Title: TESTING FOR SEPSIS

The invention relates to the field of experimental medical research in humans and more particularly to clinical trials in human medicine and to prospective studies of human subjects designed to answer questions about biomedical interventions, e.g., drugs, treatments, devices, or new ways of using known treatments with the purpose to determine whether they are safe and/or effective.

BACKGROUND

The field of experimental medical research in human subjects is rapidly evolving due to the fact that on the one hand, experiments done in

(experimental) animals or done in vitro do not always give the answers required to further understanding about biomedical interventions in human beings, while on the other hand, new drugs, new devices and new treatments require intensive study before they are approved by the respective regulatory bodies for patient use. In particular, the number regulatory clinical trial studies in humans is rapidly increasing. Most clinical trials are designated as phase I, II, III, or IV, based on the type of questions that the study is seeking to answer. Design and outcome of clinical trials generally has to be reported to the various regulatory authorities and in order to get approval to proceed with further development and the final prospect of marketing.

Generally, in phase I clinical trials, researchers test a new drug or treatment in a small group of people for the first time to evaluate its safety, determine a safe dosage range, and identify side effects. This is the first phase of clinical drug development where the evaluation of a drug is primarily for safety and tolerability in healthy individuals. In one or more clinical trials, safety, tolerability, dose range, and pharmacokinetic profiles may be explored.

In phase II clinical trials, the study drug or treatment is given to a larger group of people to see if it is effective and to further evaluate its safety. This is the second phase of clinical drug development in which the evaluation of a drug is primarily in patients for safety, tolerability and efficacy. Dosing regimens are tested for magnitude and duration of effect. Positive efficacy is often referred to as clinical "proof of concept." This phase should preferably conclude with an indication of whether the drug works, which patient population to target, and what is the optimal dose (or dose range) at which beneficial effects are maximized and side effects are minimized ("therapeutic window"). Sometimes, phase II is subdivided in phases Ha and lib; phase iia being a clinical trial involving the administration of a drug to human patients in which the primary objective is to obtain a preliminary evaluation of the efficacy of the drug, typically in small numbers of patients, and phase lib being a clinical trial involving the administration of a drug to human patients in which the primary objective is to establish the appropriate therapeutic dose for the drug.

In phase III studies, the study drug or treatment is given to large groups of people to confirm its effectiveness, monitor side effects, compare it to commonly used treatments, and collect information that will allow the drug or treatment to be used safely. This is the third phase of clinical drug development in which the drug undergoes an extensive test of its ultimate proposed use on the market. Trials evaluate whether the drug presented at a particular dose, to a particular population and in a particular formulation has sufficient clinical and statistically significant effects along with an appropriate side effect profile. These trials are often carried out in large patient populations and are regarded as pivotal in the regulatory assessment of safety and efficacy.

In the United States, an NIH-defined phase III clinical trial is a broadly based prospective investigation, including community and other population-based trials, usually involving several hundred or more people, to evaluate an experimental intervention in comparison with a standard or

control, or to compare two or more existing treatments. Often, the aim is to provide evidence for changing policy or standard of care. It includes pharmacologic, non-pharmacologic, and behavioral interventions for disease prevention, prophylaxis, diagnosis, or therapy. A phase IV is a study done after an intervention has been marketed to monitor its effectiveness in the general population and collect information about adverse effects associated with widespread use.

These clinical trial studies are generally conducted in accordance with Good Clinical Practice (GCP) as required by the International Conference on Harmonization (ICH) guidelines and in accordance with country-specific laws and regulations governing clinical studies of investigational products. Compliance with these requirements also constitutes conformity with the ethical principles of the Declaration of Helsinki.

