This invention relates to the prevention or treatment of graft-versus-host disease.
Since the 1930's, the increasing use of immunosuppressive therapy and invasive devices, as well as the increased incidence of antibiotic resistance in bacteria, have led to a gradual rise in the occurrence of sepsis and septic shock. Currently, the estimated incidences in the U.S. of sepsis and septic shock are 400,000 and 200,000 patients/year, respectively. This results in about 100,000 fatalities/year, making septic shock the most common non-coronary cause of death in the hospital Intensive Care Unit (ICU). Currently, ICU therapy for septic shock is limited to antibiotic therapy, cardiovascular resuscitation, vasopressor/ionotrope therapy, and ventilatory support. This ICU care can cost up to $1,500/day and average a total of $13,000 to $30,000 per patient.
It is likely that antibiotics themselves can worsen morbidity associated with sepsis; their bactericidal action can result in the release of endotoxin from gram negative bacteria, which are believed to induce many pathophysiological events such as fever, shock, disseminated intravascular coagulation (DIC), and hypotension. Further, endotoxin can be detected in the blood of patients regardless of pathogen. Consequently, medicines for the treatment of gram-negative sepsis have been desired for some time, especially drugs capable of blocking endotoxin or cytokines derived from endotoxin-mediated cellular stimulation. To this end, various strategies for treatment have included use of antibodies against LPS or cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1. For various reasons, these approaches have failed.
While endotoxin itself is a highly heterogenous molecule, the expression of many of the toxic properties of endotoxin is attributed to a highly conserved hydrophobic lipid A portion. An effective drug that acts as an antagonist to this conserved structure is known as E5564 (also known as compound 1287 and SGEA). This drug is described as compound 1 in U.S. Pat. No. 5,681,824, which is hereby incorporated by reference. E5564 has the formula:
(α-D-Glucopyranose, 3-O-decyl-2-deoxy-6-O-[2-deoxy-3-O-[(3R)-3-methoxydecyl)-6-O-methyl-2-[[(11Z)-1-oxo-11-octadecenyl)amino]-4-O-phosphono-β-D-glucopyranosyl]-2-[(1,3-dioxotetradecyl)amino]-,1-(dihydrogen phosphate), which can be provided as a tetrasodium salt. E5564 has a molecular weight of 1401.6.
We have discovered that administration of E5564 by continuous infusion over a relatively long period of time overcomes an unexpectedly short pharmacodynamic half-life of the drug, which surprisingly has been observed even though E5564 demonstrates a long pharmacokinetic half-life in circulation in the blood.
Accordingly, the invention features methods of treating patients suffering from medical conditions amenable to treatment with E5564. Examples of such conditions include endotoxemia (e.g., surgery-related endotoxemia), sepsis, septic shock, HIV infection, and immunological disorders, such as allograft rejection and graft-versus-host disease. The methods of the invention can also be used with patients suffering from damage to the gastrointestinal tract due to chemotherapy or radiation, and patients that have undergone bone marrow transplantation.
In the methods of the invention, E5564 is administered to patients by intravenous infusion over a period of 12-100, e.g., 60-80 or 72 hours. Activity in the ICU is often hectic, and minor variations in the time period of infusion of the drug are included within the scope of the invention.
The infusion dosage rate can be, for example, 0.001-0.5 mg/kg body weight/hour, e.g., 0.01-0.2 mg/kg/hour or 0.03-0.1 mg/kg/hour. The infusion of E5564 can, if desired, be preceded by a bolus injection of E5564, which can be given at a dosage of 0.001-0.5 mg/kg. The total amount of E5564 administered to a patient can be, for example, 50-600 mg of drug, e.g., 150-500 mg, by infusion over a period of 60-80 hours.
