WO2012140109A1

WO2012140109A1 - Biomarkers for pain intensity

Info

Publication number: WO2012140109A1
Application number: PCT/EP2012/056625
Authority: WO
Inventors: Hellmut Samonigg; Walter Schippinger; Ellen Heitzer
Original assignee: Medizinische Universitaet Graz
Current assignee: Medizinische Universitaet Graz
Priority date: 2011-04-12
Filing date: 2012-04-12
Publication date: 2012-10-18
Anticipated expiration: 2013-10-12
Also published as: EP2697651A1

Abstract

The present invention is directed to a method of determining the intensity of pain in a patient, a diagnostic kit for determining the intensity of pain in a patient and a pharmaceutical composition for use in the treatment of pain. Further, the present invention is concerned with the use of the proteins IL-7, IL-18, MCP-1, MIP-1b and OPG as biomarkers for pain intensity.

Description

Biomarkers for pain intensity

FIELD OF THE INVENTION

The present invention is concerned with a method of determining the intensity of pain in a patient comprising the determination of the amounts of the biomarkers IL-7, IL-18, MCP-1, MIP-lb and OPG in a sample derived from said patient. The present invention is further concerned with a diagnostic kit for determining the intensity of pain, a pharmaceutical composition for use in the treatment of pain as well as the use of IL-7, IL-18, MCP-1, MIP-lb and OPG as biomarkers for pain intensity.

BACKGROUND OF THE INVENTION

Pain is the most common symptom for which patients seek medical advice and treatment. According to the International Association for the study of pain (IASP), pain can be defined as "an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage." Pain can lead to significant changes in a patient's personality, lifestyle, functional ability and quality of life.

There are several classifications for pain. Thus, pain may be classified as either "somatic" (also referred to as "somatogenic") pain or "psychogenic" pain. Somatic pain arises from a perturbation of the body and the underlying cause can be detected using inter alia physical examinations, imaging and laboratory tests. In contrast thereto, psychogenic pain is assumed to be the product of psychic conflict or psychopathology. The differentiation between said two types may be crucial for further decisions on the therapy. Pain may further be classified as being acute or chronic.

Somatic pain is usually divided into "nociceptive" pain and "neuropathic" pain. Nociceptive pain includes tissue injury-induced pain and inflammatory pain such as that associated with arthritis. Neuropathic pain is caused by damage to the peripheral or central nervous system and is maintained by aberrant somatosensory processing.

Pain is usually treated by the administration of a suitable analgesic. The choice of the analgesic depends inter alia on the intensity, the cause, the duration and the type of pain. For certain types of pain, such as e.g. chronic malignant pain, titration of the analgesic can become necessary in order to determine the efficient amount of the analgesic. Pain intensity represents one of the main factors important for the decision on the type of pain management, including inter alia the choice of the analgesic, the dosage form, the amount of the analgesic, etc. Thus, pain intensity should be assessed as exact as possible. Thus far, patient's self reports are typically used in order to determine the intensity of pain. In this respect, numeric rating scales ranging from 0 to 10 or questionnaires are routinely used in order to assess pain intensity. Clearly, the patients must be able to somehow communicate their degree of pain by indication or answering a

questionnaire and the determination is based on a subjective impression. If a patient is incapable of communicating her/his pain intensity, the pain intensity is usually estimated by observing the patient's body language or specific behaviours of the patient. However, this estimation appears to be less accurate than other detection methods. The group of patients incapable of communicating their pain intensities are estimated to be around 80.000 patients per year in Austria.

In case children suffer from pain but lack the language needed to report it, a nonverbal pain assessment can be carried out. In this case, the assessment often also includes the parent's assessment of changes in a child and thus the involvement of a further party for the assessment. In case elderly and/or palliative patients suffer from severe chronic (e.g. malignant) pain, such patients may also be incapable of communicating their pain intensities such that pain management can be based on observations only. Thus, there is a strong need in the field to base the assessment of the pain intensity experienced by a patient on endogenous, "objective" factors such that the assessment is independent of the patient's ability to communicate. Clearly, there is also a continuing need for novel pain therapies and endogenous factors, which may be used as biomarkers for pain.

OBJECTS AND SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide a method for determining the intensity of pain in a patient by determining in a sample from said patient the amount(s) of at least one specific biomarker for pain intensity.

In a further object of the present invention, a diagnostic kit is provided for determining the intensity of pain in a patient, wherein said diagnostic kit comprises at least one detecting agent specific for at least one biomarker for pain intensity. Further, it is an object of the present invention to provide a pharmaceutical composition for use in the treatment of pain, wherein said composition comprises at least one active agent directed to at least one biomarker for pain intensity. It is also an object of the present invention to provide for the use of at least one specific biomarker as biomarker for pain intensity and/or as target for pain therapy.

Thus, in one embodiment, the present invention is directed to a method of

determining the intensity of pain in a patient comprising the step of determining in a sample from said patient the amounts of IL-7, IL-18, MCP-1, MIP-lb and OPG.

In another preferred embodiment, a method of determining the intensity of pain in a patient comprising the step of determining in a sample from said patient the amount of a protein selected from the group consisting of IL-7, IL-18, MCP-1, MIP-lb and OPG is provided.

In another embodiment, a method of determining the intensity of pain in a patient comprising the step of determining in a sample from said patient the amount of at least one, preferably of at least two, more preferably of at least three, and most preferably of at least four proteins selected from the group consisting of IL-7, IL-18, MCP-1, MIP-lb and OPG is provided. Preferably, said at least one protein is MCP-1 or MIP-lb or OPG. Preferably, said at least two proteins are MCP-1 and MIP-lb, or MCP-1 and OPG, or MIP-lb and OPG. Preferably, said at least three proteins are MCP-1, MIP-lb and OPG. Preferably, said at least four proteins are MCP-1, MIP-lb, OPG and IL-7.

Thus, in a particularly preferred embodiment, a method of determining the intensity of pain in a patient comprising the step of determining in a sample from said patient the amount of MIP-lb is provided. It can be preferred that such a method comprises the step of determining the amount of MIP-lb and the amount of at least one additional cytokine selected from the group consisting of IL-7, IL-18, MCP-1 and OPG. It can be preferred that the above group consists of IL-18, MCP-1 and OPG.

