The complement system in a subject can be activated through three distinct enzymatic cascades, referred to as the “classical pathway”, “Lectin/MBL”, and “alternative” pathway” (CP, MBL, and AP respectively). The classical pathway is responsible for aiding in host defense against antigens to prevent infection of cells. The lectin pathway is a variation of the classical pathway. The alternative pathway is responsible for 80-95% of total complement activity in cases where trigger of complement activation is the classical pathway (“AP amplification loop”). The alternative pathway by itself is activated in a number of disease indications where complement components have been found in elevated state.
There are three “alternative pathway specific proteins”; Factors B, D, and P, which play a major role in the a) initiation and propagation of the alternative pathway and b) classical pathway propagation via the alternative pathway amplification loop. Proteins C3 and C3b, the key players in complement system, are common to all classical and alternative complement pathways.
The classical pathway (CP) is initiated by antigen-antibody complex. The CP progression involves proteins such as C1Q, C1r/C1s, C4, and C2. The CP C3 convertase consists of C3bC4b2a. This complex can cleave the C3 into C3b and C3a. This C3b is derived from classical pathway convertase and is usually required for opsonization of various pathogens and bacteria. Inhibition of this C3b is undesirable. C3b coated cells are removed via complement receptors present on various cells.
Both complement pathways independently produce C3a, C3b, C5a, C5b, C5b-9, and sC5b-9 as complement activation byproducts.
During classical pathway triggered activation of the alternative pathway, Classical pathway C3 convertase also cleaves C3 into C3b which can work independent of the alternative pathway with full amplification of the classical pathway in 1% normal human serum in the presence of Ca2+/Mg2+ ions. Classical pathway C5 convertase can cleave C5 to generate C5a and C5b. The C5b molecule then inserts into the lipid bilayer of the cell to initiate the formation of C5b-9 or sC5b-9.
In alternative pathway activation, C3b produced by the complement system can bind properdin and Factor B to form the complex “PC3bB”. Factor D then cleaves Factor B, within the complex, into Bb and Ba. This cleavage results in the release of Ba from the complex and the formation of the AP convertase PC3bBb. PC3bBb cleaves C3 into C3a and C3b, thereby perpetuating the amplification loop of the alternative pathway for the benefit of the alternative pathway. PC3bBb can then cleave C5 to make C5b and C5a. The C5b molecule then inserts into a lipid bilayer of a cell and forms the nucleus for MAC deposition.
Classical pathway can also initiate the propagation of the alternative pathway known as amplification loop of the classical pathway. Within the amplification loop, C3b binds properdin and Factor B to form the complex “PC3bB”. Factor D then cleaves Factor B, within the complex, into Bb and Ba. This cleavage results in the release of Ba from the complex and the formation of the AP convertase PC3bBb. PC3bBb cleaves C3 into C3a and C3b, thereby perpetuating the amplification loop.
As noted above, C3b is therefore both a component and a byproduct of the complement system irrespective of the type of complement pathway activation. During the amplification of the AP, as the PC3bBb (AP C3 Convertase) generates increasing amounts of C3b, an amplification loop is established so that activation of the alternative pathway can continue. Furthermore, the classical pathway can also generate C3b, which can bind factor B and thereby engage the alternative pathway, even though the trigger is CP mediated. This allows more C3b to deposit on a target, which leads to enhanced amplification of AP activation.
Addition of newly formed C3b to the existing AP C3 convertase PC3bBb generates the AP C5 convertase. Addition of newly formed C3b to the existing CP C3 convertase generates CP C5 convertase. Both C5 convertases have the ability to cleave C5 to produce C5b and C5a. The terminal complex produced as a result of complement activation is known as the MAC complex (also known as C5b-9 or sC5b-9), which is responsible for lysis of cells in a subject. Both C3a and C5a are potent anaphylatoxins that are responsible for activating platelets, neutrophils, and monocytes. As a result, inflammatory molecules such as elastase, TNF-α, IL-1, VEGF, and peroxides are released. Formation of C5b-9/sC5b-9 is responsible for tissue damage and tissue injury/tissue damage seen in “other diseases”.