Sepsis, SIRS and Septic Shock

Sepsis can be simply defined as a spectrum of clinical conditions caused by the immune response of a patient to infection that is characterized by systemic inflammation and coagulation. It includes the full range of response from systemic inflammatory response syndrome (SIRS) to organ dysfunction to multiple organ failure and ultimately death. This is a very complex sequence of events and much work still needs to be done to completely understand how a patient goes from SIRS to septic shock. Clinicians often use the terms sepsis and septic shock without a commonly understood definition. The American College of Chest Physicians and the Society of Critical Care Medicine developed the following definitions to clarify the terminology used to describe the spectrum of disease that results from severe infection. The basis of sepsis is the presence of infection and the subsequent physiologic alterations in response to that infection, namely, the activation of the inflammatory cascade. Systemic inflammatory response syndrome (SIRS) is a term used to define this clinical condition and it is considered present if abnormalities in

two of the following four clinical parameters exist: (1) body temperature, (2) heart rate, (3) respiratory rate, and (4) peripheral leukocyte count. Sepsis is defined as the presence of SIRS in the setting of infection. Severe sepsis is defined as sepsis with evidence of end-organ dysfunction as a result of hypoperfusion. Septic shock is defined as sepsis with persistent hypotension despite fluid resuscitation and resulting tissue hypoperfusion. Bacteremia is defined as the presence of viable bacteria within the liquid component of blood. Bacteremia may be primary (without an identifiable focus of infection) or, more often, secondary (with an intravascular or extravascular focus of infection). While sepsis is commonly associated with bacterial infection, bacteremia is not a necessary ingredient in the activation of the systemic inflammatory response that results in severe sepsis. In fact, fewer than 50% of cases of sepsis are associated with bacteremia and severe sepsis or septic shock may develop in patients that undergo SIRS due to trauma, severe burns and other inflammatory stimuli wherein no infection can be detected. Patients with septic shock may have a biphasic immunological response. Initially, they manifest an overwhelming inflammatory response to the infection. This is most likely due to the pro-inflammatory cytokines Tumor Necrosis Factor (TNF), IL-I, IL- 12, Interferon gamma (IFN-gamma), and IL-6. The body then regulates this response by producing anti-inflammatory cytokines (IL-10), soluble inhibitors (TNF receptors, IL-I receptor type II, and IL-IRA (an inactive form of IL-I)), which is manifested in the patient by a period of immunodepression. Persistence of this hyporesponsiveness is associated with increased risk of nosocomial infection and death. Septic shock, the most severe complication of sepsis or SIRS, is a deadly disease. In recent years, exciting advances have been made in the understanding of its pathophysiology and treatment (D. Annane et al., Lancet 2005; 365:63-78). Pathogens trigger immune cells, epithelium, endothelium and the neuroendocrine system. The resulting inflammatory response leads to damage to host tissue. The time window for interventions is short and

treatment must promptly control the source of infection and restore hemodynamic homeostasis. Septic shock is the most common cause of mortality in the intensive care unit. It is the tenth leading cause of death overall (2000) and is the most common cause of shock encountered by internists in the United States. Despite aggressive treatment, mortality ranges from 15% in patients with sepsis to 40% to 60% in patients with septic shock. There is a continuum of clinical manifestations from SIRS to sepsis to severe sepsis to septic shock to Multiple Organ Dysfunction Syndrome (MODS). The first attempts to combat inflammation in patients with septic shock relied on non-selective drugs, i.e., high dose corticosteroids (D. Annane et al., BMJ 2004; 329:480) and non-steroidal inflammatory drugs (G.R. Bernard, N. Engl. J. Med. 1997; 336:912-918). These drugs failed to improve survival. Monoclonal antibodies (HA-IA, E5) targeting Mucopolysaccharide (LPS) were also tested, but proved ineffective because of their weak biological activity (E.J. Ziegler et al., N. Engl. J. Med. 1991; 324:429-436). Second-generation drugs for septic shock blindly and systemically block one factor in the inflammatory cascade, for instance, TNF-α, interleukin-1, platelet-activating factor, adhesion molecules or NO synthase. Around 80 clinical trials were done, resulting in several phase III studies with the most promising agents. All failed to improve survival (J. C. Marshall, Nat. Rev. Drug Discov. 2003; 2:391-405). No wonder the development of compounds for treatment of sepsis is sometimes called "the graveyard of bio-technology ." One of the problems in developing new therapeutic compounds for sepsis is the lack of adequate human models, in particular, for early phase II studies. There are only limited methodologies that can be used in human volunteers to model physiologic alterations similar to those that are present in critically ill, septic patients. Exposure of volunteers to bacterial products, such as LPS, or pro-inflammatory cytokines whose release is increased in severe infection, can duplicate some of the relevant clinical findings in sepsis. The

most common protocols have used a single intravenous LPS dose of 1 to 4 ng/kg or an intrapulmonary LPS dose of 4 ng/kg. Also, intravenous or intrapulmonary endotoxin administration to volunteers has been used to simulate the inflammatory response associated with sepsis.

