WO2012162215A1 - Promotion of fracture healing using complement inhibitors - Google Patents

Abstract

Methods for promoting healing of a fracture in the presence of other traumatic injuries are disclosed. The methods involve administration of a complement inhibitor to inhibit systemic C5a receptor signaling resulting from the trauma. Pharmaceutical compositions comprising a complement inhibitor and at least one other agent for treating the fracture are also disclosed.

Description

PROMOTION OF FRACTURE HEALING USING COMPLEMENT INHIBITORS

Pursuant to 35 U.S.C. §202(c), it is acknowledged that the United States government may have certain rights in the invention described herein, which was made in part with funds from the National Institutes of Health under Grant Nos. GM-62134 and AI-068730.

FIELD OF THE INVENTION

This invention relates to the field of trauma and healing. Methods for stimulating fracture healing, particularly in the presence of other systemic trauma, are provided. The methods involve administration of a complement inhibitor to inhibit C5a receptor signaling to promote fracture healing.

BACKGROUND OF THE INVENTION

Various publications, including patents, published applications, technical articles and scholarly articles are cited throughout the specification. Each of these cited publications is incorporated by reference herein, in its entirety. Full citations for publications not cited fully within the specification are set forth at the end of the specification.

A severe trauma such as a blunt chest trauma is considered a potent initiator of a systemic inflammatory response, being characterized by a strong systemic activation of the complement and coagulation cascades, and the release of pro-inflammatory cytokines and prostanoids.^1"3 It has been reported that fracture healing is delayed and more non-unions occur in severely injured patients.⁴'⁵ In confirming the clinical evidence, it was recently demonstrated experimentally in rats that a blunt chest trauma, which induced a post-traumatic systemic inflammation, considerably impaired fracture healing.⁶

One trigger of post-traumatic systemic inflammation is the complement system.¹'⁷'⁸ The complement cascade, consisting of over 30 proteins, is an important component of innate immunity and can be activated by four pathways, the classical, the lectin, the alternative and the extrinsic pathways. In all cases, the activation pathways lead to the production of the anaphylatoxin C5a. ⁹'¹⁰ In trauma victims, systemic C5a was reported to be increased within minutes after trauma, and was strongly correlated with injury severity.¹¹'¹² C5a induces, for example, the migration of phagocytes, degranulation of mast cells, systemic cytokine release, respiratory burst induction and the regulation of apoptosis in inflammatory cells, thus acting at the very first line of defense in the post-traumatic systemic inflammatory response.¹ The excessive activation of complement, however, can also cause harmful effects, for example immunoparalysis and organ dysfunction.¹²'¹³ Due to its strong pro-inflammatory character, C5a is regarded as being a hazardous molecule if the complement cascade is over- activated.¹'¹³'¹⁴

A recent study reported that the cellular receptor for C5a, C5aR, was locally expressed in a distinct spatial and temporal pattern in the fracture callus of rats not only by inflammatory cells but also by osteoblasts, chondroblasts and osteoclasts in zones of intramembranous and enchondral bone formation.¹⁵ In vitro studies revealed that in osteoblasts C5aR activation could induce cell migration¹⁵ and cytokine release,¹⁶ and could also modulate osteoclast formation¹⁷ This suggests that the anaphylatoxin C5a could influence bone healing by acting on osteoblasts and osteoclasts, as well as possibly influencing the fine local inflammatory balance of the bone healing process.

In view of the discussion above, the relationship between systemic complement activation and local complement activation on the fracture healing process is unclear.

Complement activation appears to be involved in fracture healing, yet complement activation from systemic trauma has been observed to delay fracture healing. Advances in the art are needed to provide a practical link between the complement system and its modulation for the purpose of promoting the bone healing process.

SUMMARY OF THE INVENTION

One aspect of the invention features a method for promoting healing of a fracture in an individual who has suffered a fracture and another trauma. The method comprises first identifying an individual who has suffered a fracture and another trauma, and then administering to the individual a therapeutically effective amount of a complement inhibitor to the individual, wherein the complement inhibitor reduces or prevents systemic C5a receptor signaling (including C5L2 signaling) resulting from the trauma, thereby promoting healing of the bone fracture. In one embodiment, the individual is a human. In other embodiments, the individual is a non-human animal.

The complement inhibitor can be any complement inhibitor that inhibits complement activation in a manner resulting in a decrease in production or activity of C5a. Thus, the complement inhibitor can be selected from one or more of a C5a inhibitor, a C5aR inhibitor, a C3 inhibitor, a C3aR inhibitor, a factor D inhibitor, a factor B inhibitor, a C4 inhibitor, a Clq inhibitor, or any combination thereof. In certain embodiments, the complement inhibitor is a C5a inhibitor or a C5aR inhibitor. Nonlimiting examples of C5a inhibitors or C5aR inhibitors include acetyl-Phe-[Orn-Pro-D-cyclohexylalanine-Trp-Arg] (PMX-53), PMX-53 analogs, neutrazumab, TNX-558, eculizumab, pexelizumab or ARC1905, or any combination thereof. In certain embodiments, the complement inhibitor is a C3 inhibitor. In particular embodiments, the C3 inhibitor is Compstatin (SEQ ID NO: l), a Compstatin analog, a Compstatin peptidomimetic, a Compstatin derivative, or any combinations thereof. Specific examples of Compstatin analogs include peptides comprising SEQ ID NO.:2, SEQ ID NO:3 or SEQ ID NO:4. In another embodiment, the complement inhibitor is a C4 inhibitor.

The complement inhibitor can be administered systemically, by different selected routes. Combinations of complement inhibitors can be utilized. In one embodiment, the complement inhibitor is administered together or concurrently with, or sequentially before or after, at least one other treatment for the fracture.

Another aspect of the invention features a pharmaceutical composition for promoting healing of a fracture in an individual who has suffered a fracture and another trauma. The pharmaceutical composition comprises one or more complement inhibitors and at least one other agent for treating the fracture, in a pharmaceutically acceptable medium. In various embodiments, the complement inhibitor comprises one or more of a C5a inhibitor, a C5aR inhibitor, a C3 inhibitor, a C3aR inhibitor, a factor D inhibitor, a factor B inhibitor, a C4 inhibitor, a Clq inhibitor, or any combination thereof. In certain embodiments, the complement inhibitor is a C5a inhibitor or a C5aR inhibitor. Nonlimiting examples of C5a inhibitors or C5aR inhibitors include acetyl-Phe-[Orn-Pro-D-cyclohexylalanine-Trp-Arg] (PMX-53), PMX-53 analogs, neutrazumab, TNX-558, eculizumab, pexelizumab or

ARC 1905, or any combination thereof. In certain embodiments, the complement inhibitor is a C3 inhibitor. In particular embodiments, the C3 inhibitor is Compstatin (SEQ ID NO: l), a Compstatin analog, a Compstatin peptidomimetic, a Compstatin derivative, or any combinations thereof. Specific examples of Compstatin analogs include peptides comprising SEQ ID NO.:2, SEQ ID NO:3 or SEQ ID NO:4. In another embodiment, the complement inhibitor is a C4 inhibitor. The pharmaceutical composition typically is formulated for systemic administration, using a variety of routes of administration. Other features and advantages of the invention will be understood by reference to the drawings, detailed description and examples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Flexural rigidity (EI) of the fracture callus of rats without (Control) or with treatment with a C5aR-antagonist immediately and 12 h after the blunt chest trauma. * = p < 0.05.