Patients that can be treated according to the methods of the invention include, for example, surgical patients (e.g., cardiac surgical patients), patients that have or are at risk of developing endotoxemia, sepsis, or septic shock, patients that are infected with HIV, and patients that are suffering from an immunological disorder, such as allograft rejection or graft-versus-host disease. The methods of the invention can also be carried out with any patients that have had, will have, or are having any type of transplant. For example, the methods can be carried out with patients having leukemia (e.g., chronic myeloid leukemia, acute myeloid leukemia, or acute lymphocytic leukemia) or another cancer, and that are treated by bone marrow or stem cell transplantation. The patients can also be kidney, liver, heart, or lung transplant patients. The graft-versus-host disease that is prevented or treated, according to the invention, can be acute or chronic.
The total dosage of drug used in the methods of the invention can be advantageously quite high, providing a maximum therapeutic effect, but not be accompanied by unacceptable toxicity. In particular, as is described further below, it has been found that, although injected or infused E5564 remains present in the blood for a relatively long period of time (i.e., E5564 has a relatively long pharmacokinetic half-life), the period during which it is active (i.e., its pharmacodynamic half-life) is relatively short. Thus, it is advantageous to administer the drug by continuous infusion over a prolonged period of time.
It is unexpected that such prolonged administration is possible, because a related, three- to ten-fold less active anti-endotoxin compound, B531 (U.S. Pat. No. 5,530,113, which is hereby incorporated by reference), could not be safely administered to patients in such a manner, due to its lack of safety margin in animal studies. Surprisingly, E5564 is about twenty-fold less toxic than B531, and thus can be administered at relatively high levels, for relatively long periods of time, according to the methods of the invention. Thus, the methods of the invention provide significant therapeutic benefits, with acceptably low toxicity. An additional advantage of the methods of the invention is that they are easily carried out, as many of the patients treated according to the methods of the invention already have intravenous lines inserted, as part of their treatment in the ICU. Further, the methods of the invention provide an approach to preventing and treating graft-versus-host disease.
Other features and advantages of the invention will be apparent from the following detailed description, the drawings, and the claims.
We have discovered that administration of E5564 by continuous infusion over a relatively long period of time overcomes the short pharmacodynamic half-life of the drug, which has been observed even though E5564 demonstrates a long pharmacokinetic half-life in circulation in the blood. The methods of the invention, as well as experimental data related to these methods, are described further, as follows.
The methods of the invention can be used to prevent or treat endotoxemia and related conditions and disorders (e.g., sepsis) in humans. For example, the methods can be used in conjunction with any type of surgery or medical procedure that could lead to the occurrence of endotoxemia or related complications (e.g., sepsis syndrome). For example, the methods of the invention can be used in conjunction with cardiac surgery (e.g., coronary artery bypass graft, cardiopulmonary bypass, or valve replacement), transplantation (of, e.g., liver, heart, kidney, lung, or bone marrow), cancer surgery (e.g., resection of a tumor), or any abdominal surgery. Additional examples of surgical procedures with which the methods of the invention can be used include surgery for treating acute pancreatitis, inflammatory bowel disease, placement of a transjugular intrahepatic portosystemic stent shunt, hepatic resection, burn wound revision, and burn wound escharectomy. The methods of the invention can also be used in conjunction with non-surgical procedures in which the gastrointestinal tract is compromised. For example, the methods of the invention can be used in association with chemotherapy or radiation therapy in the treatment of cancer.
The methods of the invention can also be used in the treatment of conditions associated with human immunodeficiency virus (HIV) infection, and immunological disorders, such as allograft rejection and graft-versus-host disease (GVHD), in particular, acute GVHD. GVHD is the most common complication of patients who have undergone allogeneic bone marrow or stem cell transplantation. These patients include, for example, patients that have chronic myeloid leukemia (CML), acute myeloid leukemia (AML), or acute lymphocytic leukemia (ALL). In GVHD, immune cells (T lymphocytes) from the donor attack cells of the transplant recipient, which the donor immune cells recognize as being foreign. Any types of cells in the recipient can be recognized as being foreign, and thus attacked, by the donor T lymphocytes. These cells include cancer cells, in which case the effect, referred to as graft-versus-leukemia (GVL) effect, is beneficial to the recipient. The recognized and attacked cells can also include normal cells of, e.g., the skin, stomach, intestines, liver, and mucosal surfaces, and this recognition can lead to very severe or even lethal damage. Acute GVHD occurs shortly after transplantation and is caused by T lymphocytes present in the donor preparation, while chronic GVHD occurs 2-3 months after the transplant, and may be caused by T lymphocytes that have grown in an adverse manner from the graft.