DISCLOSURE OF THE INVENTION

The invention provides a method of conducting an experiment in a human test group to determine the safety or efficacy of a pharmaceutical test composition for future treatment of at least one subject suffering or believed to be suffering of sepsis or systemic inflammatory response syndrome (SIRS) with THE composition comprising providing at least one test individual of the group with an antibody-like substance capable of inducing systemic release of cytokines from cells of the individual, possibly further comprising providing at least one test individual with the composition, possibly further comprising providing at least one control individual of the group with an antibody-like substance capable of inducing systemic release of cytokines from cells of the control individual, possibly further comprising providing at least one control individual with a control composition other than the pharmaceutical test composition. For the further development of pharmaceutical test compositions (herein also called anti-sepsis compounds) capable of treating a sepsis/SIRS condition, an early phase II proof of concept study is often required. Sepsis is envisioned to be the primary goal for clinical development. However, clinical studies in patients with sepsis are characterized by extensive heterogeneity in the patient populations necessitating large test groups to be used, making sepsis trials very costly. Among other factors, patients with sepsis may differ regarding baseline disease, causative microbiological agent, co-morbidity, interval between onset of symptoms and initiation of treatment and type of treatment. As a result of this heterogeneity, patients with sepsis also do not form an attractive population for demonstration of a beneficial effect of pharmaceutical test compositions, for example in a phase II setting, simply

because the number of patients to be tested to demonstrate similar clinical effects is very large, again making such studies very, and sometimes prohibitively, costly. The invention provides an inexpensive, or at least less costly, solution for this problem by testing anti-sepsis compositions in groups of human volunteers that are brought in a state of sepsis-SIRS-like condition by inducing a systemic cytokine release in individuals to be tested. The term "antibody-like substance" as used herein encompasses antibodies and antibody fragments comprising comprising at least one, and preferably two, heavy chain variable regions (VH) and/or at least one, preferably two, light chain variable regions (VL). For example, it is a polyclonal or monoclonal antibody capable of activating T cells, thereby inducing systemic cytokine release. An antibody fragment is typically an antigen-binding fragment. The term "antigen-binding fragment" refers to one or more fragments of a full- length antibody that are capable of specifically binding to an antigen. Examples of binding fragments include (i) a Fab fragment (a monovalent fragment consisting of the VL, VH, CL and CHl domains; (ii) a F(ab')2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulphide bridge at the hinge region; (iii) a Fd fragment (consisting of the VH and CHl domains); (iv) a Fv fragment (consisting of the VH and VL domains of a single arm of an antibody); (v) a dAb fragment (consisting of the VH domain); (vi) an isolated CDR; (vii) a single chain Fv (scFv) (consisting of the VH and VL domains of a single arm of an antibody joined by a synthetic linker using recombinant means such that the VH and VL domains pair to form a monovalent molecule); (viii) diabodies (consisting of two scFvs in which the VH and VL domains are joined such that they do not pair to form a monovalent molecule; the VH of each one of the scFv pairs with the VL domain of the other scFv to form a bivalent molecule); (ix) bi-specific antibodies (consisting of at least two antigen binding regions, each region binding a different epitope). It is preferred that the antibody-like substance comprises an antibody or fragment thereof directed against a cell or cell marker of the individual, preferably

wherein the cell is a white blood cell such as a macrophage, granulocyte, lymphocyte or thymocyte, more preferably, wherein the antibody-like substance is capable of inducing lysis of a cell of the test individual(s). Preferred cells to be lysed are white blood cells, such as a macrophage, granulocyte, lymphocyte or thymocyte.