Figure 2. Relative amounts of osseous tissue (TOT), cartilage tissue (Cg) and fibrous tissue (FT) within the callus of rats without (Ctrl) or with treatment with a C5aR-antagonist.

Figure 3. Absolute amounts of osseous tissue (TOT), cartilage tissue (Cg) and fibrous tissue (FT) within the callus of rats without (Ctrl) or with treatment with a C5aR- antagonist.

Figure 4. Bone stiffness (N x mm⁴) as measured by the three-point bending test in normal versus C3- or C5-deficient strains of mice after 21 days of healing from fracture.

Figure 5. Micro-computed tomography (μΟΤ) analysis in normal versus C3- or C5- deficient strains of mice after 21 days of healing from fracture. Top panel: callus volume (mm³); bottom panel: moment of inertia of ostomy gap (mm⁴).

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Various terms relating to the methods and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein.

Definitions

Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization are those well known and commonly employed in the art.

Standard techniques are used for nucleic acid and peptide synthesis. The techniques and procedures are generally performed according to conventional methods in the art and various general references (e.g., Ausubel et al, 2011, Current Protocols in Molecular Biology, John Wiley & Sons, NY), which are provided throughout this document.

The nomenclature used herein and the laboratory procedures used in analytical chemistry and organic syntheses described below are those well known and commonly employed in the art. Standard techniques or modifications thereof, are used for chemical syntheses and chemical analyses.

As used herein, each of the following terms has the meaning associated with it in this section.

The singular form of a word includes the plural, and vice versa, unless the context clearly dictates otherwise. Thus, the references "a", "an", and "the" are generally inclusive of the plurals of the respective terms. For example, reference to "a compound" or "a method" includes a plurality of such "compounds" or "methods." Similarly, the words "comprise", "comprises", and "comprising" are to be interpreted inclusively rather than exclusively. Likewise the terms "include", "including" and "or" should all be construed to be inclusive, unless such a construction is clearly prohibited from the context.

The terms "comprising" or "including" are intended to include embodiments encompassed by the terms "consisting essentially of and "consisting of. Similarly, the term "consisting essentially of is intended to include embodiments encompassed by the term "consisting of.

Dosages expressed herein are in units per kilogram of body weight (e.g., μg/kg or mg/kg) unless expressed otherwise.

Ranges are used herein in shorthand, to avoid having to list and describe each and every value within the range. Any appropriate value within the range is intended to be included in the present invention, as is the lower terminus and upper terminus, independent of each other.

The term "about" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, in some embodiments ±5%, in some embodiments ±1%, and in some embodiments ±0.1% from the specified value, as such variations are appropriate to practice the disclosed methods or to make and used the disclosed compounds, compositions or articles of manufacture.

The term "antibody" refers to an immunoglobulin molecule that is able to bind specifically to a particular epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. The antibodies useful in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies ("intrabodies"), Fv, Fab and F(ab)2, as well as single chain antibodies (scFv), camelid antibodies and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

A "complement inhibitor" is a molecule that prevents or reduces activation and/or propagation of the complement cascade that results in the formation of C3a or signaling through the C3a receptor, or C5a or signaling through the C5a receptor. A complement inhibitor can operate on one or more of the complement pathways, i.e., classical, alternative or lectin pathway. A "C3 inhibitor" is a molecule or substance that prevents or reduces the cleavage of C3 into C3a and C3b. A "C5a inhibitor" is a molecule or substance that prevents or reduces the activity of C5a. A "C5aR inhibitor" is a molecule or substance that prevents or reduces the binding of C5a to the C5a receptor. A "C3aR inhibitor" is a molecule or substance that prevents or reduces binding of C3a to the C3a receptor. A "factor D inhibitor" is a molecule or substance that prevents or reduces the activity of Factor D. A "factor B inhibitor" is a molecule or substance that prevents or reduces the activity of factor B. A "C4 inhibitor" is a molecule or substance that prevents or reduces the cleavage of C4 into C4b and C4a. A "Clq inhibitor" is a molecule or substance that prevents or reduces Clq binding to antibody-antigen complexes, virions, infected cells, or other molecules to which Clq binds to initiate complement activation. Any of the complement inhibitors described herein may comprise antibodies or antibody fragments, as would be understood by the person of skill in the art.

A "fracture" is a break or discontinuation in bone or cartilage. Fractures are classified according to their character and location as, for example, a transverse fracture of the tibia. Clinically most fractures may be classified by the AO-classification system.

A "subject", "individual" or "patient" refers to an animal of any species. In various embodiments, the animal is a mammal. In one embodiment, the mammal is a human. In another embodiment, the mammal is a non-human animal.

"Systemic complement activation" or "systemic C5a formation" refers to an activation of the complement system in the circulating blood measured by an increase of complement components in the blood and decrease in complement hemolytic activity. In contrast "local complement activation" occurring at the site of injury (fracture) does not lead to a systemic activation of the complement cascade in the circulating blood, as reflected by unchanged complement activation products and unaltered complement hemolytic activity in the peripheral blood.

"Trauma" or "traumatic injury" as used herein refers to a severe physical injury, including for example open wounds, dismemberment, blunt injury, crush injury and major burns.

"Treating" refers to any indicia of success in the treatment or amelioration of the disease or condition, or promotion of the healing process. Treating can include, for example, reducing or alleviating the severity of one or more symptoms of the disease or condition, or it can include reducing the frequency with which symptoms of a disease, defect, disorder, or adverse condition, and the like, are experienced by a patient, or it can include speeding, promoting or otherwise improving the healing process following injury to cells, tissues or organs. "Preventing" refers to the partial or complete prevention of the disease or condition in an individual or in a population, or in a part of the body, such as a cell, tissue or bodily fluid (e.g., blood). "Promoting," such as promoting the healing process, refers to improving or accelerating the rate at which healing of a wounded cell, tissue or organ occurs. The term "prevention" does not establish a requirement for complete prevention of a disease or condition in the entirety of the treated population of individuals or cells, tissues or fluids of individuals. Nor does the term "promotion" establish a requirement that the healing of an entire population of injured cells, tissues or organs will be accelerated or improved.