Classical complement pathway activation provides a valuable first-line defense against potential pathogens and can generate C3a/C3b, C5a/C5b, and C5b-9/sC5b-9. Therefore, exacerbation of the classical pathway can produce large amounts of complement byproducts. As described elsewhere, both C3a and C5a are potent anaphylatoxins, C3b mediates opsonization, and C5b is responsible for wanted killing of the pathogens. Here, both C3a and C5a would generate beneficial responses and are produced to kill the invaders. This pathway is required for host defense and therefore must not be inhibited.
Alternative pathway activation in Mg++ ions without the calcium ions guarantees only the AP activation. In disease state, this pathway is activated independent of the classical pathway. This pathway is not required for host defense and therefore can be inhibited in its entirety.
It is known that the progression of arthritis or arthritic condition in a subject is mediated by activation of the alternative pathway (Price, Experimental Arthritis: Targeting the Complement Alternative Pathway, Nature Reviews Rheumatology 6, 4 (January 2010)). An “arthritic condition,” in this context can include, but is not limited to, rheumatoid arthritis, osteoarthritis, juvenile arthritis, psoriatic arthritis, gouty arthritis, septic arthritis, Lupus, gout, and ankylosing spondylitis.
Rheumatoid arthritis is a chronic inflammatory disorder with an unknown origin that affects the joints on both sides equally in a subject. Osteoarthritis is caused by normal “wear and tear” on the joint. Juvenile arthritis refers to an arthritic condition that develops in subjects under the age of 18. Gout is an acute arthritic condition in which one joint, such as the base of the big toe of a subject, is attacked repeatedly. Septic arthritis is an arthritic condition is the result of an infection caused by a bacteria or fungus. Psoriatic arthritis is an arthritic condition that occurs as a result of psoriasis of the skin of a subject. Ankylosis arthritis is an arthritic condition which causes a joint to harden.
Rheumatoid arthritis is an arthritic condition that can cause inflammation of the joints and of the surrounding tissues of the joint of a subject. Symptoms of rheumatoid arthritis can include, but are not limited to, swelling, pain, and stiffness of a joint. In RA: a) a tissue known as pannus tissue forms over the joint, and causes the joint to lose mobility, b) the synovial membrane of a joint becomes inflamed by the infiltrating leukocytes neutrophils and monocytes, and c), bone erosion, cartilage loss, and bone and tissue damage occurs. Collectively, the joint weakens and losses mobility.
Juvenile idiopathic arthritis (JIA) is characterized by persistent arthritis that begins in a child less than 16 years of age and is present for at least six weeks. Approximately 50% of children with JIA will have arthritic disease as adults. Alternative complement pathway plays a role in JIA based on the clinical study (56 patients) that suggested elevated levels of AP specific protein Bb. These data suggest that AP C3 convertase is elevated.
Osteoarthritis is an arthritic condition that can cause pain and stiffness in a joint of a subject. There are two known types of osteoarthritis. Primary osteoarthritis occurs as a result of aging. As a subject ages, the cartilage in the joint of the subject can degenerate. Secondary osteoarthritis can be caused by several factors including, but not limited to, previous injury to a joint, surgery of a joint, a genetic disorder, obesity, or physical activity. As a result of the degeneration of cartilage in the joint of a subject, the bones of the joint can rub together, causing bone erosion and bone damage. In addition, small bone spurs called osteophytes can form around the area of the joint. Osteophytes can cause pain and limit joint mobility in the subject.
Osteoarthritis can occur in a subject through a variety of repeated stresses and strains on the joint via physical activity. In one example, activities, such as sports or dancing, place additional stresses and strains on the joints, such as the knee, shoulder, wrist, etc. of a subject, which may, over time, lead to an increase of the risk of the subject being diagnosed with arthritic conditions. In another example, laborers, such as electricians, carpenters etc., are at an increased risk of getting arthritic conditions because of repetitive stresses and strains on the joints of the worker, such as the knee, wrist, fingers etc.
Osteoarthritis (OA) is also an inflammation driven arthritis in which the cartilage of the joint, or joints, degenerates with age. Secondary OA refers to OA which is thought to be caused by previous injury to a joint, surgery of a joint, a genetic disorder, obesity, or other repeated physical stresses. As a result of the degeneration of cartilage in the joint, the bones of the joint can begin to rub together, causing bone erosion and bone damage. In addition, small bone spurs called osteophytes can form around the area of the joint. Osteophytes can cause pain and severely limit joint mobility. In those afflicted with OA, complete loss of cartilage can occur very rapidly.
