WO1993011793A1

WO1993011793A1 - Use of the combination of anti-tumor necrosis factor plus interleukin-6 to treat septic shock

Info

Publication number: WO1993011793A1
Application number: PCT/US1992/010596
Authority: WO
Inventors: Beverly E. Barton; James V. Jackson
Original assignee: Schering Corporation
Priority date: 1991-12-17
Filing date: 1992-12-15
Publication date: 1993-06-24
Also published as: AU3244693A

Abstract

Methods and compositions are provided for treating or preventing septic shock in a mammal. The methods comprise administering to a mammal afflicted with or at high risk for developing septic shock and effective amount of a combination of an anti-TNF antibody and IL-6.

Description

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USE OF THE COMBINATION OF ANTI-TUMOR NECROSIS FACTOR PLUS INTERLEUKIN-6 TO TREAT SEPTIC SHOCK

BACKGROUND OF THE INVENTION

5 Septic shock is an often fatal condition usually resulting from gram-negative bacteremia. Despite the use of potent antibiotics and intensive care, the mortality with sepsis and gram-negative bacteremia remains high (20-60% depending on the specific population) [Ziegler et al., New Eng.

1 0 J. Mecf. 324:429 (1991); Bone et al., New Eng. J. Med. 377:653 (1987); and Kreger et al., Am. J. Med 65:344 (1980)]. Approximately 100,000-300,000 cases of gram-negative bacteremia caused sepsis are reported per year, with the resulting deaths estimated at 30,000 to 100,000 [Wolff, New

1 5 Eng. J. Med. 524:486 (1991)]. Sepsis requires prompt treatment, since the patient's condition often deteriorates rapidly. It is a leading cause of morbidity and mortality among hospitalized patients. The symptoms of septic shock include fever or hypothermia, tachycardia, tachypnea,

20 hypotension, peripheral hypoperfusion or systemic toxicity. [Ziegler et al., supra].

Tumor necrosis factor (TNF), a pro-inflammatory cytokine, is thought to play a major role in the pathogenesis of septic shock [Franks et al., Infec. Immunol. 59:2609 (1991)].

25 Administration of neutralizing antibodies to murine or human TNF has been shown to protect mice, rabbits, and primates against death from the experimentally-induced manifestations of septic shock. [Dinarello et al., J. Infec. Dis. 163 :1111 (1991); Tracey et al, Nature 330:66 (1987); Beutler et al.. Science

30 229:869 (1985); Mathison et al., J. Clin. Invest. 87 : 1925 (1988)]. The role of Interleukin-6 (IL-6) in septic shock has not been as clearly defined. One investigator has reported a new link between TNF and IL-6; IL-6 can reduce the experimentally-induced release of TNF in the monocytoid cell line U937, cultured human peripheral blood monocytes, and intact mice [Aderka et al., J. Immunol. 745:3517 (1989)]. Other investigators have demonstrated that TNF is a potent inducer of IL-6 in cultured fibroblasts [Kohase et al., Cell 45:659 (1986)], in various tumor cell lines [Defilippo et al., Proc. Natl. Acad. Sci. (USA) 84:4557 (1987)], and also in man [Jablons et al, J. Immunol. 742:1542 (1989)].

The existence of a reciprocal stimulatory/inhibitory interaction between TNF and IL-6 suggests a complex relationship of considerable potential importance in the regulation of the many varied biologic actions of these two cytokines [Aderka et al, supra]. The inhibitory effect of IL-6 on TNF production possibly reflects a predominantly anti-inflammatory function of IL-6 [Aderka et al, supra]. IL-6 has also been shown to induce ACTH release and thereby induces cortisol synthesis, which further enforces the suggestion that IL-6 sometimes serves an anti-inflammatory function [Woloski et al, Science 230: 1035 (1985)].