A "prophylactic" treatment is a treatment administered to a subject (or sample) that does not exhibit signs of a disease or condition, or in advance of signs of the condition that are expected to manifest, such as symptoms of inflammation or stress after a trauma. This term may be used interchangeably with the term "preventing," again with the understanding that such prophylactic treatment or "prevention" does not establish a requirement for complete prevention of a disease in the entirety of the treated population of individuals or tissues, cells or bodily fluids.

As used herein, a "therapeutically effective amount" or simply an "effective amount" is the amount of a composition sufficient to provide a beneficial effect to the individual to whom the composition is administered, or who is otherwise treated using a method involving the composition. Description

It was previously known from clinical and experimental observations that trauma, such as a blunt chest trauma, considerably impairs fracture healing; however, the underlying mechanisms were unknown, so a pharmacological intervention was not available. The present invention springs in part from the inventors' determination that systemically activated complement significantly contributes to the impairment of bone healing observed after severe trauma, and their demonstration that pharmacological interference with systemic posttraumatic C5a signaling substantially alleviated the impairment of fracture healing in the presence of severe trauma. In view of the fact that C5a appears to play an important role in the healing process at the fracture site, the fact that a systemic reduction in C5a actually promoted fracture healing was surprising. Thus, C5a and C5a signaling has been determined to be a pharmacological target prevent delayed bone healing in patients with severe trauma.

One aspect of the invention provides a method for promoting healing of fractures accompanied by one or more other traumatic injuries that result in systemic complement activation. The method comprises identifying or determining that an individual has suffered a fracture and another trauma, and administering a complement inhibitor to the individual.

A typical candidate for the method of the invention is an individual who has experienced a fracture in the course of a more complex traumatic injury. For instance, someone injured in a moving vehicle collision may suffer one or more fractures, as well as a blunt trauma to the head or chest, or a crushing trauma to the pelvis. In such instances, the trauma experienced by the individual is expected to stimulate a complement activation response, resulting in systemic formation and activity of C5a; therefore, the individual is a suitable candidate for practice of the method.

For complement inhibition, any inhibitor of C5a formation or activity may be used in the method of the invention. Inhibition of C5a formation or activity may be accomplished in a variety of ways. For instance, C5a activity may be inhibited directly by preventing or significantly reducing the binding of C5a to its receptor, C5aR. A number of C5aR inhibitors are known in the art. Acetyl-Phe-[Orn-Pro-D-cyclohexylalanine-Trp-Arg]

(AcF[OPdChaWR]; PMX-53; Peptech) is a small cyclic hexapeptide that is a C5aR antagonist and is exemplified herein. Analogs of PMX-53 (e.g., PMX-201 and PMX-205) that also function as C5aR antagonists are also available (see for instance Proctor et al., 2006, Adv Exp Med Biol. 586:329-45 and U.S. Pat. Pub. No. 20060217530). Neutrazumab (G2 Therapies) binds to C5aR, thereby inhibiting binding of C5a to C5aR. Neutrazumab (G2 Therapies) binds to extracellular loops of C5aR and thereby inhibits the binding of C5a to C5aR. TNX-558 (Tanox) is an antibody that neutralized C5a by binding to C5a.

C5a activity may also be inhibited by reducing or preventing the formation of C5a. Thus, inhibition of any step in the complement cascade that contributes to the downstream formation of C5a is expected to be effective in practicing the invention. Formation of C5a may be inhibited directly by inhibiting the cleavage of C5 by C5-convertase. Eculizumab (Alexion Pharmaceuticals, Cheshire, CT) is an anti-C5 antibody that binds to C5 and prevents its cleavage into C5a and C5b. Pexelizumab, a scFv fragment of Eculizumab, has the same activity. Similarly, ARC 1905 (Archemix), an anti-C5 aptamer, binds to and inhibits cleavage of C5, inhibiting the generation of C5b and C5a.

In another embodiment, formation of C5a is reduced or prevented through the use of a C3 inhibitor. Preferably, the C3 inhibitor is Compstatin or a Compstatin analog, derivative, aptamer or peptidomimetic. Compstatin is a small molecular weight cyclic peptide having the sequence Ile-Cys-Val-Val-Gln-Asp-Trp-Gly-His-His-Arg-Cys-Thr (SEQ ID NO. 1). Examples of Compstatin analogs, derivatives and peptidomimetics are described in the art. See, for instance, U.S. Pat. No. 6,319,897, U.S. Patent No. 7,888,323, WO/1999/013899, WO/2004/026328 and WO/2010/127336.

An exemplary Compstatin analog comprises a peptide having a sequence: Xaal - Cys

- Val - Xaa2 - Gin - Asp - Trp - Gly - Xaa3 - His - Arg - Cys - Xaa4 (SEQ ID NO. 2); wherein:

Xaal is He, Val, Leu, Ac -He, Ac -Val, Ac-Leu or a dipeptide comprising Gly-Ile; Xaa2 is Trp or a peptidic or non-peptidic analog of Trp;

Xaa3 is His, Ala, Phe or Trp;

Xaa4 is L-Thr, D-Thr, He, Val, Gly, or a tripeptide comprising Thr-Ala-Asn, wherein a carboxy terminal -OH of any of the L-Thr, D-Thr, He, Val, Gly or Asn optionally is replaced by -NH₂; and the two Cys residues are joined by a disulfide bond. Xaal may be acetylated, for instance, Ac -He. Xaa2 may be a Trp analog comprising a substituted or unsubstituted aromatic ring component. Non-limiting examples include 2-naphthylalanine, 1 -naphthylalanine, 2-indanylglycine carboxylic acid, dihydrotryptophan or

benzoylphenylalanine.

Another exemplary Compstatin analog comprises a peptide having a sequence: Xaal

- Cys - Val - Xaa2 - Gin - Asp - Xaa3 - Gly - Xaa4 - His - Arg - Cys - Xaa5 (SEQ ID NO. 3); wherein:

Xaal is He, Val, Leu, Ac -He, Ac -Val, Ac-Leu or a dipeptide comprising Gly-Ile;

Xaa2 is Trp or an analog of Trp, wherein the analog of Trp has increased hydrophobic character as compared with Trp, with the proviso that, if Xaa3 is Trp, Xaa2 is the analog of Trp;

Xaa3 is Trp or an analog of Trp comprising a chemical modification to its indole ring wherein the chemical modification increases the hydrogen bond potential of the indole ring;

Xaa4 is His, Ala, Phe or Trp;

Xaa5 is L-Thr, D-Thr, He, Val, Gly, a dipeptide comprising Thr-Asn or Thr-Ala, or a tripeptide comprising Thr-Ala-Asn, wherein a carboxy terminal -OH of any of the L-Thr, D- Thr, He, Val, Gly or Asn optionally is replaced by -NH₂; and the two Cys residues are joined by a disulfide bond. The analog of Trp of Xaa2 may be a halogenated trpytophan, such as 5- fluoro-l-tryptophan or 6-fluoro-l-tryptophan. The Trp analog at Xaa2 may comprise a lower alkoxy or lower alkyl substituent at the 5 position, e.g., 5-methoxytryptophan or 5- methyltryptophan. In other embodiments, the Trp analog at Xaa 2 comprises a lower alkyl or a lower alkenoyl substituent at the 1 position, with exemplary embodiments comprising 1 - methyltryptophan or 1 -formyltryptophan. In other embodiments, the analog of Trp of Xaa3 is a halogenated tryptophan such as 5-fluoro-l-tryptophan or 6-fluoro-l-tryptophan.