Thus, the amounts of MIP-lb and IL-7, or MIP-lb and IL-18, or MIP-lb and MCP- 1, or MIP-lb and OPG may be determined in said step. Further, the amounts of MIP- lb, IL-7 and IL-18, or MIP-lb, IL-7 and MCP-1, or MIP-lb, IL-7 and OPG may be determined in said step. Also, one may determine the amounts of MIP-lb, IL-18 and MCP-1, or MIP-lb, IL-18 and OPG, or the amounts of MIP-lb, MCP-1 and OPG in said step. Further, the amounts of MIP-lb, IL-7, IL-18 and MCP-1, or MIP-lb, IL-7, IL-18 and OPG, or MIP-lb, IL-7, MCP-1 and OPG, or the amounts of MIP-lb, IL- 18, MCP-1 and OPG may be determined in said step. As disclosed above, it can be particularly preferred to determine in a sample from said patient the amounts of MIP- lb, IL-7, IL-18, MCP-1 and OPG. In a further preferred embodiment, the method further comprises the step of comparing the amount(s) of the determined protein(s) in said sample from the patient to the amount(s) of said protein(s) determined in a reference and the step of assigning the intensity of pain based on said comparison, wherein a decrease in the amount(s) determined in said sample relative to the amounts determined in said reference is indicative for a decrease in pain intensity.

Thus, in a further preferred embodiment of said method, the method further comprises the step of comparing the amounts of IL-7, IL-18, MCP-1, MIP-lb and OPG determined in said sample from the patient to the amounts of IL-7, IL-18, MCP-1, MIP-lb and OPG determined in a reference and the step of assigning the intensity of pain based on said comparison, wherein a decrease in the amounts determined in said sample relative to the amounts determined in said reference is indicative for a decrease in pain intensity. In a further preferred embodiment of said method, the method further comprises the step of comparing the amount of a protein selected from the group consisting of IL-7, IL-18, MCP-1, MIP-lb and OPG determined in said sample from the patient to the amount of the respective protein, i.e. the protein selected from the group consisting of IL-7, IL-18, MCP-1, MIP-lb and OPG, determined in a reference and the step of assigning the intensity of pain based on said comparison, wherein a decrease in the amount determined in said sample relative to the amount determined in said reference is indicative for a decrease in pain intensity. Further, in another preferred embodiment of said method, the method further comprises the step of comparing the amount(s) of the at least one, preferably of the at least two, more preferably of the at least three, and most preferably of the at least four proteins selected from the group consisting of IL-7, IL-18, MCP-1, MIP-lb and OPG determined in said sample to the amount(s) of the respective proteins, i.e. the at least one, preferably of the at least two, more preferably of the at least three, and most preferably of the at least four proteins selected from the group consisting of IL- 7, IL-18, MCP-1, MIP-lb and OPG, determined in a reference and the step of assigning the intensity of pain based on said comparison, wherein a decrease in the amount(s) determined in said sample relative to the amount(s) determined in said reference is indicative for a decrease in pain intensity. As for the step of determining, said at least one protein is preferably MCP-1 or MIP-lb or OPG. Preferably, said at least two proteins are MCP-1 and MIP-lb, or MCP-1 and OPG, or MIP-lb and OPG. Preferably, said at least three proteins are MCP-1, MIP-lb and OPG. Preferably, said at least four proteins are MCP-1, MIP-lb, OPG and IL-7.

Thus, with respect to the above method relating to the determination of MIP-lb, such a method preferably comprises the step of comparing the amount of MIP-lb determined in said sample from the patient to the amount of MIP-lb determined in a reference and the step of assigning the intensity of pain based on said comparison, wherein a decrease in the amount determined in said sample relative to the amount determined in said reference is indicative for a decrease in pain intensity. Since the amount of at least one additional cytokine may in preferred embodiments be determined in addition to the determination of the amount of MIP-lb, the step of comparing and assigning also relates to the above outlined combinations of cytokines, i.e. MIP-lb and at least one additional cytokine selected from the group consisting of IL-7, IL-18, MCP-1 and OPG, preferably consisting of IL-18, MCP-1 and OPG.

Further, the step of comparing and the step of assigning also relates in preferred embodiments to the combinations stated above, i.e. MIP-lb and IL-7, or MIP-lb and IL-18, or MIP-lb and MCP-1, or MIP-lb and OPG; MIP-lb, IL-7 and IL-18, or MIP-lb, IL-7 and MCP-1, or MIP-lb, IL-7 and OPG; MIP-lb, IL-18 and MCP-1, or MIP-lb, IL-18 and OPG, or MIP-lb, MCP-1 and OPG; MIP-lb, IL-7, IL-18 and MCP-1, or MIP-lb, IL-7, IL-18 and OPG, or MIP-lb, IL-7, MCP-1 and OPG, or MIP-lb, IL-18, MCP-1 and OPG. As disclosed above, it can be particularly preferred to determine in a sample from said patient the amounts of MIP-lb, IL-7, IL-18, MCP-1 and OPG.

In a particularly preferred embodiment of the method as outlined above, the intensity of pain is determined after administration of at least one analgesic to said patient.

Said analgesic may be selected from the group of analgesics comprising nonsteroidal anti-inflammatory, COX-2 inhibitors and particularly opioids. Opioids may be selected from the group consisting of morphine, oxycodone, hydromorphone, propoxyphene, nicomorphine, dihydrocodeine, diamorphine, papaveretum, codeine, ethylmorphine, phenylpiperidine, methadone, dextropropoxyphene, buprenorphine, pentazocin, tilidine, tramadol, hydrocodone and pharmaceutically acceptable salts thereof. It can be preferred to determine the intensity of pain about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes or about 50 minutes after

administration of the at least one analgesic. It can also be preferred to determine the intensity of pain about 1 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours or about 12 hours (later time points e.g. for twice-a-day analgesics) after administration of the at least one analgesic. It can also be preferred to determine the intensity of pain in regular intervals, such as e.g. 30 minutes or 1 hour, in order to be able to monitor analgesia. The timing of the determination may of course depend on the characteristics of the analgesic chosen and its formulation; thus, if the analgesic is e.g. formulated as sustained release dosage form, the intensity of pain may be determined later and/or in longer intervals compared to a situation where an immediate release dosage form of an analgesic is administered. Preferably, said sample is provided outside the human or animal body and said step of determining is performed outside the human or animal body. Further, said step of comparing and said step of assigning is preferably performed outside the human or animal body. Thus, the method according to the present invention is preferably carried out in vitro, i.e. outside the human or animal body and not on the human or animal body.

