WO2014009779A1 - Selective inhibition of the membrane attack complex of complement and c3 convertase by low molecular weight components of the aurin tricarboxylic acid synthetic complex - Google Patents

Abstract

Selective inhibition of the membrane attack complex of complement in a mammalian species, preferably human, with components of the aurin tricarboxylic acid complex of less than 1 kilodalton in molecular weight. Preferred inhibitors are aurin tricarboxylic acid, aurin quadracarboxylic acid and aurin hexacarboxylic acid. Preferred disease to be treated by elective inhibition of the membrane attack complex of complement are age-related macular degeneration, Alzheimer's disease, atherosclerosis, rheumatoid arthritis, paroxysmal nocturnal hemoglobinemia, malaria infection and multiple sclerosis.

Description

Selective inhibition of the membrane attack complex of complement and C3 convertase by low molecular weight components of the aurin tricarboxylic acid synthetic complex Technical Field

Synthesis of the aurin tricarboxylic acid complex

Synthesis of the aurin tricarboxylic acid complex was carried out according to the published standard procedure (Cushman and Kanamathareddy, 1990).

1. Synthesis of 3,3'-dichloro-5,5'-dicarboxy-4,4'-dihydroxydiphenylmethane: 3-Chlorosalicylic acid (1 g) was dissolved in methanol (10 ml). Water (2.5 ml) was added and the flask was cooled to -5°C in an ice-salt (NaCl) bath. Concentrated sulfuric acid (30 ml) was slowly added over 20 min with the temperature being maintained at -5ºC. The reaction mixture was then stirred at this temperature for 1 h while a solution of 37% formaldehyde (4 ml) was added. The temperature was maintained at 0°C for 1 h and then the mixture was left at room temperature for a further 24 h. The reaction mixture was poured into crushed ice (150 g) and the precipitate filtered and dried to give the product,3,3'-dichloro-5,5'-dicarboxy-4,4'-dihydroxydiphenylmethane (yield 0.92g, 92%) as a powder. The sample was recrystallized from methanol.
2. Synthesis of 3,3'- dicarboxy-4,4'-dlhydroxydiphenylmethane: 3,3'-Dichloro-5,5'-dicarboxy-4,4'-dihydroxydiphenylmethane (0.92 g) was dissolved in ethanol (18 ml) and triethylamine (10 ml). Pallidiun on carbon was added to the solution and the mixture was stirred under an atmosphere of hydrogen for 48 h. The catalyst was filtered off, the solvent evaporated, and water (100 ml) added to the residue. The solution was cooled, and concentrated hydrochloric acid (5 ml) added. The white precipitate was filtered and dried to give the product, 3,3'-dicarboxy-4,4'-dihydroxydiphenylmethane (0.75 g, 90%) as a solid. It was dissolved and recrystallized from methanol. Powdered sodium nitrite (4 g) was added with vigorous stirring to concentrated sulfuric acid (4 ml). A mixture of the compound 3,3'-Dicarboxy-4,4'-dihydroxydiphenylmethane (0.75 g) and salicylic acid (0.38 g) was stirred until it was homogeneous. It was then poured into the solution of sodium nitrite-sulfuric acid. Stirring was continued at room temperature for an additional 18 h. The mixture was poured into crushed ice (100 g) with stirring. The dark orange precipitate was filtered and dried to give the crude product (0.6 g, yield 60 %). The powder was dissolved in 2% ammonium hydroxide for analysis.
3. 3,3',3'-tricarboxy-4,4',4'-trihydroxytriphenylcarbinol complex (aurin tricarboxylic acid complex): Powdered sodium nitrite (4 g) was added with vigorous stirring to concentrated sulfuric acid (4 ml). A mixture of the compound 3,3'-Dicarboxy-4,4'-dihydroxydiphenylmethane (0.75 g) and salicylic acid (0.38 g) was stirred until it was homogeneous. It was then poured into the solution of sodium nitrite-sulfuric acid. Stirring was continued at room temperature for an additional 18 h. The mixture was poured into crushed ice (100 g) with stirring. The dark orange precipitate was filtered and dried to give the crude product (0.6 g, yield 60 %). The powder was dissolved in 2% ammonium hydroxide for analysis.