The primary route by which donor T lymphocytes cause GVHD is by priming inflammatory cells (monocytes and macrophages) to secrete cytopathic amounts of cytokines when stimulated by bacterial lipopolysaccharide (LPS). The cytokines in turn directly damage tissues and organs, as well as provoke T cell expansion and increases in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, responses that can also damage tissues and organs. Thus, according to the present invention, patients that have or are at risk of developing acute or chronic GVHD (e.g., patients with CML, AML, or ALL that are treated by bone marrow or stem cell transplant) can be treated by the administration of an LPS antagonist, which blocks such stimulation. The LPS antagonist can be administered just before, during, and/or shortly after (e.g., during the first 4-7 days after) transplantation to prevent GVHD. GVHD has been detected with other types of transplantations as well, for example, with kidney, liver, heart, and lung transplants. The methods of the invention can be used in the prevention and treatment of GVHD occurring with these types of transplantations as well.
In the case of preventing or treating GVHD, antiendotoxin compounds can be administered using the doses and regimens described herein, or by use of other approaches determined to be appropriate by those of skill in the art.
The drug can be formulated according to standard pharmaceutical practice. A specific example of a formulation of the drug is described in detail in U.S. Ser. No. 60/452,022, the contents of which are incorporated herein by reference.
Analysis of Anti-Endotoxin Drug Activity
Many of the signs and symptoms of sepsis can be mimicked in vivo by administration of endotoxin to an animal model system. The physiological effects of endotoxin can vary depending on dose, route of administration, and species tested, but generally include symptoms such as elevated temperature (fever), hypotension, changes in cellular composition of blood (decreased white blood cells, etc.), and elevation of proinflammatory cytokines, such as TNF-α and IL-6, and some anti-inflammatory cytokines. The activity of a drug designed to antagonize the effects of endotoxin can be tested in animal model studies by determining if it blocks any or all of these physiological markers of endotoxin activity.
In general, the candidate antagonist is administered to a test species of animal, and an appropriate dose of endotoxin (lipopolysaccharide (LPS)) is administered to test the ability of the candidate antagonist to block the effects of endotoxin. Some of the experiments described below use an in vivo challenge of LPS given intravenously both during and after intravenous infusion of E5564. Activity of an antagonist can also be assayed ex vivo by removing blood samples from animals treated with the candidate antagonist and testing that blood to determine if the drug is active and/or present in sufficient quantities to inhibit cellular activation by LPS. In both assays, activity of the antagonist is quantitated by analysis of the cytokines induced by LPS administration. In addition, other physiological symptoms of endotoxin poisoning can be used as readouts of activity. Studies described herein use TNF-α and/or IL-6 as readouts of cellular activation, but a variety of other cytokines and cellular mediators can also be used for this purpose.
Pharmacodynamic Analysis of E5564 in vivo
As is shown in
In vivo Pharmacokinetic Analysis of E5564 After Bolus Injection
As is shown in
In vivo Pharmacokinetic Analysis of E5564 During Infusion
To assess the activity of E5564 over multiple time points from a single treated animal, we employed an ex vivo assay, as is mentioned above, to test for active drug in samples of blood drawn from a treated animal. Samples of blood were drawn from beagle dogs infused with E5564 over a period of 24 hours. One dog of each sex was tested under each of two dose regimens: a low dose of 0.24 mg/kg/hour and a high dose of 2.4 mg/kg/hour.