In a preferred embodiment of the present invention, said sample is a body fluid sample, preferably a blood sample, a urine sample, a saliva sample or a liquor cerebrospinalis sample. Most preferably, said sample is a blood sample.

In a preferred embodiment of the present invention, the patient referred to in the method is a patient incapable of communicating. An exemplary patient for patients incapable of communicating is inter alia a child, an unconscious patient, a mentally disabled patient, a disabled patient, a palliative patient or an elderly patient. Preferably, said pain referred to in the methods of the invention is somatic pain.

If the method of the present invention is used for determining the intensity of somatic pain in a patient, said method may also be used to differentiate between somatic pain and non-somatic pain. Thus, should no somatic pain be detectable in a patient using a method of the present invention (i.e. no decrease in pain intensity can be detected although analgesic therapy is applied), and the patient still suffers from pain, said patient appears to suffer from non-somatic pain and corresponding treatment methods may be initiated. It should be noted in this respect that it appears that the pain experienced by a patient is based inter alia on her/his subjective judgment without a clear differentiation between somatic and non-somatic pain.

In another preferred embodiment of the present invention, said method is used to determine the efficacy of pain therapy. Thus, should the decrease in pain intensity determined according to the present invention not be pronounced, the applied pain therapy appears to be inefficient.

In another preferred embodiment of the present invention, said method is used to adjust pain therapy by selecting an appropriate analgesic and/or selecting an appropriate amount of an analgesic. The selection of an appropriate analgesic and/or of an appropriate amount of an analgesic clearly corresponds to a routine task for a person skilled in the art.

The present invention thus also refers to the use of a method according to the invention for determining the efficacy of pain therapy. The present invention also refers to the use of a method according to the invention for adjusting pain therapy, preferably by selecting an appropriate analgesic and/or selecting an appropriate amount of an analgesic. The step of determining the amount of a specific protein in the method according to the present invention may be carried out employing techniques such as multiplex fluorescent bead immunoassay, ELISA-assays, FACS-assays, Western-blot analysis and further immuno-based assays known to the skilled person.

In another embodiment, the present invention relates to a diagnostic kit for determining the intensity of pain in a patient, wherein said kit comprises detecting agents specific for IL-7, IL-18, MCP-1, MIP-lb and OPG. Preferably, said pain is somatic pain. In a further embodiment, said kit consists of detecting agents specific for MIP-lb, IL-7, IL-18, MCP-1 and OPG or detecting agents specific for MIP-lb, IL-18, MCP-1 and OPG.

In a preferred embodiment, the present invention refers to a diagnostic kit for determining the intensity of pain in a patient, wherein said kit comprises a detecting agent specific for a protein selected from the group consisting of IL-7, IL-18, MCP- 1, MIP-lb and OPG. Preferably, said pain is somatic pain.

In another embodiment, the present invention refers to a diagnostic kit for determining the intensity of pain in a patient, wherein said kit comprises detecting agents specific for at least one, preferably for at least two, more preferably for at least three, and most preferably for at least four proteins selected from the group consisting of IL-7, IL-18, MCP-1, MIP-lb and OPG. Preferably, said at least one protein is MCP-1 or MIP-lb or OPG. Preferably, said at least two proteins are MCP- 1 and MIP-lb or MCP-1 and OPG or MIP-lb and OPG. Preferably, said at least three proteins are MCP-1, MIP-lb and OPG. Preferably, said at least four proteins are MCP-1, MIP-lb, OPG and IL-7. Preferably, said pain is somatic pain.

Thus, in a preferred embodiment, the present invention refers to a diagnostic kit for determining the intensity of pain in a patient, wherein said kit comprises a detecting agent specific for MIP-lb. In other embodiments, said diagnostic kit may comprise or consist of detecting agents specific for MIP-lb and IL-7, or MIP-lb and IL-18, or MIP-lb and MCP-1, or MIP-lb and OPG. Said kit may also comprise or consist of detecting agents specific for MIP-lb, IL-7 and IL-18, or MIP-lb, IL-7 and MCP-1, or MIP-lb, IL-7 and OPG; or detecting agents specific for MIP-lb, IL-18 and MCP-1, or MIP-lb, IL-18 and OPG, or MIP-lb, MCP-1 and OPG. Alternatively, said kit may comprise or consist of detecting agents specific for MIP-lb, IL-7, IL-18 and MCP-1, or MIP-lb, IL-7, IL-18 and OPG, or MIP-lb, IL-7, MCP-1 and OPG, or MIP-lb, IL-18, MCP-1 and OPG.

Preferably, the detecting agent of said kit is an antibody and/or an aptamer specific for the respective protein selected from the group consisting of IL-7, IL-18, MCP-1, MIP-lb and OPG.

In another embodiment, the present invention relates to a pharmaceutical

composition for use in the treatment of pain, wherein said composition comprises active agents directed to IL-7, IL-18, MCP-1, MIP-lb and OPG. Preferably, said pain is somatic pain.

In a preferred embodiment, the present invention relates to a pharmaceutical composition for use in the treatment of pain, wherein said composition comprises an active agent directed to a protein selected from the group consisting of IL-7, IL-18, MCP-1, MIP-lb and OPG. Preferably, said pain is somatic pain.

In another embodiment, the present invention refers to a pharmaceutical composition for use in the treatment of pain, wherein said composition comprises an active agent / active agents directed to at least one, preferably to at least two, more preferably to at least three, and most preferably to at least four proteins selected from the group consisting of IL-7, IL-18, MCP-1, MIP-lb and OPG. Preferably, said at least one protein is MCP-1 or MIP-lb or OPG. Preferably, said at least two proteins are MCP- 1 and MIP-lb or MCP-1 and OPG or MIP-lb and OPG. Preferably, said at least three proteins are MCP-1, MIP-lb and OPG. Preferably, said at least four proteins are MCP-1, MIP-lb, OPG and IL-7. Preferably, said pain is somatic pain.