It is preferred that the antibody -like substance is (polyclonal or monoclonal) anti-thymocyte globulin (ATG), or Fab fragments thereof. ATG is an infusion of rabbit-derived antibodies against human T cells which is used, among others, in the prevention and treatment of acute rejection in organ transplantation and therapy of aplastic anemia.

Other examples are OKT3 antibodies or similar antibodies directed against cell or cell markers of humans that are capable of inducing cytokine release from human cells. OKT3 ( also called muromonab) is an immunosuppressant drug given to reduce acute rejection in transplant patients. OKT3 is a murine monoclonal IgG2a antibody that specifically reacts with the T cell receptor-CD3 complex on the surface of circulating human T cells. The T cell has 2 molecules on its surface which function primarily in antigen recognition. These antigen recognition structures are associated with 3 polypeptide chains (the CD-3 complex). The CD-3 complex transduces the signal for the T cell to react to the foreign antigen, proliferate, and attack the foreign matter. OKT3 is a monoclonal antibody that specifically reacts with the T-3 complex by blocking the function of T cells.

Among others, the invention provides a method for evaluating the safety or efficacy of a pharmaceutical test composition for the treatment of a human subject suffering from, or suspected to be suffering from, sepsis or SIRS, comprising the steps of: a) administering to at least a first and a second human test individual an antibody-like substance capable of inducing cytokine release from cells of the individuals;

b) administering to at least the first test individual the pharmaceutical test composition; c) monitoring whether there is a difference in at least one parameter between at least the first and second test individuals that is indicative of (i) the safety of the pharmaceutical test composition and/or (ii) the efficacy of the test composition for the treatment of systemic cytokine release from cells of the individuals; and d) using the information obtained in step c) to determine whether the composition is suitable for the treatment of a human subject suffering from or suspected to be suffering from sepsis or SIRS.

It is preferred to administer to at least a second test individual a reference control compound and not the test composition. Parameters to be studied are body temperature, heart rate, respiratory rate, and peripheral leukocyte count and sepsis-like conditions in test individuals are considered present when in at least two of the above four abnormalities exist. In particular, the invention provides a method wherein the antibody-like substance comprises an antibody or fragment thereof directed against a cell or cell marker of the individual. The cell is preferably a white blood cell, such as a macrophage, granulocyte, lymphocyte or thymocyte and the antibody-like substance is preferably capable of inducing lysis of a cell (preferably a white blood cell such as a macrophage, granulocyte, lymphocyte or thymocyte) of the individual.

The invention also provides use of an antibody-like substance capable of inducing release of cytokines from cells of an individual in a human test group for the preparation of an agent for evaluating the safety or efficacy of a pharmaceutical test composition for future treatment of a subject suffering from, or believed to be suffering from, sepsis or systemic inflammatory response syndrome in an experiment conducted in the human test group, the experiment comprising: a) providing at least one individual of the group with the agent, and

b) providing at least one individual with the pharmaceutical test composition.

Also, the invention provides a test kit for evaluating the safety or efficacy of a pharmaceutical test composition for future treatment of a subject suffering from, or believed to be suffering from, sepsis or systemic inflammatory response syndrome in an experiment conducted in a human test group, comprising: a) an antibody-like substance capable of inducing release of cytokines from cells, and b) an instruction indicating that the experiment comprises the steps of:

1) providing at least one individual of the group with the antibody-like substance, and

2) providing at least one individual with the pharmaceutical test composition.

An example of such an instruction can be found in the experimental part herein.