An exemplary Compstatin analog of this type is Ac-I[CVW(Me)QDWGAHRCT]I- NH₂ (SEQ ID NO:4), which can be synthesized as described by Katragadda M, et ah, 2006, J Med Chem. 49: 4616-4622.

Another set of exemplary Compstatin analogs features Compstatin or any of the foregoing analogs, in which Gly at position 8 is modified to constrain the backbone conformation at that location. In one embodiment, the backbone is constrained by replacing the Gly at position 8 (Gly8) with Not-methyl Gly.

Other C3 inhibitors include vaccinia virus complement control protein (VCP) and antibodies that specifically bind C3 and prevent its cleavage.

In other embodiments, formation of C5a is reduced or prevented through the use of an inhibitor of complement activation prior to C3 cleavage, e.g., in the classical or lectin pathways of complement activation. Non-limiting examples of such inhibitors include, but are not limited to: (1) factor D inhibitors such as diisopropyl fluorophosphates and TNX-234 (Tanox), (2) factor B inhibitors such as the anti-B antibody TA106 (Taligen Therapeutics), (3) C4 inhibitors (e.g., anti-C4 antibodies) and (4) Clq inhibitors (e.g., anti-Clq antibodies). Likewise, inhibitors of signaling via the C3a receptor are also contemplated as being useful in the present invention.

Antibodies useful in the present invention, such as antibodies that specifically bind to either C4, C3 or C5 and prevent cleavage, or antibodies that specifically bind to factor D, factor B, Clq, or the C3a or C5a receptor, can be made by the skilled artisan using methods known in the art. See, for instance, Harlow, et al. (1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor, NY), Tuszynski et al. (1988, Blood, 72: 109-115), U.S. patent publication 2003/0224490, Queen et al. (U.S. Patent No. 6, 180,370), Wright et al., (1992, Critical Rev. in Immunol. 12(3,4): 125-168), Gu et al. (1997, Thrombosis and Hematocyst 77(4):755-759) and Burton et al., (1994, Adv. Immunol. 57: 191-280). Anti-C3 and anti-C5 antibodies are also commercially available.

The complement inhibitor can be administered immediately upon identifying the individual as a target candidate, i.e., the individual having experienced a fracture and another trauma that is expected to stimulate systemic complement activation. Alternatively, complement inhibitors can be administered as a prophylactic measure, if the nature and extent of an individual's injuries are not fully determined. Since traumatic injury typically occurs outside the setting of a health care facility, the complement inhibitor may be administered "in the field", for instance, at or near the location where the injury occurred or during transport of the patient to a health care facility such as a hospital, clinic, or physician's office.

Accordingly, the complement inhibitor can be administered any time from immediately following the injury, to within minutes, or an hour, or several hours, or within 24 hours following occurrence of the injury, and typically until a point at which the systemic complement activation would be expected to subside to a point that it no longer interferes with the local balance of inflammation and healing at the fracture point.

During that period, a single dose or multiple doses of complement inhibitor can be administered, as would be understood by the skilled practitioner. For example, a sufficient dose (or multiple doses) of complement inhibitor can be administered to reduce complement activation in the individual to within e.g., 1, 2, or 5 times the average level in individuals who have not suffered trauma.

The skilled artisan will appreciate that numerous biomarkers of complement activation can be measured for the purpose of determining when to initiate or when to cease administration of complement inhibitor. The complement inhibitors can be administered singly or in combination with one another. They may also be administered as part of a treatment regimen to promote healing of a fracture. For example, complement inhibitors can be administered within the first hours after fracture has occurred or before, during or after operative (e.g., external fixator, K-wires, screws, intramedullary nail, plate osteosynthesis) or conventional fracture treatment (cast, splint, brace) once or several times within several days up to one week.

The invention encompasses the use of pharmaceutical compositions comprising a complement inhibitor to practice the methods of the invention. Such a pharmaceutical composition may consist of the active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The active ingredient may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single-or multi-does unit.

As used herein, the term "pharmaceutically-acceptable carrier" means a chemical composition with which a complement inhibitor may be combined and which, following the combination, can be used to administer the complement inhibitor to a mammal.

As used herein, the term "physiologically acceptable" ester or salt means an ester or salt form of the active ingredient that is compatible with any other ingredients of the pharmaceutical composition, which is not deleterious to the subject to which the composition is to be administered.

The pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day. In one embodiment, the invention envisions administration of a dose that results in a concentration of a complement inhibitor between 1 μΜ and 10 μΜ in an individual identified as an appropriate candidate for treatment, i.e., having one or more fractures and a concomitant trauma causing a systemic inflammatory response. While the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of patient and type of disease state being treated, the age of the patient and the route of administration. Preferably, the dosage of the compound will vary from about 1 mg to about 10 g per kilogram of body weight of the patient. More preferably, the dosage will vary from about 10 mg to about 1 g per kilogram of body weight of the patient.

In a particular embodiment, fixed dose formulations containing sufficient complement inhibitor to significantly inhibit complement activation in individuals of different size or maturity; e.g., a child or adult human, following a single administration (which may take the form of an IV bolus or infusion or, in the case of orally bioavailable agents, oral

administration). For example, such formulations may be designed to reduce systemic complement activation by between 50% and 99%, e.g., by at least 50%, 60%, 70%, 80% or 90%, relative to levels present prior to administration or relative to levels that would have been expected in the individual under the circumstances, in the absence of the complement inhibitor.

A single complement inhibitor may be administered, or two or more different complement inhibitors may be administered, in the practice of the method of the invention. In one embodiment of the invention, the method comprises administration of only a complement inhibitor. In other embodiments, other biologically active agents are administered in addition to the complement inhibitor in the method of the invention. Non- limiting examples of other biologically active agents useful in the invention include other modulators; e.g., bisphosphonates (such as aledronate), and cathepsin-K inhibitors, modulators of the coagulation cascade (such as factor XIII, Xa), or growth factors (e.g., bone morphogenetic proteins such as BMP-2 and -7, and growth differentiation factor (GDF) -5).