SUMMARY OF THE INV-ENTION

This invention provides a method for treating septic shock in a mammal comprising administering to a mammal afflicted with septic shock an effective amount of a combination of an anti-TNF antibody and IL-6. This invention also provides a method for preventing septic shock in a mammal which comprises administering to a mammal susceptible to or at high risk for developing septic shock, an effective amount of a combination of an anti-TNF antibody and IL-6. A pharmaceutical composition comprising a combination of an anti-TNF antibody and IL-6, and a physiologically acceptable carrier, is also provided by this invention.

BRIEF DESCRIPTION OF THE FIGURES

This invention can be more readily understood by reference to the accompanying Figures, in which:

Fig. 1 is a graphical representation of the effects of various treatments administered to groups of 20 mice 18 hours prior to challenge with LPS-gal. Mortality in the groups of mice 24 hours after LPS-gal challenge is shown as a function of pre-treatment with control Dulbecco's phosphate buffered saline (DPBS) and monoclonal antibodies against IL-6 (20F-3) and IL-5 (TRFK-5).

Fig. 2 is a graphical representation of the effects of various treatments administered to groups of 20 mice 18 hours prior to challenge with LPS-gal. Mortality in the groups of mice 24 hours after LPS-gal challenge is shown, from left to right, as a function of pre-treatment with control Dulbecco's phosphate buffered saline (DPBS), 500 μg of hamster gamma globulin (HGG), and 50 μg and 100 μg of an anti-TNF antibody (TN3) (hamster origin).

Fig. 3 is a graphical representation of the effects of varying doses of anti-TNF antibody administered to groups of 20 mice 18 hours prior to challenge with LPS-gal. Mortality in the groups of mice 24 hours after challenge is shown as a function of pre-treatment antibody dose.

Fig. 4 is a graphical representation of the effects of various treatments administered to groups of 20 mice prior to challenge with LPS-gal. Mortality in the groups of mice 24 hours after LPS-gal challenge is shown as a function of pre- treatment with 25 μg/mouse anti-TNF antibody (TN3) with or without 1 mg/mouse anti-IL-6 antibody (20F-3, or with Dulbecco's phosphate buffered saline (DPBS) or 1 mg/mouse hamster gamma globulin (HGG). The results from two experiments are shown using TN3 with or without 20F-3; the DPBS and HGG values shown are the averages from the two experiments. For the combination treatments, p<0.05 as determined by the Student's t-test.

Fig. 5 is a graphical representation of the effects of varying doses of recombinant IL-6 or 0.57 μg/mouse control hamster gamma globulin (HGG) administered to groups of 20 mice 1 hour prior to challenge with LPS-gal. The mice had also been treated with 25 μg/mouse anti-TNF antibody prior to LPS-gal challenge. The mortality in the groups of mice 24 hours after challenge is shown as a function of IL-6 dose.

Fig. 6 is a graphical representation of the effects of varying doses of recombinant IL-6 administered to groups of 20 mice 1 hour prior to challenge with LPS-gal. Mortality in the groups of mice 24 hours after challenge is shown as a function of IL-6 dose. The two bars for each IL-6 dose represent the results of two separate experiments. For both IL-6 doses, p > 0.07 as determined by the Student's t-test.

DESCRIPTION OF THE INVENTION

All references cited herein are hereby incorporated in their entirety by reference.

The term "septic shock" as used herein is defined as a state of morbidity manifesting one or more of the following symptoms: fever or hypothermia [temperature above 38.7°C (101° F) or below 35.6° C (96° F)]; tachycardia (heart rate above 90 beats per minute in the absence of a beta-blockade), tachypnea (respiratory rate above 20 breaths per minute or the requirement of mechanical ventilation); and either hypotension (systolic blood pressure below 90 mm Hg or a sustained drop in systolic pressure above 40 mm Hg in the presence of adequate fluid challenge and the absence of anti-hypertensive agents) or two of the following six signs of systemic toxicity or peripheral hypoperfusion: unexplained metabolic acidosis (blood pH below 7.3, base deficit of greater than 5 mmol per liter, or an elevated plasma lactate level); arterial hypoxemia (partial pressure of oxygen below 75 mm Hg or ratio of the partial pressure of oxygen to the fraction of inspired oxygen less than 250); acute renal failure (urinary output- of less than 0.5 ml per kilogram of body weight per hour); elevated prothrombin or partial thromboplastin time or reduction of the platelet count to less than half the baseline value or less that 100,000 platelets per cubic milliliter; sudden decrease in mental acuity; and cardiac index of more than 4 liters per m .ute per square meter of body-surface area with systemic vascular resistance of less than 800 dyn • sec • cm^-5; and serum elevation of TNF [Ziegler et al, supra] .