In a preferred embodiment, said active agent of said pharmaceutical composition is a neutralizing and/or depleting and/or inhibitory agent and preferably selected from the group comprising antibodies, aptamers, and the like, specific for the respective protein selected from the group consisting of IL-7, IL-18, MCP-1, MIP-lb and OPG.

The present invention is also concerned with the use of IL-7, IL-18, MCP-1, MIP-lb and OPG as biomarkers for pain intensity and/or as targets for pain therapy. It can be preferred that said pain intensity is a somatic pain intensity and that said pain therapy is a somatic pain therapy. The present invention is particularly concerned with the use of MIP-lb, optionally in combination with at least one cytokine selected from the group consisting of IL-7, IL-18, MCP-1 and OPG, particularly IL-18, MCP-1 and OPG, as biomarker(s) for pain intensity and/or for an analgesic response.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the concentrations of IL-7 in serum (y-axis of each box blot in pg/ml, with deviations) for sample A (left value for each box blot corresponding to sample taken prior to analgesic therapy) and sample B (right value of each box blot corresponding to sample taken after successful pain therapy [for A and B] and control sample [for C]). In figure A, the concentrations for the interim patient population are depicted; figure B shows the concentrations for the final patient population, whereas figure C shows the concentrations in the control group.

Figure 2 shows the concentrations of IL-18 in serum (y-axis of each box blot in pg/ml, with deviations) for sample A (left value for each box blot corresponding to sample taken prior to analgesic therapy) and sample B (right value of each box blot corresponding to sample taken after successful pain therapy [for A and B] and control sample [for C]). In figure A, the concentrations for the interim patient population are depicted; figure B shows the concentrations for the final patient population, whereas figure C shows the concentrations in the control group.

Figure 3 shows the concentrations of MCPl in serum (y-axis of each box blot in pg/ml, with deviations) for sample A (left value for each box blot corresponding to sample taken prior to analgesic therapy) and sample B (right value of each box blot corresponding to sample taken after successful pain therapy [for A and B] and control sample [for C]). In figure A, the concentrations for the interim patient population are depicted; figure B shows the concentrations for the final patient population, whereas figure C shows the concentrations in the control group. Figure 4 shows the concentrations of MlPlb in serum (y-axis of each box blot in pg/ml, with deviations) for sample A (left value for each box blot corresponding to sample taken prior to analgesic therapy) and sample B (right value of each box blot corresponding to sample taken after successful pain therapy [for A and B] and control sample [for C]). In figure A, the concentrations for the interim patient population are depicted; figure B shows the concentrations for the final patient population, whereas figure C shows the concentrations in the control group.

Figure 5 shows the concentrations of OPG in serum (y-axis of each box blot in pg/ml, with deviations) for sample A (left value for each box blot corresponding to sample taken prior to analgesic therapy) and sample B (right value of each box blot corresponding to sample taken after successful pain therapy [for A and B] and control sample [for C]). In figure A, the concentrations for the interim patient population are depicted; figure B shows the concentrations for the final patient population, whereas figure C shows the concentrations in the control group. DETAILED DESCRIPTION OF THE INVENTION

The present invention is inter alia based on the surprising finding that pain intensity in a patient correlates with the levels of specific biomarkers in the blood serum of said patient.

1. Definitions including a general description of the invention Before some of the embodiments of the present invention are described in more detail, the following definitions are introduced.

As used in the specification and the claims, the singular forms of "a" and "an" also include the corresponding plurals unless the context clearly dictates otherwise.

The term "about" in the context of the present invention denotes an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates a deviation from the indicated numerical value of ±10% and preferably ±5%.

It needs to be understood that the term "comprising" is not limiting. For the purposes of the present invention, the term "consisting of is considered to be a preferred embodiment of the term "comprising of. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also meant to encompass a group which preferably consists of these embodiments only.

The term "pain" as referred to herein is used in the general meaning in the field. Thus, pain may be described as a symptom experienced by an individual resulting from a perturbation in the body (e.g. a cut or a tumor) or from a different, not localizable origin (i.e. have psychogenic origin). Pain resulting from a perturbation in the body is referred to as "somatic" pain in the present invention. Accordingly, "non- somatic" pain as used herein describes pain, which is not based on a perturbation of the body. The term "intensity of pain" as used herein is meant to describe a certain degree of pain experienced by an individual. Pain experience may also be referred to as pain perception in the present invention. Pain intensities can differ among individuals but may generally be referred to as covering a range from "no pain at all" to "the worst pain I can imagine". Depending on the intensity of pain within this range, an appropriate pain therapy should be applied. As described in the Example section, a numerical rating scale ( RS) for pain is usually used in order to determine the intensity of pain (if the patient is able to communicate).

"Pain therapy" as used herein refers to the administration of a specific analgesic in a specific amount for a specific time. Thus, in patients suffering from chronic malignant pain, opioids such as morphine may be administered in amounts of from less than 1 mg per day to at least 1000 mg per day in a continuous way, e.g. via infusion or in the form of sustained release dosage forms over a period of several months to years. In patients suffering from acute pain (such as postoperative pain), different analgesics such as nonsteroidal anti-inflammatory drugs (NSAIDs) may be administered in amounts of from less than 1 mg per day to at least 1000 mg per day for a time period of several days.

Should the applied pain therapy be unsuccessful (i.e. should the patient still suffers from pain), a different analgesic and/or a different amount of the analgesic may inter alia be used. This is referred to in the present invention as "adjusting" the pain therapy, wherein "appropriate" refers to an analgesic and/or an amount thereof, which is typically applied in a comparable situation. The term "patient" as used herein refers to an individual suffering from pain

(optionally from concomitant diseases as well), preferably from pain to such a degree that therapy needs to be applied in order to abolish or at least reduce the pain experienced by the patient. Such a patient may e.g. be a cancer patient suffering from tumor pain.