Pharmaceutical compositions that are useful in the methods of the invention may be administered systemically in oral solid formulations, parenteral, ophthalmic, suppository, aerosol, topical/transdermal or other similar formulations. Such pharmaceutical compositions may contain pharmaceutically-acceptable carriers and other ingredients known to enhance and facilitate drug administration. Other formulations, such as nanoparticles, liposomes, resealed erythrocytes, and immunologically based systems may also be used to administer a complement inhibitor according to the methods of the invention.

As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, intravenous, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral

administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable or infusible aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3 -butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di- glycerides. Other useful parentally-administrable formulations include those comprising the active ingredient in microcrystalline form, in a liposomal preparation, in microbubbles for ultrasound-released delivery or as a component of a biodegradable polymer systems.

Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt. As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surface active agents including replacement pulmonary surfactants; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents;

preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents;

emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" that may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Genaro, ed., 1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, which is incorporated herein by reference.

In certain embodiments, the pharmaceutical composition may be a liquid formulation provided in a vial, a prefilled syringe, and the like. Such fixed dose formulations can be assembled into an article of manufacture containing a fixed dose of complement inhibitor in a convenient form for rapid administration to an individual. For instance, the formulation may be prepared for adding directly to an IV fluid solution.

The pharmaceutical compositions comprising complement inhibitors and/or other active agents or additional ingredients, can be conveniently packaged together in kits. Such kits comprise at least the complement inhibitor and instructions for its use in treating fractures accompanied by one or more other traumatic injuries. Such kits may also comprise the complement inhibitor and another treatment agent, along with instructions for their use. The kits may also comprise one or more of the diluents, excipients, carriers and other ingredients referred to above.

One embodiment features a kit containing at least one fixed dose formulation comprising a complement inhibitor. Also provided are kits containing multiple fixed dose formulations of a complement inhibitor, with at least two of the fixed dose formulations containing different amounts of the complement inhibitor. The different amounts can be selected to achieve a desired amount of complement inhibition depending on the size and/or maturity of the patient being treated; e.g., infants, children, and adults.

The following examples are provided to describe the invention in greater detail. They are intended to illustrate, not to limit, the invention. Example 1

Experiments were performed to show that the impairment of fracture healing by a severe trauma results at least in part from systemically activated complement, and to demonstrate that systemic pharmacological inhibition of C5a signaling through C5aR ameliorated the deleterious effect of a blunt chest trauma on bone healing in a rat model. The C5aR-antagonist was applied after the thoracic trauma to prevent the immediate C5a- dependent systemic inflammation. The fracture healing outcome was investigated after 35 days.

Materials and Methods:

Animals and experimental treatments:

The animal experiment was performed according to international regulations for the care and use of laboratory animals, and approved by the local ethical committee

(Regierungsprasidium Tubingen, Germany). Sixteen male Wistar rats (weight 400-450 g) received a blunt chest trauma combined with a femur osteotomy that was stabilized with an external fixator. Then the animals received either a C5aR-antagonist (n=8) or a control peptide (control group, n=8).

Surgery was performed as described previously.⁶'¹⁸ Briefly, a standardized osteotomy gap of 1 mm was created at the mid-shaft of the right femur and fixated with a custom-made external fixator. The offset of the fixator block was 6 mm, resulting in an axial stiffness of 1 19 N/mm.⁶ Immediately after surgery the rats received an additional blunt chest trauma under general anesthesia using a blast wave generator as previously described in detail.¹'¹⁹ This model allows a bilateral, isolated lung contusion by the application of a standardized single blast wave centered on the middle of the thorax and induces a reproducible transient systemic inflammation.¹'⁶ An analgesic (20 mg/kg, Tramal^®, Gruenenthal GmbH, Aachen, Germany) was administered subcutaneously during the operation and was diluted in the drinking water (25 mg/1) for the first 3 days following surgery. Each animal was individually housed, given unrestricted access to food and monitored daily for infection and mobility.

Immediately after the blunt chest trauma, one group received a C5aR-antagonist ([Ac- F[OPdChaWR]; PMX-53) at a dosage of 1 mg/kg intravenously into the penis vein.²⁰'²¹ The injection was repeated 12 h after the trauma to prevent the C5a-dependent systemic inflammation, which was detectable during the first 12-24 hours after the blunt chest trauma in rats. Control animals received a peptide ([Ac-F[OPdChaAdR]), which does not have antagonistic activity and thus does develop any biological effect at the same concentration and at the same time pomts.²²Biomechanical testing:

After 35 days the rats were sacrificed and the operated as well as the contralateral intact femora were explanted. Biomechanical testing was performed using a non-destructive, three-point bending test, as described previously. Briefly, after removing the fixators, the distal end of each bone was potted in a cylinder using polymethylmethacrylate (Technovit^® 3040, Heraeus Kulzer GmbH, Wertheim, Germany) and fixed in a hinge joint whereas the proximal end of the femur rested on the bending support. A quasistatic load was applied in a three-point bending mode with a materials testing machine (1454, Zwick GmbH, Ulm, Germany) using a 500 N load cell (System-Technik GmbH, Germany) and the flexural rigidity (EI) was calculated from the slope of the force deflection curve. The absolute values of the operated femora were related to the contralateral values of the un-operated femora to eliminate individual differences.

Micro-computed tomography :

The femora were scanned using a μΟΤ scanning device (skyscan 1172, Kontich, Belgium), operating at a peak voltage of 50 kV and 200 μΑ at a resolution of 15 μιη. The former osteotomy gap was segmented and the total tissue volume and the bone volume fraction (BV/TV) were calculated by global thresholding to distinguish between mineralized and non-mineralized tissue.²³ The maximum moment of inertia was calculated based on the tissue area on the transversal slices in the fracture gap. The apparent modulus of elasticity was calculated as the flexural rigidity divided by the maximum moment of inertia.²⁴

According to the standard clinical evaluation of x-rays the number of bridged cortices per callus were evaluated in two planes at right angles to one another by using an CT analyzing software (Data viewer, Skyscan, Kontich, Belgium). The distal pin hole served as orientation for the exact positioning of the specimens. At least three bridged cortices per callus were considered as a "healed fracture". Two observers evaluated the cortical bridging independently.

Histomorphometry :

After fixating the femora in buffered 4% formaldehyde they were dehydrated with ethanol (40-100%) and embedded in methyl methacrylate (Merck KGaA, Darmstadt, Germany). 70 μιη slices were cut and stained with Paragon (Paragon C&C; New York, NY, USA). In the now filled osteotomy gap the newly formed tissue was evaluated by using a light microscope (Leica DMI6000B, Heerbrugg, Switzerland) at a 5 -fold magnification. The relative amount of bone, cartilage and fibrous tissue was assessed by circumscribing the corresponding areas with image analysis software (Leica MMAF 1.4.0 Imaging System, Leica, Heerbrugg, Switzerland powered by MetaMorph®).