Separation and analysis of ATAC

The powder we obtained from synthesis, or commercially purchased 'aurin tricarboxylic acid' from Sigma-Aldrich, or Aluminon from GFS Chemicals Inc. (Columbus, OH) were separated into high and low molecular weight components. In a typical experiment, five grams of material were dissolved in 0.2 % ammonium hydroxide (45 ml) and forced through a 1kDa filter (PLAC04310, Millipore, Ballerica, MA) under air pressure (70-75 Psi, 5.3 kg/cm² for 6 h). The filtered material was recrystal l ized by lyophilization. The filtrate (4.5 mg in 1 ml) was then loaded onto a size exclusion chromatography column (Sephadex LH-20 packed in 60% ethanol, GE healthcare, Piscataway, NJ). Three different eluant fractions were collected. The three fractions, as well as the starting mixture, were analyzed by mass spectrometry on a Waters ZQ apparatus equipped with an ESCI ion source and a Waters Alliance Quadrupole detector. All samples were exposed to electron spray ionization in positive and negative modes, as well as atmospheric pressure chemical ionization. Scans ranged from m/z 0-1100 and m/z 500-1500. Three molecules were detected of MW 422, 572, and 858. These molecular weights correspond to ATA, AQA, and AHA respectively as shown in Figure 3. There was no other derivative of less than 1.5 kDa detected. The components were separated and analyzed by mass spectroscopy. Results from the three sources were almost identical. The low MW components made up only 12- 16% of the total. They all contained three molecules of MW 422, 572, and 858.

Evaluation of the low molecular weight products as selective inhibitors of the membrane attack complex and C3 convertase

To evaluate the strength of blockade of the classical complement pathway by the low molecular weight products of the aurin tricarboxylic acid complex, (i.e. ATA plus AQA plus AHA), the standard CH50 assay was employed. Sheep red blood cells were sensitized by incubation overnight with rabbit anti sheep red blood cell antibody. Then dilutions of serum, with and without various amounts of the low molecular weight aurin tricarboxylic acid fraction (ATAC), were incubated with the sensitized red blood cells for 1 hour at 37°C. The incubates were centrifuged at 5,000 rpm for 10 min. The hemoglobin released into the serum from red blood cells that had been destroyed by complement attack, was determined by reading the optical density (OD) at 405 nm. As a positive control, red blood cells were 100% lysed with water, and as a negative control, no serum was added to the incubate.

The results are shown in Figure 3. Each of these components inhibited human complement-mediated red blood cell hemolysis almost identically. IC₅₀ values were for ATA 544 nM, for AQA 576 nM, for AHA 559 nM and for ATAC 580 nM. The IC₅₀ for ATAC in rat serum was 268 nM. These data establish that inhibition of complement activation by low molecular weight aurin tricarboxylic acid derivatives is in the nanomolar range and includes rodent as well as human serum

To determine at which stage of the complement cascade blockade was occurring, a variation of the CH50 assay was carried out. Instead of measuring hemolysis, western blot analyses were run to determine which serum complement proteins were consumed and converted into activated complement products on susceptible membranes. Complement proteins are consumed and converted only up to the stage of blockade. At stages beyond the blockade, they remain unchanged in the serum but their activated products appear on cell membranes. Results are shown in Figure 4. Human serum was diluted 16 fold. It was then treated for 30 min with ATA, AQA, AHA or ATAC. Then antibody-conjugated sheep red blood cells in an equal volume were added. The mixtures were incubated at 37⁰C for 1 h. They were then treated with a lysis buffer followed by a loading buffer for western blots. Equal amounts of protein from each sample were loaded onto gels and separated by 10% SDS. Following SDS, proteins were transferred to a PVDF membrane. The membranes were then treated with various primary antibodies followed by labeled secondary antibodies using well established techniques (Lee et al ., 2011). The list of antibodies that were utilized is shown in Table 1. Bands recognized by the antibodies were visualized by use of an enhanced chemiluminescence system and exposure to photographic film. For probing the same membrane with different antibodies, the membranes were treated with stripping buffer (Lee et al., 2011) and then treated as before with a different primary antibody.

Typical results are shown in Figure 4a. The left lane was loaded with serum only and shows that bands for C1q, C3, C4, and C5 were readily detected. The adjacent lane illustrates the effect of adding sensitized red blood cells, which then become hemolyzed by complement attack. Native serum proteins are consumed and become incorporated into the red cell membranes. C1q was not metabolized, but the band was intensified due to its dissociation from the C1 complex. Native C3 was no longer detected because it had been cleaved, and the C3b fragment had become covalently attached to the membrane. The degradation product C3d was detected. C4 was no longer detected because it had similarly been cleaved and the C4b fragment attached to the membrane and metabolized into the degradation product C4d. This fragment was also detected. C5 was cleaved and a band for the C5a product detected. Finally, the C5b-9 membrane attack complex, which had formed on the red cell membrane causing its hemolysis, was detected.