The term "sample" as used herein refers to a sample obtained from the patient's body, which has been separated from the body of the patient. The sample is preferably liquid and may thus be referred to as "body fluid sample" including blood, saliva, urine and liquor cerebrospinalis. In this respect, it should be noted that e.g.

OPG and MCP1 have been detected in urine samples. Generally, proteins of the class of cytokines are detectable in the body fluid samples as listed above.

Preferably, the sample is a blood sample from said patient. Depending on the analysis method used for determining the amounts of biomarkers, said sample may be purified and/or concentrated prior to the step of determination.

Thus, if e.g. a blood plasma sample is used, blood may be obtained from the patient's vein by using a needle and collecting the blood in a suitable container. Prior to analysis, cooling steps, centrifugation steps and/or concentrating steps may then be carried out to provide the final blood plasma sample. Preferably, up to about 10 ml of blood are collected in a suitable container and then centrifuged in order to obtain blood serum (e.g. at about 3000xg for about 10 minutes) which may subsequently be frozen (optionally in aliquots) prior to the analysis of the biomarkers in said serum.

The term "amount" as used herein is meant to describe a normalized amount of an agent, such as a protein. The amount can be normalized against a specific volume of a sample resulting in a concentration of the agent to be analyzed. Alternatively, the amount of the agent to be analyzed can be normalized against an internal standard, such as a specific number of a different agent. For the present invention, the term "amount" is interchangeable with the term "concentration" and the term "level" and preferably refers to an amount normalized against a specific volume.

The term "IL-7" refers to the interleukin-7 protein, a cytokine. IL-7 is comprised of SEQ ID Nol (database sequences: UniProt: P13232; NCBI: AAC63047.1).

The term "IL-18" refers to the interleukin-18 protein, a cytokine. Alternative names are Iboctadekin, Interferon gamma-inducing factor, IFN-gamma-inducing factor and Interleukin-1 gamma. IL-18 is comprised of SEQ ID No2 (database sequences:

UniProt: Q14116; NCBI: CAG46798.1).

"MCP-1" („monocyte chemoattractant protein-1") is also a cytokine/chemokine. MCP-1 is comprised of SEQ ID No3 (database sequences: UniProt: Q6UZ82; NCBI: AAQ75526.1).

"MlP-lb" („monocyte chemoattractant protein-1 -beta") is a cytokine also referred to as C-C motif chemokine 4, G-26 T-lymphocyte-secreted protein, HC21,

Lymphocyte activation gene 1 protein, MIP-l-beta(l-69), PAT 744, Protein H400, SIS-gamma, Small-inducible cytokine A4 and T-cell activation protein 2. MIP-lb is comprised of SEQ ID No4 (database sequences: UniProt: P13236; NCBI:

NP_002975.1).

The protein "OPG" („osteoprotegerin"), a cytokine, may also be referred to as tumor necrosis factor receptor superfamily member 1 IB or Osteoclastogenesis inhibitory factor. OPG is comprised of SEQ ID No5 (database sequences: UniProt/NCBI:

000300).

All five proteins belong to the family of cytokines. Their general functions may be referred to as the control of survival, growth, differentiation and effector function of tissues and cells. It should be noted that both MCP-1 and MlPlb belong to the same cytokine subfamily, namely the family of cysteine-cysteine (C-C) chemokines. Thus, it may be preferred to generally use the family of cysteine-cysteine (C-C)

chemokines as biomarkers for pain intensity. It can be particularly preferred to use MCP-1 and MlPlb as members of said family if the method for determining the intensity of pain according to the present invention is based on the determination in a sample from a patient the amounts of at least two proteins.

The determination of the amount of the protein of interest can e.g. be achieved by contacting the sample from the patient with a detecting agent specific for said protein. In some embodiments, the amount of more than one protein selected from the group as mentioned above may be detected in parallel within one sample by applying detecting agents with corresponding specificities.

The amount of the protein of interest may be determined using specific techniques known to the skilled person, such as multiplex fluorescent bead immunoassay, ELISA-assay, immunofluoresence microscopy, flow cytometry or luminometry.

The term "reference" as used herein can refer to a sample previously gained from said patient (e.g. prior to the analgesic therapy or at an earlier stage using a different analgesic therapy) in order to be able to compare the amounts of the analyzed biomarkers over time. In a situation, where the intensity of pain is determined after administration of at least one analgesic to the patient, the reference most preferably corresponds to a sample gained from said patient before the administration of the analgesic therapy, preferably shortly (such as e.g. about 1 to about 30 minutes) before the analgesic therapy.

Thus, in a preferred embodiment, the method according to the invention may be used to monitor the pain intensity in a patient over time, thereby also monitoring e.g. the efficacy of pain therapy. One may also refer in this respect to monitoring the "analgesic response" in a patient (see also hypothetical example 2). However, the term "reference" can also refer to database entries based on previous results of a comparable population of pain patients. Thus, the database entries may have been gained by determining in one or more reference sample(s) from patients with known pain intensities the amounts of said proteins of interest. Said amounts may further be averaged such that the reference is a predetermined value

corresponding to a specific intensity of pain.

The step of "comparing" said two amounts results in the information whether the amounts determined in said sample are higher or lower relative to the amounts determined in said reference.

The "assignment" of pain intensity is then based on the finding of the present inventors that a decrease in the amounts determined in said sample correlates with a decrease in pain intensity, i.e. is indicative for a decrease in pain intensity. In this respect, the term "decrease" can be understood as referring to the amounts determined in the sample from the patient as being at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 10 fold, at least about 20 fold, at least about 30 fold, at least about 40 fold, at least about 50 fold, at least about 60 fold, at least about 70 fold, at least about 80 fold, at least about 90 fold, at least about 100 fold, at least about 500 fold, at least about 1000 fold or at least about 10000 fold lower than in the reference, correlating with respective lower pain intensities. The "diagnostic kit" according to the present invention comprises detecting agents specific for the biomarkers for pain intensity as described herein. If the diagnostic kit according to the invention comprises more than one detecting agent, said detecting agents are preferably each specific for the respective proteins selected from the group consisting of IL-7, IL-18, MCP-1, MIP-lb and OPG. The diagnostic kit may further comprise additional components or reagents that may be suitable for performing the methods according to the invention, such as e.g. buffers or controls. The components contained in the diagnostic kit may be comprised in one or more containers. The diagnostic kit according to the present invention may also comprise an instruction leaflet, which indicates how to use the diagnostic kit and its components.