The symptoms listed above are illustrative of specific selection criteria to be used in determining candidates for the proposed method of treatment. States of morbidity which can cause the foregoing symptoms include but are not limited to: acute gram-negative bacteria infections, endotoxemia, purpura fulminans, severe psoriasis, acute rheumatoid arthritis, burns, organ transplant rejection, and physical traumas, such as abdominal wounds. Candidates for abdominal surgery (especially bowel surgery) are at high risk for developing septic shock [Debets et al, Crit. Care Med. 7(6):489 (1989)] and could benefit from prophylactic administration of the combination of an anti-TNF antibody and IL-6. The effectiveness of treatment can be assessed by monitoring the above mentioned manifestations of septic shock.

Anti-TNF antibodies are available commercially, e.g., Boehringer Mannheim Biochemicals, Indianapolis, IN. IL-6 is commercially available from Genzyme Corporation, Cambridge, MA. Both can also be prepared by known methods using natural sources or recombinant DNA methodologies [Sheehan et al, J. Immunol. 742:884 (1989); Starnes et al, J. Immunol. 745:4185 (1990)].

These materials are generally supplied in lyophilized form and can be reconstituted just prior to use in a pharmaceutically acceptable carrier such as phosphate buffered saline or any of the other well known carriers. The pharmaceutical compositions of the invention can be injected directly into the bloodstream intravenously or via an I.V. drip solution such as Ringer's lactate. Parenteral preparations that can be used include sterile solutions or suspensions. These preparations can be prepared with conventional pharmaceutically acceptable excipients and additives such as stabilizers and carriers. The solutions to be administered may be reconstituted lyophilized powders which may additionally contain, e.g., preservatives, buffers and dispersants. Preferably, the compositions are administered by intravenous injection.

Kits are also provided by this invention comprising anti-TNF antibodies and IL-6 in physiologically acceptable carriers, in separate containers.

Of course, all of the monoclonal antibodies can be modified by standard recombinant DNA techniques. Such techniques include but are not limited to the production of antibody variants that combine the rodent variable or hypervariable regions with the human constant or constant and variable framework regions [Rudikoff et al, Proc. Natl. Acad. Sci. USA 79:979 (1982), Morrison and Oi, Adv. Immunol. 44:65 (1989), Queen et al, Proc. Natl. Acad. Sci. USA 86: 10029 (1989)]. Humanized antibodies can be generated in which the antigen binding complementarity determining regions (CDRs) from the parent rodent monoclonal antibody are grafted into a human antibody framework. These antibodies are less immunogenic [Queen et al, supra]. Humanized rodent antibodies also demonstrate a longer half-life in humans in vivo than their unmodified rodent counterparts [LoBuglio et al, Proc. Natl. Acad. Sci. USA 86:4220 (1989)].

An alternative approach to the production of monoclonal antibodies entails the cloning of the V-region genes from B -cells using the polymerase chain reaction technique. Antibody derivatives are then expressed in a microbial system (e.g. E. coli) and screened for antigen binding ability [Winter and Milstein, Nature 549:293 (1991); Mullinax et al, Proc. Natl. Acad. Sci. USA 87:8095 (1990)]. Heavy and light chain libraries can be prepared in phage lambda and used to generate a large array of random heavy plus light chain pairs expressed in bacteria [Mullinax et al, supra, and Wald ann, Science 252 :1657 (1991)].