The next membrane shows the effect of incubation of serum plus sensitized red blood cells in the presence of the ATAC. Identical bands for the opsonization steps were detected, but the red cells were not hemolyzed and the membrane attack complex was not detected.

To determine at which stage of assembly of the membrane attack complex was being blocked, additional analyses were carried. The incubations were the same as before except that the red blood cells were separated from the residual serum and washed prior to being treated for western blot analysis. The blots were probed with antibodies to C6, C7, C8 and C9. The results are shown in Figure 4b for ATAC, 4c for ATA, for 4d for AQA and 4e for AHA. The results were identical for each component. Lane 1 for human serum alone shows that C6, C7, C8 and C9 were readily detected in the untreated serum. Lane 2 shows that in unprotected red blood cells that have become hemolyzed by complement attack, these antibodies detected only C5b-9, the fully formed membrane attack complex. Lane 3, in which the cells have been protected by ATAC, shows that the membrane attack complex does not fully form but becomes arrested at the C8 stage. The C6 antibody detected C5b6, C5b67, and C5b678. The C7 antibody detected C5b67 and C5b678, while the C8 antibody detected C5b678. These data establish that ATAC arrests formation of the membrane attack complex at the stage where C9 attaches to C5b678. Since C9(n) is required for creating the membrane destroying holes, this blockade is highly specific to preventing C9 attachment.

To determine the effects of ATAC on the alternative pathway, experiments were carried out where the classical pathway was blocked with C1 inhibitor (1.8 micrograms/ml) or with a C4b antibody (1,1000 dilution). For these experiments, human serum (15-fold dilution) was incubated with C1 inhibitor and ATA (5 microM, lane 3), or ATA with either properdin (1 microgm/ml, lane 4) or Factor D (0.1 microgm/ml, lane 5) for 1 h before opsonized zymosan (1 microgm/ml) was added. The mixtures were incubated for 1 h at 37ºC and centrifuged at 5,000 rpm for 10 min. The pellets were washed two times with Hank's balanced salt solution (HBSS) and treated with sample loading buffer for SDS-PAGE and immunoblotting. The buffer consisted of 50 mM Tris (pH 6.8), 0.1% SDS, 0.1% bromophenol blue and 10% glycerol. To preserve the molecular complexes that had formed, mild conditions for SDS-PAGE were followed. For C1q blotting, conventional sample loading buffer (50 mM Tris (pH 6.8), 1% SDS, 0.1% bromophenol blue and 10% glycerol and 2% beta-mercaptoethanol) was used.

Figure 5a shows the results when western blots of these erythrocyte membranes were developed with monoclonal antibodies to properdin (1/2,000), C3b (1/2,000), Factor B/Bb (1/2,000) and Factor D (1/2,000) respectively. Lane 1 in each blot shows that the native proteins were detected in untreated serum. Lane 2 shows that, in red blood cells that have become hemolyzed by complement attack mediated by zymosan in the presence of C1 inhibitor, similar bands were detected by antibodies to properdin, C3b and Factor B/Bb corresponding in MW to PC3b (~240kDa), PC3bB (~340 kDa), PC3bBb (~300 kDa) and PC3bBbC3b (>410 kDa). These data show that C3 convertase and C5 convertase were present on the membranes. However an independent band for C3b was not detected. This result indicates that C3b required properdin to bind and direct its binding to the erythrocyte membranes. The antibody to Factor D did not detect any bands for Factor D, indicating that Factor D did not form any SDS stable complexes on the membranes. Lane 3 shows the results obtained in the presence of 5 microM ATA. Bands for PC3bBb and PC3bBbC3b did not form. Instead, strong bands for the earlier steps of PC3b and PC3bB appeared. These results indicate that arrest of activation occurred at the stage where PC3bB becomes cleaved by Factor D to form the C3 convertase enzyme. Lanes 4 and 5 illustrate the effect of supplementing the serum with properdin (1 microgm/ml) or Factor D (0.1 microgm/ml). The effect of ATA was partially overcome. Weak bands for PC3bBb and PC3bBbC3b reappeared, although the band for PC3bB persisted. No bands Factor D were observed. This result provides further evidence that Factor D does not form a stable bond attached to membranes but remains in the serum.

Figure 5b illustrates the effects on the residual serum as shown by western blots developed with an antibody to C5/C5a. Treatment with zymosan and C1 inhibitor resulted in disappearance of the C5 band and appearance of the activation product C5a (lane 2). The addition of ATA and C1 inhibitor (lane 3) prevented cleavage of C5, which was partially antagonized by treatment with properdin (lane 4) and Factor D (lane 5). Weaker bands for C5 appeared as well as faint bands for C5a indicating partial activation of serum C5.