Blood samples were taken from these dogs at predose, 4 hours after initiation of infusion, and 24 hours after initiation of infusion. The blood samples were challenged with 0, 1, 10, or 100 ng/ml LPS, and then incubated for three hours. The samples were then analyzed for activation by LPS, using induction of cytokine response as a readout. As is shown in
Inhibition of LPS-Induced IL-6 Release in ex vivo Blood Samples by Intravenous Infusion of E5564 in Beagle Dogs
Effect of Infusion of E5564 at 0.24 mg/kg/hour
As is shown in
Effect of Infusion of E5564 at 2.4 mg/kg/hour
As is shown in
These results show that infusion of E5564 at a dose of either 0.24 mg/kg/hour or 2.4 mg/kg/hour inhibits LPS response in blood over the period of infusion. Inhibition of LPS response is dose dependent for both E5564 and for the concentration of LPS used as challenge.
1Response to LPS was measured in groups of four beagle dogs for each treatment.
2Plasma levels of TNF-α induced at one and two hours after LPS administration. All TNF-α measured in units/ml.
1Rounded to the nearest whole number
2T½ after end of infusion
Materials and Methods
(1) In vivo Assays
(1.1) Reagents
E5564 was synthesized by Eisai Research Institute of Boston, Andover, Mass., U.S.A. E5564 drug product was manufactured at the Eisai Preclinical Laboratory (Tsukuba, Japan) by dissolving 35.4 mg of drug substance in 52.1 ml 0.01 N NaOH, stirring for one hour at room temperature, and diluting into phosphate-buffered lactose. After adjusting the pH to 7.3 and diluting to a final concentration of 0.1 mg/ml E5564, the solution was filter-sterilized and lyophilized.
The formulation of drug product in 1 ml vials is shown below.
Escherichia coli LPS (Serotype 0111:B4; phenol extracted, Cat. # L-2630) was purchased from Sigma Chemical Co. Ltd., St. Louis, Mo., U.S.A. Lyophylized E5564 was solubilized in 5 ml of sterile water (Otsuka Pharm. Co. Ltd., Tokyo, Japan). LPS was weighed to an accuracy of 1/10 mg and solubilized in 5% glucose (Otsuka Pharm. Co. Ltd., Tokyo, Japan). The LPS solution was sonicated with a bath-type sonicator for 15 minutes after which aliquots were immediately prepared and stored at −20° C. Prior to use, the solution was sonicated for one or two minutes, and then dilutions were prepared in 5% glucose.
(1.2) Animals
Nine month-old beagles were obtained from Kawashima-shoji Co. Ltd., Gifu, Japan) and housed in stainless steel wire cages (W 800 mm×D 680 mm×H 680 mm; one dog per cage) in a room with a constant temperature of 20-24° C., humidity of 45-65%, and 12 hour light-dark cycle. The animals were provided with pellet food (DS, Oriental Yeast Co., Tokyo, Japan) and water ad libitum. LPS endotoxin (300 ng/0.1 ml/kg) was injected into the vein of the right foreleg at a rate of 1-2 ml/minute, and E5564 was injected into the vein of the left foreleg at a rate of 10-20 ml/minute.
(1.3) Blood Collection and Treatment
Immediately before or 1, 2, or 4 hours after intravenous injection of LPS and E5564, 1.5 ml of blood was drawn from the left cephalic vein. One milliliter was transferred into a tube containing 10 U of heparin (Mochida Pharm. Co. Ltd., Tokyo, Japan), centrifuged (2000×g, 5 minutes, 4° C.), and the plasma was used for bioassays for TNF-α and IL-6.
(1.4) TNF Bioassay
Aliquots of the plasma were tested for TNF in a bioassay based on the TNF-dependent cell death of L-P3 cells in the presence of actinomycin D. The L-P3 cell line is more sensitive to TNF-induced cell death than the L-929 cell line that is more commonly used.