IL-6 can be made if desired using standard recombinant DNA methods. For example, oligonucleotide probe mixtures based on known IL-6 nucleotide sequences can be used to identify DNA encoding IL-6 in genomic or cDNA libraries prepared by standard methods. DNA thus identified can be excised from the library by restriction endonuclease cleavage or prepared using appropriate primers and the polymerase chain reaction (PCR) method [Saiki et al, Science 259:487 (1988)], sequenced and expressed in a eukaryotic expression system or (following intron deletion by standard methods if necessary) in a prokaryotic or eukaryotic expression system. Of course, both cDNA and genomic DNA libraries can be screened by the application of standard expression cloning methods, instead of by the use of oligonucleotide probes or PCR. IL-6 thus produced is detected through the use of known immunochemical or bioassay methods.

The anti-TNF and the IL-6 used will preferably be those of the mammalian species being treated (e.g., anti-human TNF and human recombinant IL-6 are preferred for treating human beings). It is also preferred that glycosylated IL-6 be used (e.g., recombinant IL-6 produced in a eukaryotic expression system).

In accordance with the present invention, mammals that are in need of treatment for septic shock as defined above are administered an effective amount of anti- TNF antibodies in combination with IL-6 to accomplish the above-described results. A dose of from about 0.5 μg to about 250.0 μg anti-TNF antibodies per kilogram of body weight and about 1.0 μg to about 3.0 mg IL-6 per kilogram of body weight is preferably administered. More preferably, mammals are administered a dose from about 1.0 μg to about 3.0 μg anti-TNF antibody per kilogram of body weight and from about 5.0 μg to about 30.0 μg E -6 per kilogram of body weight. In humans, administration of the anti-TNF antibody can be concomitant with or prior to administration of the EL-6. The precise amount of the combination of the anti-TNF antibody and the IL-6 to be administered would be determined by the attending clinicians, taking into account the etiology and severity of the disease, the patient's condition, sex, age, and other factors. In the Example below, overnight treatment of mice with the antibodies and cytokines investigated was done in order to facilitate adequate circulating concentrations of these materials in the bloodstream at the time of LPS-gal administration, because intraperitoneal injection of these materials requires a longer diffusion period to enter the bloodstream than other routes of administration (such as intravenous injection). Intraperitoneal injection was selected in this model due to the difficulty of intravenous injection in the mouse. The preferred route of administration would normally be intravenous injection, where bioavailability of the circulating therapeutic agents would be as rapid as 10 minutes.

EXAMPLE

The present invention can be illustrated by the following, non-limiting Example.

Materials

Male C57BL/6J mice (5 weeks of age) were obtained from Jackson Laboratories, Bar Harbor, ME. Endotoxin-free phosphate buffered saline (PBS) was purchased from GIBCO, Grand Island, NY. LPS (£. coli 0111 :B4) was purchased from List Biological Laboratories, Inc., Campbell, CA. D-galactosamine was obtained from Sigma Chemicals, St. Louis, MO. Purified Recombinant TNF is available from Genzyme, Cambridge, MA and IL-6 is available from Biosource, Camarillo, CA; both were used as standard proteins. Monoclonal rat anti-mouse IL-6 , 20F-3 was obtained from DNAX Research Institute of Cellular and Molecular Biology, Palo Alto, CA. Anti-mouse TNF (purified TN3-19.12 Ab) was obtained from Dr. Robert D. Schreiber, Washington University, St. Louis, MO. Purified hamster gamma globulin (HGG), a protein control, was purchased from Cappel, Durham, NC. The purified rat anti-mouse IL-5 was obtained from DNAX Institute of Cellular and Molecular Biology, Palo Alto, CA.

Demonstration of Protective Effects

LPS, or endotoxin, a component of the outer membrane of gram-negative bacteria, is responsible for the toxic manifestations associated with septic shock. Administration of purified LPS is a well-established method for experimentally inducing septic shock in animal models [Calandra et al, Diag. Microbiol. Infect. Dis. 75:377]. D-galactosamine is a hepatotoxin shown to potentiate the lethal effects of endotoxin (LPS) up to 100,000 fold [Galanos et al, Proc. Natl. Acad. Sci. USA 76:5939 (1979)]. This model is predictive of clinical utility in mammals afflicted with septic shock [Franks et al, supra] .

