leaks, a mixture of fluorescein-labeled PEG-Alb and Texas Red labeled albumin was administered. Fluorescence microscopy of lung sections demonstrated co-localization of the Texas Red and fluorescein signals in rats subjected to sham surgery whereas in the CLP rats, Texas Red fluorescence (PEG-Alb) was detected only within vascular structures; colocalization of the dyes would indicate that both were retained while the presence of one of the dyes in the interstitial space would indicate leak of the labeled species.
Both fluorescein labeled PEG-Alb and Texas Red labeled albumin were seen only in the intravascular space of control animals. These results are consistent with the retention of PEG-Alb in blood vessels during capillary leak.
vFluorescence concentration of PEG-Alb/albumin in both normal and CLP rats was not significantly different at the first part of the curve, up until 40 hours after tracer injection or 60 hours after CLP. The upward slope of the curve suggests more retention of PEG-Alb or loss of albumin. In normal rats significant albumin loss it is not expected, decreased clearance of PEG-Alb is responsible for the increase in the ratio in normal and CLP rats at this relatively early phase of CLP. In the CLP rats, severe capillary leak was expected to occur after 48 hours after the onset of CLP. At this stage, PEG-Alb/albumin ratio in the CLP rats progressively increased after 60 hours, suggesting albumin loss consistent (with capillary leak) in addition to decreased clearance of PEG-Alb. The area under the CLP curve and above the normal rats curve measures the capillary leak or the organ dysfunction index. Quantification of capillary leak is important to predict pateitns destined to develop MODS. In relation to this, the use of this index can guide the use of expensive treatments for sepsis (example activated protein C or Xigris™) early before the overt development of MODS. What guides activated protein C use in severe sepsis is the measured APACHE II score where if >25 have shown to decrease absolute mortality by 6%. Although APACHE II scores are an indication of the severity of illness in populations of patients, they may be less useful in predicting the outcome of individual patients.
This invention uses multiple proteins or molecules with different molecular weights and tagged with different fluorophores each with distinct and distant emission and excitation wavelengths. These are administered to a patient at risk of developing multiorgan dysfunction. The, the process follows their concentrations (under the same pathophysiological processes such as hemoconcentration and capillary leak) serially at multiple times.
While this invention has been described with emphasis upon preferred embodiments, it would be obvious to those of ordinary skill in the art that preferred embodiments may be varied. It is intended that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and claims spirit and scope of the appended claims.
This application claims the benefit of U.S. patent applications: Ser. No. 10/985,798 filed Nov. 9, 2004, which is a CIP of Ser. No. 10/106,793, filed Mar. 26, 2002 now U.S. Pat. No. 7,037,895.
This invention was made with government support under Cooperative Agreement No. DAMD 17-97-2-7016 awarded by the U.S. Army Medical Research Acquisition Activity. The government has certain rights in the invention.
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| Number | Date | Country | |
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| Child | 11258646 | US | |
| Parent | 10106793 | Mar 2002 | US |
| Child | 10985798 | US |