L-P3 cells were cultured in RPMI 1640 medium containing 10% heat inactivated fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. Plasma samples to be assayed were diluted 5-100 fold, and 0.1 ml of each was serially diluted into 96-well culture plates. 7×104 L-P3 cells in 100 μl medium containing 1 μg/ml actinomycin D mannitol (Sigma Chemical Co. Ltd., St. Louis, Mo., U.S.A.) were added to each well containing the plasma samples and incubated for 15 hours at 37° C. in 5% CO2. TNF-induced cell toxicity was measured using methylene blue as follows. Wells were washed with water at least 5 times to remove dead cells, after which cells were fixed with 50 μl glutaraldehyde and stained with 0.1 ml of a 0.05% methylene blue solution in water for 15 minutes. Excess methylene blue was removed by washing at least 5 times, after which the plate was dried. Methylene blue was then re-extracted from cells by addition of 0.2 ml of 0.33 N HCl to each well, and absorbance was read with dual wavelengths of λ1405 and λ2660 nm on a microplate reader (ImmunoReader NJ-2000; Japan InterMed Co. Ltd., Tokyo, Japan).
(1.5) IL-6 Bioassay
Aliquots of the plasma were tested for IL-6 activity by measuring IL-6-dependent proliferation of the mouse-derived lymphoma cell line, B9. Cells were cultured in RPMI 1640 medium containing 10% heat inactivated fetal calf serum, 50 μM 2-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM glutamate. Plasma samples diluted ten-fold or 500 pg/ml of IL-6 standard (human recombinant IL-6; Genzyme Corporation, Boston, Mass.) were added to each well of a 96-well culture plate and then diluted serially. 1.5×103 B9 cells in 50 μl medium were added to each well and the plates were incubated for three days at 37° C. in 5% CO2.
B9 cell proliferation was measured by the MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Sigma Chemical Co., St. Louis, Mo, USA) staining method. Twenty microliters of 6 mg/ml of MTT in Dulbecco phosphate-buffered saline were added to each well and the plates were incubated for 3 hours at 37° C. in 5% CO2. Next, 100 μl/well of 10% SDS (sodium dodecyl sulfate; Nacalai Tesque Co. Ltd., Kyoto, Japan) in 1 mM NH4OH was added and the cells were then solubilized overnight. Absorbance of each well was read by a plate reader (Model 3550, Bio-Rad Labs, Richmond, Calif., U.S.A.) with dual wavelengths of λ1540 and λ2660 nm. One unit/ml of human IL-6 is equivalent to 100 pg/ml.
(2) Ex vivo Assays
(2.1) Reagents
LPS from Escherichia coli (0111:B4) was purchased from List Biologicals (Campbell, Calif.). LPS was dissolved in sterile water at 1 mg/ml and stored at −20° C. Prior to use, LPS was sonicated in a bath sonicator (VW-380; Heat Systems-Ultrasonics Inc., Farmingdale, N.Y.) for 1-2 minutes immediately before use and diluted into Ca2+, Mg2+ free Hanks balanced salt solution (HBSS; Sigma).
(2.2) Origin of Samples and Study Design
Dogs were treated with E5564 (0.24 or 2.4 mg/kg/hour) dissolved in a mixture of placebo solution (10% lactose monohydrate, 0.045% Na2HPO4.7H2O, 0.035% NaH2PO4.H2O, 0.006% NaOH; pH 7.4±0.3) and 5% dextrose (1:4) by intravenous infusion via indwelling catheter for 24 hours at a rate of 2 mg/kg/hour. The study design is shown in the following table:
(2.3) Analysis of E5564 Activity in Dog Whole Blood
Prior to and during infusion of E5564, blood was drawn into heparinized syringes, and either aseptically reduced to plasma by centrifugation and frozen to −80° C. (for time zero samples), or incubated with the indicated concentrations of LPS for three hours. Plasma was then prepared and immediately frozen at −80° C. Samples were stored at −80° C. until assay.