A "detecting agent" is specific for a given target, if it binds said target with a higher affinity than any other compound in a sample (i.e. a non-target). Preferably, a detecting agent specific for a given target binds to said target only and does not bind at all to a non-target. In the context of the present invention, which is inter alia directed to the detection of proteins, it is clear to the skilled person that the specificity of a detecting agent is preferably achieved via the binding of said detecting agent to the specific structure of the respective protein, which is mainly determined by its amino acid sequence. Thus, a detecting agent specific for IL-7 is preferably directed to SEQ ID Nol, a detecting agent specific for IL-18 is preferably directed to SEQ ID No2, a detecting agent specific for MCP-1 is preferably directed to SEQ ID No3, a detecting agent specific for MTP-lb is preferably directed to SEQ ID No and a detecting agent specific for OPG is preferably directed to SEQ ID No5.

A suitable "detecting agent" according to the present invention may be an antibody and/or an aptamer specific for the respective protein selected from the group consisting of IL-7, IL-18, MCP-1, MIP-lb and OPG.

The term "aptamer" as used herein refers to a polynucleotide that has a specific binding affinity for a target compound or molecule of interest, e.g. a protein.

Aptamers may e.g. be RNA, single stranded DNA, modified RNA or modified DNA molecules. The preparation of aptamers is well known in the art and may involve, inter alia, the use of combinatorial RNA libraries to identify binding sites (reference may e.g. be made to Gold (1995), Ann. Rev. Biochem 64, 763-797). The term "antibody" is used herein as common in the field and preferably refers to a monoclonal or polyclonal antibody directed to a specific antigen. In some embodiments the detecting agent may also be selected from antibody variants or fragments such as e.g. single chain antibodies, diabodies, minibodies, single chain Fv fragments (sc(Fv)), sc(Fv)₂ antibodies, Fab fragments or a F(ab')₂ fragments.

In a preferred embodiment, a detecting agent as described herein may comprise a detectable label. Any suitable label which can be attached to the detecting agent may be used. In a preferred embodiment the detectable label is covalently or non- covalently attached to the detecting agent. Examples of labels that may be attached to the detecting agent include e.g. fluorescent dyes such as e.g. Cyanine dyes, e.g. Cyanine 3, Cyanine 5 or Cyanine 7, Alexa Fluor dyes, e.g. Alexa 594, Alexa 488 or Alexa 532, fluorescein family dyes, R-Phycoerythrin, Texas Red and rhodamine. Detecting agents may also be labeled with enzymes such as e.g. horseradish peroxidase, alkaline phosphatase or beta-lactamase, radioisotopes such as e.g. ³H,

14 32 33 35 125

C, P, P, S or I or metal such as e.g. gold. In another preferred embodiment the detecting agent may also be detected by a secondary detecting agent comprising a label as described above. Preferably a secondary detecting agent is capable of specifically binding to the above described detecting agent. In a particularly preferred embodiment a secondary detecting agent is an antibody.

"Active agent" as used herein is interchangeable with "pharmaceutically active agent" and means that a compound is potent of modulating a response in a human or animal being in vivo, in the present case a reduction of pain intensity. The active agent of the present invention is directed to a modulation of at least one of the biomarkers as described herein and may also be referred to as neutralizing and/or depleting and/or inhibitory agent.

The major goal of an active agent according to the present invention is to be seen in a decrease of the amount and/or the activity of at least one biomarker according to the present invention such that a reduction of pain intensity is achieved thereby. It can be especially preferred that said active agents are selected from the group consisting of antibodies and/or aptamers directed to the respective protein selected from the group consisting of IL-7, IL-18, MCP-1, MIP-lb and OPG. Said agents are directed to the proteins themselves. However, one may also apply active agents directed to the regulation of said proteins, i.e. agents downregulating the expression of said proteins. In this regard, e.g. siRNAs or the like may be used.

The term "pharmaceutically acceptable excipient" as used herein refers to

compounds commonly comprised in pharmaceutical compositions, which are known to the skilled person. Such compounds or excipients are exemplary listed below. In view of the definition "pharmaceutically active agent" as given above, a

pharmaceutically acceptable excipient may thus be defined as being

pharmaceutically inactive.

A pharmaceutical composition may be formulated for oral, buccal, nasal, rectal, topical or parenteral application. Parenteral application may include intravenous, intramuscular or subcutaneous administration. The pharmaceutical compositions of the invention may be made from the active agents of the invention alone or they may comprise pharmaceutically acceptable excipients. Pharmaceutical dosage forms may be solid or liquid dosage forms or may have an intermediate, e.g. gel-like character depending inter alia on the route of administration.

In general, the inventive dosage forms will comprise various pharmaceutically acceptable excipients which will be selected depending on which functionality is to be achieved for the dosage form.

A "pharmaceutically acceptable excipient" in the meaning of the present invention can be any substance used for the preparation of pharmaceutical dosage forms, including but not limited to coating materials, film-forming materials, fillers, disintegrating agents, release-modifying materials, carrier materials, diluents, binding agents and other adjuvants. The term carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being commingled with the compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.

As regards human patients, the active agents directed to the proteins of interest may be administered to a subject in an amount of less than 1 μΜ.

The invention is now illustrated with respect to specific examples. The examples are, however, not to be construed as limiting. Example 1 : Identification of biomarkers for pain intensity

In order to identify possible biomarkers, a study according to the following general setup was conducted. Palliative patients suffering from strong cancer pain (classification of > 5/10 on a numerical rating scale (NRS) for pain) were selected for the present study. Prior to analgesia, blood samples of the patients were taken (samples A). Prior to and following the administration of an opioid analgesic such as Vendal, Hydal, Dipidolon or morphine, the patients were asked to classify their pain intensity on a NRS. One hour after initiation of pain therapy and when reaching a reduction of the pain intensity by factor > 3 on the NRS, another set of blood samples was taken from the patients (samples B). Typically, such a reduction could be observed after a period of 1 to 2 hours. Patients which failed to display a reduction by factor > 3 within three hours after initiation of pain therapy were classified as non-responder. Samples A and B were then analyzed for the concentration of cytokines present in the blood serum.