LPS was dissolved in PBS at 1 mg/ml and frozen at

-80°C until use. Prior to freezing it was sonicated for 5 minutes in a sonifying bath. It was re-sonicated for 5 minutes after thawing. Appropriate dilutions were made in PBS in polypropylene tubes. D-galactosamine was dissolved at 32 mg/ml in PBS and mixed with an equal volume of diluted, sonciated LPS. The LPS-galactosamine mixture was used immediately and fresh batches were made for each experiment. Each mouse received 0.5 ml of LPS-gal mixture intraperitoneally (i.p.) between 1 and 3 pm. Animals were scored for mortality 24 hours later. All therapeutic agents were administered at the times indicated.

Blood samples were collected from anesthetized mice in serum separator tubes and allowed to clot overnight at 4^CC. Sera were removed after microcentrifugation for 5 minutes at 2080 x g. IL-6 and TNF concentrations were measured in sera obtained 90 minutes after LPS-gal administration. Cytokine -specific enzyme linked immunosorbent assays (ELISA's) were performed essentially as described by Sheehan et al, and Starnes et al, supra.

Doses of LPS ranging from 6.25 to 200 ng/mouse were injected i.p. with D-glactosamine as described. As shown in Table I, a dose dependent relationship was observed up to 100 ng/mouse (90% mortality). At higher doses the relationship was not observed. 100 ng/ml was selected as the dose to be used in subsequent experiments because it was found to be the dose that would yield the highest mortality at the lowest dose. In parallel experiments where the

D-galactosamine was not co-administered with the LPS, animals survived doses of 1.5 mg of LPS for greater than 72 hours.

To identify the role of IL-6 in this lethal septic shock model, mice were injected i.p. with 1 and 2 mg/mouse anti-IL-6 antibody (20F-3) 1 to 2 hours prior to LPS-gal treatment. [Starnes et al, supra]. Equal amounts of the isotype control of anti-IL-5 antibody (TRFK-5) were used as control proteins. Table I shows that there was no effect on mortality with the anti-IL-6 antibody treatment.

^a Ab was anti-IL-6 antibody ^b Control Ab was anti-IL-5 antibody

Because of the long circulating half-life of the isotype of anti-IL-6 antibody (10 days to 2 weeks) the antibody was given the night before LPS-gal administration, in case 1 hour was not long enough for all of the anti-IL-6 antibody to enter the circulation from the peritoneal cavity. Results are shown in Fig. 1. Even with overnight treatment, the anti-IL-6 antibody failed to confer any protection against mortality.

As shown in Fig. 2, mice given 50 and 100 μg i.p. of anti-TNF antibody were protected from death. In this representative experiment, no mice died with overnight treatment using 100 μg anti-TNF antibody, and only 10% died after receiving 50 μg of the antibody the night before LPS-gal administration. All mice given only the vehicle control or HGG control antibody died.

Fig. 3 shows the average dose relationship of mortality vs. treatment with anti-TNF antibody. Twenty-five μg/mouse given i.p. the night before LPS-gal administration conferred about 70% protection from death in this model. Doses lower than 10 μg/mouse conferred very little protection.

Treatment with anti-IL-6 antibody potentiated mortality when TNF was partially neutralized, as shown in Fig. 4. Mice were treated simultaneously with 1 and 2 mg of anti- IL-6 antibody and 25 μg/mouse anti-TNF antibody 18 hours prior to LPS-gal administration. Surprisingly, anti-IL-6 antibody was found to enhance mortality significantly. In one experiment where anti-TNF antibody only resulted in 25% mortality, the same dose plus anti-IL-6 antibody resulted in 65% mortality. In another experiment where the anti-TNF resulted in 20% mortality, the anti-TNF antibody plus IL-6 combination resulted in 45% mortality. These data suggest a protective role of IL-6 in this lethal shock model.