Statistical analysis:

Results are presented as mean and standard deviation. For statistical analysis, the software PASW Statistics 18.0 (SPSS Inc., Chicago, USA) was used. Differences between groups were calculated using a Mann- Whitney U test. The level of significance was p < 0.05.

Results:

Biomechanical testing:

The treatment of the animals with the C5aR-antagonist after blunt chest trauma significantly increased the flexural rigidity of the callus by about 98% compared to the control group, which received the control peptide (Figure 1).

Micro-computed tomography:

The application of the C5aR-antagonist led to a tendency for higher total callus volume, maximum moment of inertia and apparent modulus of elasticity. None of the parameters showed statistical significance compared to the control group (Table 1), likely due to the small sample size. The results nevertheless indicate a somewhat larger and qualitatively superior callus in the rats treated with the C5aR-antagonist.²⁴ Furthermore, the fracture callus of rats that received the C5aR-antagonist did show significantly more bridged cortices compared to the control group (Table 2).

Table 1 : μ-computed tomography analysis of the calli of rats without (Control)

or with treatment with a C5aR-antagonist after blunt chest trauma

Measure Control C5aR-Antagonist

Total callus volume (mm^J) 14.46 ± 3.41 15.42 ± 3.77

BV/TV (%) 83.81 ± 11.75 81.25 ± 11.47

Maximum moment of inertia (mm⁴) 30.40 ± 13.40 37.31 ± 17.42

Apparent modulus of elasticity (MPa) 4097.28 ± 2814.70 5602.53 ± 3321

Table 2: Number of bridged cortices of the calli evaluated by μ-computed tomography in two planes of rats without (Control) or with treatment with a C5aR-antagonist.

Histomorphometry:

The tissue distribution within the fracture callus did not show statistical significance between groups even though in tendency the treatment with the C5aR-atagonist led to less amount of cartilage compared to the control group (Figure 2). The absolute values confirmed the results of the μΟΤ analysis showing slightly more newly formed bone in the C5aR- antagonist treated group (Figure 3).

Example 2:

Experiments were performed to compare several parameters of fracture healing between complement-deficient mice and corresponding control animals.

Materials and Methods:

Animal model and experimental procedure:

157 male mice, aged 8-12 weeks, were divided into 4 experimental groups. Group 1 and 3 comprised complement deficient animals (Group 1 : n=38 C5 deficient animals; strain number: B10.D2-HC¹ H2^d H2-T18 nSnJ) (Group 3: n=39 C3-/- animals; strain number: B6;129S4-C3^tmlCr7J) and group 2 and 4 consisted of the corresponding control animals (Group 2: n=40 C57/B110SnJ; Group 4: n=40 C57/B16J). Animals were delivered by The Jackson Laboratories Bar Harbor, Maine, USA.

All animals received analgetics in the drinking water from 2 days preoperatively to 3 days postoperatively (12.55 mg/500 ml tramalhydrochloride, Tramal^®, Gruenenthal, Aachen, Germany). After subcutaneous injection of atropine sulfate (50 μg/kg, Atropin^®, Braun, Melsungen, Germany), the mice were anesthetized with 2 % isoflurane (Forene^®, Abbott, Wiesbaden, Germany). Antibiotic prophylaxis with Clindamycin was performed for 3 days postoperatively (45 mg/kg, Sobelin 600^®, Pfizer Pharma, Karlsruhe, Germany).

The surgical procedure was conducted after skin incision and blunt, careful preparation to the right femur. After stabilization of the femur with 4 pins the osteotomy was carried out using a gigli saw wire (0,45mm) between pin 2 and 3 (RI Systems, AO

Development Institute, Davos, Switzerland). Wound closure was achieved with suture in layers using absorbable thread for the muscle (Vicryl®, Johnson & Johnson, Norderstedt, Germany) and non-absorbable thread for the skin (Resolon®, Resorba, Nurnberg, Germany) After a healing period of 1, 3, 7 and 21 days animals were sacrificed by blood withdrawal of the lower vena cava after induction of general isoflurane anesthesia as described above.

Postoperative radiographs were taken directly after the surgical procedure and after sacrificing the animals.

The protocol was in accordance with the principles of the Guide for the Care and Use of Laboratory Animals, and was approved by the local animal care and use committee (Regierungsprasidium Tubingen, No. 965).

Biomechanical testing:

As a measure of mechanical stability, the flexural rigidity of the healed femora was evaluated using a non-destructive three point bending test. The contralateral femora served as controls.

The proximal femora were embedded in aluminium cylinder using SelfCem (Heraeus Kulzer, Hanau, Germany). Then the embedded femora were inserted into a material testing machine (Mod. Z010, Zwick GmbH & Co., Ulm, Germany). The bending load (F) was applied on top of the callus and was recorded continuously versus sample deflection (d). At a maximum speed of 2 mm/min the maximum load was 1,5N. The first two tests conditioned each sample and the last test was used for the measurement.

During the tests, the bone was moistened periodically with a 0.9 % NaCl solution. From the linear region of the load/deflection curve the flexural rigidity EI was calculated a²b²

according to EI = k (in Nmm²)

Histological analysis:

After embedding the femora in 4% formaldehyde and dehydratation with ethanol, the bones were embedded in methyl methacrylate (Merck KGaA, Darmstadt, Germany).

Histological slices were harvested from longitudinal cuts through the center of the bone. The 70 μπι-thick sections from undecalcified bones were surface-stained (5μιη) with Paragon (toluidine blue and fuchsin; both Waldeck GmbH & Co KG, Munster, Germany). The histological slices were examined under a light microscope (Leica DMI6000B) at a fivefold magnification. The amount of bone, cartilage and fibrous tissue was assessed by

circumscribing the corresponding areas with an image analysis software (Leica MMAF 1.4.0 Imaging System, Leica, Heerbrugg, Switzerland powered by MetaMorphl).

Micro computed tomography (μΟΤ): Besides postoperative native radiological analysis a CT-analysis of the fractured femora was conducted. For bone characterization μΟΤ-αηαΙνβϊβ from the lumbar column as well as from the intact femora was performed.

For these purposes a μΟΤ scanning device (Skyscan 1172, Kontich, Belgium) was used with a resolution of 15μιη (voltage 50kV and 200mA).

For the fracture healing experiments two regions of interest (ROI) were defined. One consisting of the periosteal callus together with the fracture gap and the fracture gap itself. For bone characterization regions of interest were the distal femur and the mid-diaphyseal shaft of the femur.

Using a CT-analysis software (Data viewer, Skyscan, Kontich, Belgium) the callus was segmented and the undesirable parts of the callus were discarded. In all ROI's the total tissue volume and the bone volume fraction (BV/TV) was calculated. The maximum moment of inertia was calculated based on the tissue area on the transversal slices in the fracture gap. The apparent modulus of elasticity was calculated as the flexural rigidity divided by the maximum moment of inertia. For all ROI's, bone mineral density as well as structural parameters such as cortical thickness and trabecular number were calculated automatically.