In order to exclude that concentration-differences in samples A and B of the analyzed cytokines in the cancer patients of the study are due to intra-individual or circadian variations, samples were also taken from a population of healthy control individuals at time points 0, 0 + 1 h, 0 + 3 h and 0 + 24 h. Depicted in figures 1 to 5 in the "healthy individuals" are samples A at time point 0 and samples B at time point 0 + 3h, which corresponds to the latest possible time point for pain reduction by factor > 3 in patients.

Briefly, blood samples were obtained according to the following protocol: Up to 10 ml of blood was drawn and collected in a vacutainer containing a gel with intermediate density between blood cells and serum. Ideally blood was immediately processed by centrifugation at 3000xg for 10 min. Otherwise blood was stored at 4°C no longer than 1 ½ h prior to the centrifugation. Serum was then aliquoted into 500 μΐ and stored at -70°C. Collected sera were sent to a laboratory for the assessment of clinical parameters for determination of the cytokine concentrations. In the final blood samples prepared as described above, the concentrations of cytokines (in pg/ml) were determined by a multiplex fluorescent bead immunoassay or ELISA according to the following protocols:

Human cytokines IL-18, MIP-lb, MCP-1, and OPG were analyzed using a bead based Analyte Detection System for quantitative detection of human cytokines by Flow Cytometry (Human Flow Cytomix Simplex Kits, BMS). For quantitative detection of IL-18 and MCP-1, fluorescent beads coated with monoclonal antibodies were used, whereas OPG and MIP-la were analyzed with fluorescent beads coated with polyclonal antibodies. All secondary antibodies were biotin-conjugated. Each kit included a standard that was serially diluted and used to construct a standard curve to calibrate the assay and confirm assay linearity. Test procedure, measurement and calculation of results were performed according to the manufacturer's instructions. In general the system used conjugated beads with monoclonal or polyclonal antibodies specific for a target protein. For multiplex reactions the beads could be differentiated by their sizes and by their distinct spectral addresses. These antibody- coupled, color-coded beads were then incubated with the serum samples, washed, followed by addition of a biotinylated detection antibody, washed again, and finally incubated with streptavidin-phycoerythrin. Streptavidin-phycoerythrin is bound to the biotin conjugate and emits fluorescent signals.

A wide range of standards was used for each cytokine to enable quantification of the individual cytokines using a flow cytometer equipped with one laser (488 nm or 532 nm) capable of detecting and distinguishing fluorescence emissions at 575 nm and far red (700 nm).

For quantitative detection of human IL-7, an enzyme-linked immunosorbent assay (ELISA) was used. To this aim, a microwell plate coated with monoclonal antibody to human IL-7, biotin-conjugate (anti-IL-7 polyclonal antibody), sample diluent and streptavidin-URP, were used.

Briefly, a biotin-conjugated polyclonal anti-human IL-7 antibody is bound to human IL-7 captured by the first antibody. Following incubation, unbound biotin conjugated anti human IL-7 and streptavidin-URP was removed during a wash step, and substrate solution reactive with horse raddish peroxidase (HRP) was added to the wells. A coloured product was formed in proportion to the amount of soluble human IL-7 present in the sample. The reaction was terminated by addition of acid and absorbance and was measured at 450 nm. A standard curve was prepared from seven human IL-7 standard dilutions and human IL-7 sample concentration was determined. The assay was performed according to the manufacturer's instructions.

The concentrations obtained for each of the analyzed cytokines were then subjected to a statistical analysis over the populations. Thus, the concentration of each cytokine (e.g. IL-7) was averaged in samples A as well as in samples B (as depicted with error bars in figures 1 to 5, left value of each blot: sample A, right value of each blot: sample B) of the patient population (see figures 1 A and IB for IL-7 in patient groups) as well as the population of healthy individuals (see figure 1 C for IL-7 in healthy individuals). The differences in the concentrations obtained for samples A and B were then determined according to the Wilcoxon-method, wherein p-values indicate the significance of a difference. A low p-value indicates a high significance of the difference in the concentrations, whereas a high p-value indicates that there is no significant difference in the concentrations.

Briefly, the RS for pain consists of a numerical scale ranging from 0 to 10, wherein "10" indicates strongest pain imaginable and "0" indicates no pain at all.

The initial study was conducted with 20 patients suffering from cancer pain. Said first 20 patients are referred to as "interim patients" in the following. Subsequently, 18 further patients suffering from cancer pain were included in the study. The population consisting of the first 20 and the subsequent 18 patients (resulting in 38 patients in total) is referred to in the following as "final patient group" or "final patients". The control group of healthy individuals consisted of 20 individuals and is referred to as "healthy probands" in the following.

The following biomarkers were found to be significantly downregulated in samples B compared to samples A in patients and did not show a significant concentration difference in the control population: IL-7, IL-18, MCP-1, MIP-lb and OPG. The box-plots are depicted in figures 1 to 5 and the p-values are depicted in table 1 :

Table 1 : p-values indicating the significance of differences between the biomarker concentrations in samples A and B. * indicates a significant difference from sample A to sample B.

In summary, it was observed that decreasing levels of IL-7, IL18, MCP-1, MIP-lb and OPG in the blood correlate with an alleviation of pain / reduction of pain intensity in pain patients, i.e. with a decrease in the pain intensity in pain patients treated with an analgesic.

Hypothetical example 2: efficacy of pain therapy

Using the method according to the present invention, it will be possible to control the efficacy of an analgesic therapy in severely sick palliative patients suffering from strong pain, even if the patients are unable to communicate verbally or in writing with the medical professionals.

To this aim, the levels of the biomarkers according to the invention are closely monitored prior to and during an analgesic therapy. Kits according to the present invention may be used for determining said levels. Pain therapy will then be adjusted according to the levels as determined, which correlate with the pain intensity in the patients, such that a more efficient pain therapy is achieved.