Doses of recombinant IL-6 were determined as follows: mice injected with 100 ng of LPS-gal were bled 90 minutes later. Sera were collected and analyzed for TNF and IL-6 concentration at this time point because this is the time determined for peak concentrations of TNF. Table II illustrates the results allowing for maximum volume of 10 ml/mouse (circulating blood volume plus partitioning into tissue and iterstitial spaces). It was calculated that 100 ng of LPS-gal treatment resulted in 440 ng of IL-6 per mouse. Therefore, 440 ng was the selected dose, plus higher and lower doses in half-log increments to measure the effect of recombinant IL-6 when TNF was partially limited by anti-TNF treatment. TABLE II

SERUM LEVELS OF IL-6 AND TNF AFTER LPS/GAL ADMINISTRATION

Time rhpi-trs) TNF. fng/mD IL-6. Cng/mD

0 0 0

1.5 2+1 44+13

Based upon the foregoing results, doses of D -6 for therapeutic administration were selected, with the results shown in Fig. 5.

Fig. 5 shows that treatment with recombinant mouse IL-6 protected against mortality when TNF was limited by prior administration of anti-TNF antibody.

Recombinant IL-6 was given i.p. 1 hour prior to LPS-gal administration. Mice had been treated the night before with 25 μg/each of anti-TNF antibody. At doses of 132 to 570 ng/mouse, recombinant IL-6 conferred significant protection against mortality. Mortality was lowered from an average in these experiments from 20-0% (ρ< 0.05) at 132, 440, and 570 ng/mouse. At lower doses (44 ng and less) no effect was observed. These results demonstrate that the combination of anti-TNF antibody and recombinant IL-6 as a treatment for septic shock is effective in significantly reducing mortality.

In contrast to the results shown in Fig. 5, IL-6 administration 1 hour prior to LPS-gal challenge was substantially ineffective in reducing mortality when the mice did not receive prior treatment with anti-TNF antibody. This is shown in Fig. 6, where the effects observed with the administration of 0.44 or 0.57 μg/mouse IL-6 were similar to the results produced by Dulbecco's phosphate buffered saline alone (no IL-6).

The effect of the anti-TNF antibody plus IL-6 is best illustrated by comparing Fig. 3 with Fig. 5. In Fig. 3, 25 μg/ml of the anti-TNF antibody conferred only 70% protection from death, where in Fig. 5 that same dose of 25 μg/ml plus IL-6 (at 132, 440, and 570 ng/ml) conferred 100% protection from death.

Many modifications and variations of this invention can be made without departing from its spirit and scope as will become apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for treating septic shock comprising administering to a mammal afflicted with septic shock an effective amount of a combination of anti-TNF antibody and IL-6.

2. A method for preventing septic shock comprising administering to a mammal susceptible to or at risk for developing septic shock an effective amount of a combination of an anti-TNF antibody and IL-6.

..

3. A method for the manufacture of a pharmaceutical composition for treating or preventing septic shock comprising admixing a combination of an anti-TNF antibody and IL-6 with a pharmaceutically acceptable carrier.

4. The method of any one of claims 1 to 3 in which the anti-TNF antibody is an anti-human-TNF antibody and the

IL-6 is recombinant human IL-6.

5. A pharmaceutical composition for treating or preventing septic shock comprising a combination of an anti-TNF antibody and IL-6, and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition of claim 5 in which the anti-TNF antibody is an anti-human-TNF antibody and the IL-6 is recombinant human IL-6.

7. The use of a combination of an anti-TNF antibody and IL-6 for the treating or preventing septic shock.

8. The use of a combination of an anti-TNF antibody and IL-6 for the manufacture of a medicament for treating or preventing septic shock.

9. The use of either claim 7 or 8 in which the anti-TNF antibody is an anti-human-TNF antibody and the IL-6 is recombinant human IL-6.

10. A kit comprising in separate containers pharmaceutical compositions for treating or preventing septic shock, one of which containers comprises an anti-TNF antibody in a pharmaceutically acceptable carrier, another of which containers comprises IL-6 in a pharmaceutically acceptable carrier.

11. The kit of claim 10 in which the anti-TNF antibody is an anti-human-TNF antibody and the IL-6 is recombinant human IL-6.