Statistical analysis:

Data were expressed as median ± standard deviation (SD). Statistical analysis was performed using a t-test (IBM SPSS Statistics 19.0, SPSS Inc., IBM, Armonk, New York, USA). Results with p≤0.05 were considered significant.

Bone characterization:

The differentiation potential of mouse derived osteoblast- like cells was measured. To isolate osteoblast like cells the long bones were minced and digested with 300 U/ml collagenase type IV (Sigma, Germany) in alpha medium (F025, Biochrom, Germany) for 2h. The bone chips were washed with PBS twice and once with alpha medium. The chips were plated in six- well plates and cultivated with alpha medium +15%FBS. Cells grown from the bone chips were cultivated in alpha-medium (F025, Biochrom, Germany) supplemented with 15% fetal bovine serum (FBS, Biochrom, Germany), 4 mM L-glutamine, 100 U/ml penicilline, 0.1 mg/ml streptomycine and 0.25 mg/ml amphothericin B (Fungizone®, Gibco, Germany). At subconfluence, cells were sub-cultured by treatment with 0.05 % trypsin/ 0.02 % EDTA (Biochrom, Germany).

Cells of passage 3 to 6 were used to investigate osteogenic differentiation. Osteoblasts were seeded with 10.000 cells/cm² into 24-well culture plates and cultivated in differentiation medium consisting of osteoblast medium supplemented with 10 nM disodium β- glycerophosphate and 0.2 mM ascorbate -2 -phosphate. After 3 weeks matrix mineralization was analyzed by von Kossa staining. Alkaline phosphatase activity was proven by a commercially available staining kit (Sigma, Germany). Semi-quantitative PCR was used to analyze osteogenic differentiation on gene expression level. RNA was isolated on day 0 and day 21 using the RNeasy Mini Kit (Qiagen, Germany). 1 μg RNA was transcribed into cDNA using the Omniscript RT Kit (Qiagen, Germany). For amplification specific primer pairs (Table 1, all synthesized by Thermo Fisher, Germany) and HotStarTaq DNA polymerase (Qiagen, Germany) were used with a 35 cycle PCR programme (Tl Thermocycler, Biometra, Germany). The amplification products were separated on a 2% agarose gel (Life

Technologies, Germany) and visualized by ethidium bromide staining.

Formation and resorption activity of osteoclasts in vitro was measured. The bone marrow was flushed out of the long bones with alpha medium (F0925, Biochrom, Germany) supplemented with 10 % fetal bovine serum (FBS, Gibco, Germany), 4 mM L-glutamine (PAA, Germany), 100 U/ml penicillin, 0.1 mg/ml streptomycin (Biochrom, Germany) and 0.25 mg/ml amphotericin B (Fungizone®, Gibco, Germany) (osteoclast medium). Bone marrow cells were seeded with 350.000 cells/cm² in culture flasks in osteoclast medium supplemented with 10 ng/ml rh M-CSF (Chemicon, Germany) for stimulation and cultivated at 37 °C, 5 % C02 and saturated humidity for three days. Stimulated non-adherent cells were seeded with 5 x 10⁵ cells/cm² in osteoclast medium supplemented with 25 ng/ml rh M-CSF and 50 ng/ml murine RANKL (R&D Systems, USA) in 96-well Plates and plates with a synthetic calcium phosphate coating respectively. After 7 days of cell cultivation tartrate resistant acid phosphatase (TRAP) staining was performed using a commercially available kit (Acid Phosphatase Leukocyte (TRAP) Kit, Sigma, Germany) to assess osteoclast formation. TRAP -positive cells with at least three nuclei were counted as osteoclasts.

Resorption activity of osteoclasts was assessed by resorption assay (BD BioCoat™ Osteologic™ Bone Cell Culture System plates, Becton Dickinson GmbH, Germany). After 7 days cells were removed with 6% sodium hypochloride. Von Kossa staining was used to visualize the remaining calcium phosphate coating. White and grey areas represent the surface resorbed by osteoclasts, hence lacking calcium. Resorption area was quantified by using the image processing software MetaMorph AF, version 1.4.0 (Leica, Germany). Results:

Fracture healing experiments were performed with C3-/- and C5-/- mice. The femurs were osteotomized and stabilized by an external fixator. After a healing period of 21 days, the healed and contralateral femors were explanted and analyzed biomechanically (Fig. 4), by micro-computed tomography (μΟΤ) (Fig. 5) and by histological methods (not shown).

Biomechanical analysis (three-point-bending test) revealed that the bending stiffness was significantly decreased in C5-/- mice compared to the corresponding wildtype mice indicating that the callus had a decreased mechanical competence. In C3-/- mice the bending stiffness was not significantly decreased in comparison to their corresponding wild type mice.

Micro-computed tomography analysis revealed that the total callus volume (including bone, cartilage and soft tissue) in the fracture gap was significantly decreased in C5-/- mice. The maximum moment of inertia (I_max) was determined based on the tissue area on the transversal slices in the fracture gap. This geometrical parameter is an important determinant of bending stiffness. It was also significantly decreased in C5-/- mice.

These results indicate that fracture healing is disturbed in congenitally C5-deficient mice.

References;

1. Flierl MA, Perl M, Rittirsch D, et al. 2008. The role of C5a in the innate immune response after experimental blunt chest trauma. Shock 29:25-31.

2. Gebhard F, Pfetsch H, Steinbach G, et al. 2000. Is interleukin 6 an early marker of injury severity following major trauma in humans? Arch Surg 135:291-295.

3. Strecker W, Gebhard F, Rager J, et al. 2002. Interleukin-6 (IL-6) - An early marker of chest trauma. European Journal of Trauma 28:75-84.

4. Bhandari M, Tornetta P, 3rd, Sprague S, et al. 2003. Predictors of reoperation following operative management of fractures of the tibial shaft. J Orthop Trauma 17:353- 361.

5. Karladani AH, Granhed H, Karrholm J, Styf J. 2001. The influence of fracture etiology and type on fracture healing: a review of 104 consecutive tibial shaft fractures. Arch Orthop Trauma Surg 121:325-328.

6. Recknagel S, Bindl R, Kurz J, et al. 2010. Experimental blunt chest trauma impairs fracture healing in rats. J Orthop Res. 2010 Dec 23. [Epub ahead of print] PMID: 21184503. 7. Donnelly TJ, Meade P, Jagels M, et al. 1994. Cytokine, complement, and endotoxin profiles associated with the development of the adult respiratory distress syndrome after severe injury. Crit Care Med 22:768-776.