Hypothetical example 3 : classification of pain Further, it will also be possible using a method according to the present invention to gain information on the type of pain in patients suffering from pain. Patients, who cannot be treated with strong analgesics successfully, could represent such a patient group suffering from non-somatic pain. In order to classify the pain, the levels of the biomarkers according to the invention are monitored during an analgesic therapy. Kits according to the present invention may be used for determining said levels.

If strong analgesics are unsuccessfully used and the method according to the present invention does not show a reduction of pain intensity in the patient, is will be likely that the patient suffers from non-somatic pain. In this case, different treatment regimes directed to the treatment of psychogenic pain may be chosen.

Preferred embodiments of the present invention relate to:

1. Method of determining the intensity of pain in a patient comprising the step of determining in a sample from said patient the amounts of IL-7, IL-18, MCP-1, MIP-lb and OPG. 2. Method according to 1, wherein said method further comprises the step of comparing the amounts of IL-7, IL-18, MCP-1, MIP-lb and OPG determined in said sample to the amounts of IL-7, IL-18, MCP-1, MIP-lb and OPG determined in a reference and the step of assigning the intensity of pain based on said comparison, wherein a decrease in the amounts determined in said sample relative to the amounts determined in said reference is indicative for a decrease in pain intensity.

3. Method according to 1 or 2, wherein said sample is provided outside the human or animal body and said step of determining and/or said step of comparing and said step of assigning is performed outside the human or animal body.

4. Method according to any of 1 to 3, wherein said sample is a sample selected from the group consisting of a blood sample, a urine sample, a saliva sample and a liquor cerebrospinalis sample, preferably a blood sample.

5. Method according to any of 1 to 4, wherein said patient is a patient incapable of communicating, preferably a child, an unconscious patient, a mentally disabled patient, a disabled patient, a palliative patient or an elderly patient.

6. Method according to any of 1 to 5, wherein said pain is somatic pain.

7. Method according to 6, wherein said method is used to differentiate between somatic pain and non-somatic pain.

8. Method according to any of 1 to 6, wherein said method is used to determine the efficacy of pain therapy.

9. Method according to any of 1 to 6, wherein said method is used to adjust pain therapy by selecting an appropriate analgesic and/or selecting an appropriate amount of an analgesic.

10. Diagnostic kit for determining the intensity of pain, preferably somatic pain, in a patient comprising detecting agents specific for IL-7, IL-18, MCP-1, MIP- lb and OPG. 11. Diagnostic kit according to 10, wherein said detecting agents are selected from antibodies and/or aptamers.

12. Pharmaceutical composition for use in the treatment of pain, preferably somatic pain, comprising active agents directed to IL-7, IL-18, MCP-1, MIP-lb and OPG.

13. Pharmaceutical composition according to 12, wherein said active agents are selected from antibodies and/or aptamers.

14. Use of IL-7, IL-18, MCP-1, MIP-lb and OPG as biomarkers for pain intensity and/or as targets for pain therapy.

Use according to 14, wherein said pain is somatic pain.

Claims

1. Method of determining the intensity of pain in a patient comprising the step of determining in a sample from said patient the amount of MIP-lb, wherein the intensity of pain is determined after administration of at least one analgesic to said patient.

2. Method according to claim 1, wherein said method further comprises the step of comparing the amount of MIP-lb determined in said sample to the amount of MIP-lb determined in a reference and the step of assigning the intensity of pain based on said comparison, wherein a decrease in the amount determined in said sample relative to the amount determined in said reference is indicative for a decrease in pain intensity.

3. Method according to claim 1 or 2, wherein said method comprises the step of determining the amount of at least one additional cytokine selected from the group consisting of IL-7, IL-18, MCP-1 and OPG.

4. Method according to claim 3, wherein said method further comprises the step of comparing the amount of said at least one cytokine selected from the group consisting of IL-7, IL-18, MCP-1 and OPG determined in said sample to the amount of at least one cytokine selected from the group consisting of IL-7, IL-18, MCP-1 and OPG determined in a reference and the step of assigning the intensity of pain based on said comparison, wherein a decrease in the amount(s) determined in said sample relative to the amount(s) determined in said reference is indicative for a decrease in pain intensity.

5. Method according to any one of the preceding claims, wherein said sample is provided outside the human or animal body and/or said step of

determining, said step of comparing and said step of assigning is performed outside the human or animal body.

6. Method according to any one of the preceding claims, wherein said sample is a sample selected from the group consisting of a blood sample, a urine sample, a saliva sample and a liquor cerebrospinal! s sample, preferably a blood sample.

7. Method according to any one of the preceding claims, wherein said patient is a patient incapable of communicating, preferably a child, an unconscious patient, a mentally disabled patient, a disabled patient, a palliative patient or an elderly patient.

8. Method according to any one of the preceding claims, wherein said pain is somatic pain.

9. Diagnostic kit for determining the intensity of pain, preferably somatic pain, in a patient comprising a detecting agent specific for MIP-lb.

10. Diagnostic kit according to claim 9 comprising at least one additional detecting agent selected from the group of detecting agents specific for IL-7, IL-18, MCP-1 and OPG.

11. Diagnostic kit according to claim 9 or 10, wherein said detecting agents are selected from antibodies and/or aptamers.

12. Use of a method according to claim 8 or of a diagnostic kit according to any one of claims 9 to 11 for differentiating between somatic pain and non-somatic pain.

13. Use of a method according to any one of claims 1 to 8 or of a diagnostic kit according to any one of claims 9 to 11 for determining the efficacy of pain therapy.

14. Use of a method according to any one of claims 1 to 8 or of a diagnostic kit according to any one of claims 9 to 11 for adjusting pain therapy by selecting an appropriate analgesic and/or selecting an appropriate amount of an analgesic.

15. Use of MIP-lb as biomarker for pain intensity, preferably for somatic pain intensity, and/or for an analgesic response.

16. Use according to claim 15, wherein MIP-lb is used in combination with at least one cytokine selected from the group consisting of IL-7, IL-18, MCP-1 and OPG.