Elevated levels of C3a have been found in arthritis joints. C3a and C5a have potent pro-inflammatory and immuno-regulatory functions. They increase vascular permeability and serve as chemo attractants, which that promotes soft tissue swelling. The anaphylatoxins activate neutrophils and monocytes, which results in the production of pro-inflammatory mediators such as TNF-α IL-1, IL-6, IL-8, and IL-17 [47-50]. Of these, TNF-α is the major pro-inflammatory cytokine involved in amplifying and perpetuating inflammation in arthritic joints. C5a is also a potent chemotactic protein that induces neutrophil chemotaxis, de-granulation, neutrophil elastase release, and superoxide generation. Neutrophils contain a potent arsenal of vasoactive, proteolytic and cytotoxic substances, which are produced to mediate many of the manifestations of inflammation in RA. Antibodies inhibit AP induced inflammation and tissue injury.
A number of current treatments target TNF-α. These include infliximab and etanercept. Whereas infliximab is a chimeric anti-TNF-α mAb, etarnercept is a TNF-α receptor fusion protein. In addition to these two, adalimumab (Humira™) binds to TNF-α and inhibits its interaction with cell surface receptors. The success of these drugs show that TNF-α is one of the major destructive elements in RA etiology. Yet, depletion of basal levels of TNF-α is not conducive for protection against serious infections. Selective removal and/or neutralization of only the disease-induced TNF-α should be the objective of future treatments. This is an important area of urgent and unmet medical need and the search for potent and safe therapeutic continues at a fast pace.
Anakinra (Kineret™) and tocilizumab (Actemra™) target the effects of mediators IL-1 and IL-6, respectively. Anakinra competes with IL-1 for the IL-1 receptor thereby providing inhibitory effect. Tocilizumab is a mAb that targets the IL-6 receptor. Other interleukins targeted for anti-RA therapy include IL-17 and IL-8. Experience with infliximab and etanercept (Remicade™ and Enbrel™) show that removal of the total TNF-α is not ideal and should be avoided. Additionally, in terms of drug therapy strategy it would be advisable to inhibit the excess production of inflammatory mediators simultaneously rather than target each mediator individually. For example, rather than seek inhibition of IL-1β, IL-6, or IL-17 individually, it may be more rewarding to prevent excess production of each of these by inhibiting an upstream element, especially that belonging to the AP of the complement. This novel design strategy has been adopted by NovelMed by targeting upstream AP specific proteins that are selective for the alternative complement pathway.
Elevated levels of C3a, C5a, C3b, C5b, and C5b-9 can gauge the level of activation of the complement system in disease conditions. Examples of complement-associated disorders involving the musculoskeletal include, but are not limited to, osteoarthritis [127], osteoporosis [128], acute gouty arthritis [129] (where C6 and MAC are activated), spondyloarthropathy [130], polymyositis [131], dermatomyositis [131, 132] (which increases C3b and C5b-C9), ankylosing spondylitis [133] (associated with increased C3b), general arthritis [10] (where C5a levels rise), enthesitis-related arthritis [134], eosinophilic fasciitis [135], juvenile rheumatoid (idiopathic) arthritis [136] (associated with increased C1q, C4, and MAC), myositis [137] and other forms of arthritis and arthritic conditions. In cases of psoriatic arthritis [138] it has been shown that anti-C5a prevents the arthritis. Inappropriate and/or over-activation of the complement system has also been associated with Reiter's syndrome (reactive arthritis) [139] and relapsing polychondritis [140]. The role of the alternative complement pathway in OA has been recently demonstrated. Elevated levels of C3, C5b-9 and properdin suggest the involvement of the alternative complement pathway.
U.S. Pat. Nos. 6,333,034 and 7,423,128 describe antibodies that inhibit both CP and AP mediated complement activation and therefore host defense is compromised. These antibodies play in role how antibodies prevent the formation of properdin oligomer. Properdin is a thrombospondin type 1 repeat and consists of six repeats of thrombospondin type 1. These antibodies inhibit the binding of properdin to C3b and prevent the formation of C3c. C3b cleavage results in the formation of C3c. Thus these antibodies prevent the cleavage of C3b.