8. Zilow G, Joka T, Obertacke U, et al. 1992. Generation of anaphylatoxin C3a in plasma and bronchoalveolar lavage fluid in trauma patients at risk for the adult respiratory distress syndrome. Crit Care Med 20:468-473.

9. Ehrnthaller C, Ignatius A, Gebhard F, Huber-Lang M. 2010. New Insights of an Old Defense System: Structure, Function, and Clinical Relevance of the Complement System. Mol Med. 2010 Oct 29. [Epub ahead of print] PMID: 21046060

10. Ricklin D, Hajishengallis G, Yang K, Lambris JD. 2010. Complement: a key system for immune surveillance and homeostasis. Nat Immunol 11:785-797.

11. Albers S, Burk AM, Rittirsch D, et al. 2006. Impairment of the Complement Function After Multiple Trauma in Humans. Shock 26 (Suppl. 1): 14.

12. Hecke F, Schmidt U, Kola A, et al. 1997. Circulating complement proteins in multiple trauma patients-correlation with injury severity, development of sepsis, and outcome. Crit Care Med 25:2015-2024.

13. Ganter MT, Brohi K, Cohen MJ, et al. 2007. Role of the alternative pathway in the early complement activation following major trauma. Shock 28:29-34.

14. Gerard C. 2003. Complement C5a in the sepsis syndrome— too much of a good thing? N Engl J Med 348: 167-169.

15. Ignatius A, Ehrnthaller C, Brenner RE, et al. 2010. The anaphylatoxin receptor C5aR is present during fracture healing in rats and mediates osteoblast migration in vitro. J Trauma, epublication March 2011.

16. Pobanz JM, Reinhardt RA, Koka S, Sanderson SD. 2000. C5a modulation of interleukin- 1 beta-induced interleukin-6 production by human osteoblast-like cells. J Periodontal Res 35 : 137- 145.

17. Tu Z, Bu H, Dennis JE, Lin F. 2010. Efficient osteoclast differentiation requires local complement activation. Blood 116:4456-4463.

18. Claes L, Blakytny R, Gockelmann M, et al. 2009. Early dynamization by reduced fixation stiffness does not improve fracture healing in a rat femoral osteotomy model. J Orthop Res 27:22-27. 19. Knoferl MW, Liener UC, Seitz DH, et al. 2003. Cardiopulmonary, histological, and inflammatory alterations after lung contusion in a novel mouse model of blunt chest trauma. Shock 19:519-525.

20. Crane JW, Buller KM. 2007. Systemic blockade of complement C5a receptors reduces lipopolysacharride-induced responses in the paraventricular nucleus and the central amygdala. Neurosci Lett 424: 10-15.

21. Huber-Lang MS, Riedeman NC, Sarma JV, et al. 2002. Protection of innate immunity by C5aR antagonist in septic mice. FASEB J 16: 1567-1574.

22. Langer HF, Chung KJ, Orlova W, et al. 2010. Complement-mediated inhibition of neovascularization reveals a point of convergence between innate immunity and angiogenesis. Blood 116:4395-4403.

23. Morgan EF, Mason ZD, Chien KB, et al. 2009. Micro-computed tomography assessment of fracture healing: relationships among callus structure, composition, and mechanical function. Bone 44:335-344.

24. Claes L, Blakytny R, Besse J, et al. 2011. Late dynamization by reduced fixation stiffness enhances fracture healing in a rat femoral osteotomy model. J Orthop Trauma 25: 169-174.

The present invention is not limited to the embodiments described and exemplified above, but is capable of variation and modification within the scope of the appended claims.

Claims

What is Claimed:

1. A method for promoting healing of a fracture in an individual who has suffered a fracture and another trauma, the method comprising:

a) identifying an individual who has suffered a fracture and another trauma; and b) administering to the individual a therapeutically effective amount of a complement inhibitor to the individual, wherein the complement inhibitor reduces or prevents systemic C5a receptor signaling resulting from the trauma, thereby promoting healing of thefracture.

2. The method of claim 1, wherein the complement inhibitor comprises one or more of a C5a inhibitor, a C5aR inhibitor, a C3 inhibitor, a C3aR inhibitor, a factor D inhibitor, a factor B inhibitor, a C4 inhibitor, a Clq inhibitor, or any combination thereof.

3. The method of claim 2, wherein the complement inhibitor is a C5a inhibitor or a C5aR inhibitor.

4. The method of claim 3, wherein the C5a inhibitor or C5aR inhibitor is acetyl - Phe-[Orn-Pro-D-cyclohexylalanine-Trp-Arg] (PMX-53), PMX-53 analogs, neutrazumab, TNX-558, eculizumab, pexelizumab or ARC1905, or any combination thereof.

5. The method of claim 2, wherein the complement inhibitor is a C3 inhibitor.

6. The method of claim 5, wherein the C3 inhibitor is Compstatin, a Compstatin analog, a Compstatin peptidomimetic, a Compstatin derivative, or any combinations thereof.

7. The method of claim 6, wherein the C3 inhibitor comprises SEQ ID NO: 1, SEQ ID NO.:2, SEQ ID NO:3 or SEQ ID NO:4.

8. The method of claim 2, wherein the complement inhibitor is a C4 inhibitor.

9. The method of claim 1 wherein the individual is human.

10. The method of claim 1, wherein the complement inhibitor is administered systemically.

11. The method of claim 1, wherein the complement inhibitor is administered together or concurrently with, or sequentially before or after, at least one other treatment for the fracture.

12. A pharmaceutical composition for promoting healing of a bone fracture in an individual who has suffered a bone fracture and another trauma, the pharmaceutical composition comprising one or more complement inhibitors and at least one other agent for treating the fracture, in a pharmaceutically acceptable medium.

13. The composition of claim 12, wherein the complement inhibitor comprises one or more of a C5a inhibitor, a C5aR inhibitor, a C3 inhibitor, a C3aR inhibitor, a factor D inhibitor, a factor B inhibitor, a C4 inhibitor, a Clq inhibitor, or any combination thereof.

14. The composition of claim 13, wherein the complement inhibitor is a C5a inhibitor or a C5aR inhibitor.

15. The composition of claim 14, wherein the C5a inhibitor or C5aR inhibitor is acetyl-Phe-[Orn-Pro-D-cyclohexylalanine-Trp-Arg] (PMX-53), PMX-53 analogs, neutrazumab, TNX-558, eculizumab, pexelizumab or ARC1905, or any combination thereof.

16. The composition of claim 13, wherein the complement inhibitor is a C3 inhibitor.

17. The composition of claim 16, wherein the C3 inhibitor is Compstatin, a Compstatin analog, a Compstatin peptidomimetic, a Compstatin derivative, or any combinations thereof.

18. The composition of claim 17, wherein the C3 inhibitor comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

19. The composition of claim 13, wherein the complement inhibitor is a C4 inhibitor.

20. The composition of claim 12, formulated for systemic administration.