(2.4) Bioassay for IL-6
B9 cells were the gift of Dr. Mary Rodrick (Beth Israel Deaconess Hospital, Boston, Mass.). They were grown in Iscove's DMEM medium containing 5% fetal bovine serum (FBS), 20 mM 2-mercaptoethanol, 2 mM L-glutamine, and 100 U/ml penicillin/streptomycin. For maintenance of growth, these cells were kept in growth media containing 50 U/ml (or 1 ng/ml) recombinant human IL-6 (Genzyme). For growth dependence by IL-6 (IL-6 bioassay), B9 cells were washed three times in assay media and counted, cell concentration was adjusted to 4×105/ml (2×104/50 μl) in assay media, and 50 μl of media was added to each well of a 96-well tissue culture plate.
To the above-described cell suspension, 50 μl of standard or sample was added to each well, and the cells cultured for 68-72 hours at 37° C./5% CO2. Dog plasma samples were added to the assay at a 1:20 dilution (10 μl+190 μl) in assay media (in duplicate), then serially diluted 1:4 (to a final dilution of 1:327,680) in 96-well microtitre plates. Fifty microliters of each dilution were then transferred to an appropriately labeled assay microtitre plate. Standard curves were prepared (2-4 rows/plate, depending on plate space) using human rIL-6 as a standard (10 μg/ml), diluted 1:100, and then diluted another 1:10 to 10 ng/ml. Two hundred microliters of this dilution were added to a dilution plate, then each was serially diluted 1:4, and 2 blank wells received 50 μl assay media only. After the culture period, actively metabolizing cells were quantitated by adding 10 μl of a 5 mg/ml solution of MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) in sterile PBS to each well. Plates were incubated 4-5 hours at 37° C., acid-isopropanol (150 μl 40 mM HCL in isopropanol) was added to each well, plates were incubated for 1 hour at 37° C., followed by repeated trituration to solubilize crystals, and absorbance was read at 540 and 690 nm (background absorbance). IL-6 concentration was determined by calculation of a linear relationship for response to IL-6 standards that yielded the greatest dose-response region of the standard curve. (In general, this range is between 0.016 and 0.063 ng/ml IL-6, yielding net absorbances of ˜0.3 to 0.4 for the low dose and ˜0.8 to 1.0 for the high dose.) Only absorbances that fell between the above values for standards (or ±0.05 AU) were used to calculate IL-6 by interpolation from the linear curve drawn by the Four Parameter Curve Fit program (Delta Soft) through the standard points.
(2.5) Induction of IL-6 by LPS Challenge in ex vivo Blood Samples
To obtain baseline values for LPS stimulation, samples of blood were drawn at approximately one hour prior to beginning administration (predose). While we did not extensively analyze the dose response relationship of dog blood to LPS, we used 1, 10, and 100 ng/ml LPS to ensure that a measurable response could be generated. Responses to LPS in these samples resulted in 6,000 pg/ml IL-6 to as high as 40,000 pg/ml IL-6 in response to 100 ng/ml LPS in the four dogs. Some samples (particularly from the two female dogs) demonstrated a more graded response to the three different concentrations of LPS. However, all LPS-challenged predose samples generated between 3,000 pg/ml IL-6 and 32,000 pg/ml IL-6. Blood from the male beagles responded more vigorously than blood from the female dogs.
All references cited herein are incorporated by reference in their entirety. Other embodiments are within the following claims.
This application is a continuation of PCT/US03/18678, filed Jun. 13, 2003, which claims priority from U.S. Ser. No. 10/171,465, filed Jun. 13, 2002, which is a continuation-in-part of U.S. Ser. No. 09/889,274, filed Jul. 12, 2001, which claims priority from PCT/US00/01043, filed Jan. 14, 2000, which claims priority from U.S. Ser. No. 60/116,202, filed Jan. 14, 1999. The contents of each of the aforementioned applications are incorporated herein by reference.
Number | Date | Country | |
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60116202 | Jan 1999 | US |
Number | Date | Country | |
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Parent | PCT/US03/18678 | Jun 2003 | US |
Child | 11010550 | Dec 2004 | US |
Parent | 10171465 | Jun 2002 | US |
Child | PCT/US03/18678 | Jun 2003 | US |
Number | Date | Country | |
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Parent | 09889274 | Jul 2001 | US |
Child | 10171465 | Jun 2002 | US |