In short, the invention also provides a method for determining whether a pharmaceutical test composition is suitable for the treatment of a human subject suffering from, or suspected to be suffering from, sepsis or SIRS, comprising the steps of: a) Administering to at least a first and a second human test individual an antibody-like substance capable of inducing systemic cytokine release from cells of individuals, preferably wherein the substance is anti-thymocyte globulin; b) Administering to at least the first test individual the pharmaceutical test composition and preferably to at least the second test individual a reference control compound; such as a placebo or a pharmaceutical composition to which the pharmaceutical test composition is compared;

c) monitoring whether there is a difference in at least one parameter/symptom/indicator between at least the first and second test individuals that is indicative of (i) the safety of the pharmaceutical test composition and/or (ii) the efficacy of the test composition for the treatment of the effects of systemic cytokine release from cells of the individuals; and d) using the information obtained in step c) to determine whether the composition is suitable for the treatment of a human subject suffering from or suspected to be suffering from sepsis or SIRS.

Also, the invention provides a method for determining whether a pharmaceutical test composition is suitable for the treatment of a human subject suffering from, or suspected to be suffering from, sepsis or SIRS, comprising the steps of: a) Administering to at least a first test individual, the pharmaceutical test composition and to at least a second test individual, a reference control compound, wherein both test individuals suffer from an induced cytokine release condition, preferably wherein the condition is caused by treatment of the individuals by the intravenous administration of anti-thymocyte globulin (ATG); b) monitoring whether there is a difference in at least one parameter/symptom/indicator between at least the first and second test individuals that is indicative of (i) the safety of the pharmaceutical test composition and/or (ii) the efficacy of the test composition for the treatment of sepsis or SIRS; and c) using the information obtained in step c) to determine whether the composition is suitable for the treatment of a human subject suffering from or suspected to be suffering from sepsis or SIRS.

The invention also provides use of a test compound for the preparation of a pharmaceutical test composition for determining the safety or efficacy of the

composition for future treatment of at least one subject suffering, or believed to be suffering from, sepsis or systemic inflammatory response syndrome with the composition by conducting an experiment in a human test group, the experiment comprising: a) providing at least one test individual of the group with an antibody-like substance capable of inducing systemic release of cytokines from cells of the individual; and b) providing at least one test individual with the composition.

In short, the invention provides the use of anti-thymocyte globulin capable of inducing or systemic release of cytokines from cells for mimicking a pathophysiological state of sepsis or systemic inflammatory response syndrome in an individual, which pathophysiological state can subsequently be used to evaluate the safety or efficacy of a pharmaceutical test composition for the treatment of a subject suffering from or believed to be suffering from sepsis or systemic inflammatory response syndrome. As an alternative, the cytokine release syndrome associated with the intravenous administration of anti-thymocyte globulin (ATG) in a polyclonal fashion or in a monoclonal fashion such as OKT3, is herein provided as a model of instant and systemic release of systemic amounts of cytokines, wherein putative anti-sepsis compounds may be tested. Patients receiving ATG or OKT3 develop chills, fever, hypotension, diarrhea, anxiety within six hours of administration, with symptoms quenched over a 24-hour time period (D. Abramowicz et al., Transplantation 1989; 47:606-608). Following the first injection, TNFα and INF-gamma levels in serum peak within several hours (L. Chatenoud et al., Transplantation 1990; 49:697-702). In contrast to sepsis patients, in ATG administration, the cause of cytokine release is uniform, the timing of clinical events is predictable and reproducible, the exact time of onset of ATG administration can be scheduled, and interventions can be arranged accordingly. These considerations make the ATG/OKT3-associated cytokine

release syndrome an attractive model for testing the effects of therapeutic compounds or pharmaceutical test compositions for their efficacy in the treatment of cytokine release-related sepsis or SIRS. OKT3 is hardly used in clinical practice nowadays. Currently, ATG administration is mostly applied in solid organ transplant recipients as induction therapy or to treat severe acute rejections, and as part of the conditioning regimen prior to haematopoietic stem cell transplantation. The haematopoietic stem cell transplant recipients form the most attractive group for a phase II study. The occurrence of a cytokine release syndrome in this patient population is well established.

DETAILED DESCRIPTION OF THE INVENTION

In order to limit the severity of the cytokine release syndrome, a number of strategies have been explored. For example, IL-IO, anti INF-gamma and anti IL-6 have been studied in experimental models (Matthys et al, Eur. J. Immunol. 1993; 23:2209-2216; and Donckier et al., Transplantation 1994; 57:1436-1439). Oral pentoxifylline, a hemorrheologic agent that was found to inhibit TNF-alfa production, was not successful in decreasing the incidence or severity of cytokine release in patients receiving OKT3 (De Vault et al. Transplantation 1994; 57:532-540).

This study protocol concerns a double-blind, placebo-controlled intervention study in ATG-treated haematopoietic stem cell transplant recipients. Study drug will be administered immediately prior to ATG infusion. The primary objective of the study is to determine the efficacy of a TEST COMPOUND in inhibiting the severity of the ATG-induced cytokine release syndrome in haematopoietic stem cell transplant recipients, compared to placebo.

Overall Study Design and Plan: Description

This proposed study is a double-blind, prospective, placebo-controlled, single center, phase II study investigating the efficacy of the test compound in inhibiting the severity of the ATG-induced cytokine release syndrome in haematopoietic stem cell transplant recipients. Patients will be randomized in a 1:1 ratio. The total number of subjects in the study is 16.

Eligibility of patients will be screened by the treating hematologist two weeks prior to ATG treatment. If patients fulfil the in-and exclusion criteria, the treating hematologist will propose the study to the patient and explain the potential risks and benefits of participation. The patient can subsequently consider participation and discuss with relatives for at least one week. If informed consent is obtained, the patient will be randomized 48 hours prior to initiation of ATG treatment to allow time for preparation of study drug by the hospital pharmacist.

Example 1

ATG administration protocol for ANTI-SEPSIS TEST COMPOUND study. Day 0

09:00 - 09:30 0.5 mg/kg prednisolon in 100 mL NaCl 0.9% 09:30 - 09:40 stop prednisolon, flush with 50 mL NaCl 0.9% 09:40 - 09:50 start ANTI-SEPSIS TEST COMPOUND or placebo infusion: xx mg/kg in xxx mL xxxx 09:30 - 09:40 stop ANTI-SEPSIS TEST COMPOUND or placebo infusion, flush with 50 mL NaCl 0.9% 10:00 - 16:00 start ATG infusion (rabbit ATG, 2 mg/kg in

500 mL NaCl 0.9%)

16:00 - 16:10 stop ATG, flush line with 50 mL NaCl 0.9% 16:10 - 16:30 0.5 mg/kg prednisolon in 100 mL NaCl 0.9%

In case of fever, acetaminophen (oral or suppository) can be administered; in case of chills, pethidine (i.v.) can be administered.

Example 2

Patient populations in which the use of ANTI-SEPSIS TEST COMPOUND substances could be tested:

1. Patients with a hematological disease receiving ATG preparations or OKT3 as part of the conditioning regimen prior to hematopoietic stem cell transplantation.

2. Solid organ transplant patients receiving ATG preparations or OKT3 for treatment of an acute rejection episode.

3. Solid organ transplant patients receiving ATG preparations or OKT3 as induction therapy to prevent acute rejection.

4. Patients with aplastic anemia receiving ATG preparations or OKT3 as therapy.

5. Patients experiencing tumor lysis syndrome following initiation of chemotherapy. 6. Patients experiencing neuroleptic malignant syndrome.

7. Patients with sepsis or systemic inflammatory response syndrome.

8. Patients suffering from a treatment-associated inflammatory response, following administration of monoclonal antibodies directed against cell surface molecules.

9. Patients undergoing a surgical procedure eliciting an inflammatory response.

Parameters to be monitored in studies using ANTI-SEPSIS TEST substances: Clinical parameters:

urine production blood pressure heart rate body temperature (central end peripheral) - sublingual capnography

Laboratory parameters:

C reactive protein

Leukocytes - Lymphocytes

Thrombocytes

Creatinine

Urea

Electrolytes: Na, K, Ca, Mg - pH, pθ2, pCO2, Base Excess

Cytokines: IL-I, IL-2, IL-4, IL-6, IL-IO, IL-15, IFN-α,

IFN-Y, TNF-α, TGF-β - LDH

SGOT/SGPT - Ex vivo cytokine production