Figure 5c shows the effects of these treatments on erythrocyte membranes developed with antibodies to the MAC components C5/C5b, C6, C7, C8 and C9. Lane 1 shows that bands for C5, C6, C7, C8 and C9 were readily detected in untreated serum. Lane 2 of membranes following serum treatment with zymosan and C1 inhibitor, resulted in disappearance of each of the protein bands and appearance of the MAC formation components C5b6, C5b67, C5b678, and the fully formed C5b-9. Lane 3 in which ATA was added shows that complete blockade appeared with no activation bands appearing on the membranes. Lanes 4 and 5, where the serum was supplemented with properdin and Factor D respectively, demonstrated partial activation of the complement system with weaker bands for C5b6, C5b67, and C5b678 appearing, but there was still blockade at the C5b-9 stage indicating that ATA was also blocking the addition of C9 to C5b-8.

The next set of experiments directly tested the binding of ATA to properdin, Factor D and complement proteins. These proteins were immobilized on microwell plates in a concentration range of 1-32 ng/ml. ATA was then added at a concentration of 100 microgm/ml and the solution incubated as described in methods. ATA binding to the proteins was then assayed according to our previously published fluorometric method (Lee et al. 2011)). Figure 6 shows the results. There was no binding of ATA to properdin. Only background fluorescence was observed. This result is consistent with observations that properdin binding to erythrocyte membranes is unaffected by ATA. But ATA bound to both Factor D and C9 in a concentration dependent manner. Such binding explains why ATA blocks the alternative pathway at the stage where Factor D cleaves PC3B to form PC3Bb, and both the classical and alternative pathways at the stage where C9 adds to C5b678. However, other complement proteins such as C2, C3, C4, C5, C6, C7, C8 and Factor B (32 ng/ml each) did not bind with ATA .

In summary, Figure 7 is a diagram of the alternative complement pathway showing the steps where ATA interferes. Activation of the alternative pathway first requires properdin binding to a target on the membrane. C3b can then attach to the bound properdin. Subsequently Factor B can be added. The critical stage is cleavage of Factor B on that complex to form C3 convertase (PC3bBb). Only then can significant amounts of C3 still remaining in the serum be cleaved and joined to C3 convertase to form C5 convertase (PC3bBbC3b). Factor D carries out this cleavage of Factor B. Since no bands incorporating Factor D were observed on Western blots of erythrocyte membranes, Factor D in the serum is unlikely to form a stable bond with membrane bound PC3bB. It may briefly attach to and cleave bound Factor B, then dissociating and returning to the serum along with Factor Ba. ATA interferes at this step, perhaps by binding to Factor D in solution preventing its access to bound PC3bB. If this step is overcome, so that C5 convertase can form (PC3bBbC3b), then ATA still blocks the addition of C9 to C5b678, preventing formation of the MAC. Thus ATA provides a two step inhibition of the alternative pathway and may be particularly efficacious in conditions where unwanted activation of the alternative pathway occurs.

Synthesis and filtration of ATA-methylester

To illustrate that simple derivatives of ATAC also have complement inhibiting properties, the methyl ester was synthesized and tested by the CH50 assay on human serum. Briefly, ATAC (0.8 g) was dissolved in methanol (16 ml). Concentrated sulfuric acid (610 microliters) was added. The reaction mixture was refluxed at 55 °C for 1 h. The solvent was evaporated and the remaining solid collected. The product was tested in a CH50 assay compared with the non-esterified material and was found to be 29% as active (Figure 8, IC₅₀ 0. 6 4 microM vs 2.52 microM assuming a MW of 422) .

In Vivo Testing

Since the invention requires material that can be safely administered on a continuing basis, it requires testing in vivo in animals. This can be achieved by feeding to mice or other species, a mixture of the powder obtained added to their normal chow. Our example was with mice that are transgenic for Alzheimer disease mutations (B6SJL-Tg). By employing such mice, the product was tested not only for safety, but also for potential efficacy in Alzheimer disease.

Control B6SJL-Tg mice were fed normal chow, and test B6SJL-Tg mice were fed show supplemented with 0.5mg/kg ATAC. Based on chow consumption, it was calculated that test mice were receiving 5mg/kg/day of ATAC. Feeding was started at ages from 56-63 days and was continued for a further 30 days or 48 days before sacrifice. Upon autopsy, no evidence of pathology in any organ of either the ATAC fortified or normal chow fed mice was observed. These data indicate that ATAC is well tolerated and apparently safe when continuously consumed at a dose of 5 mg/kg/day for 44 days.

The results of CH50 assays are shown in Figure 9. Serum at various dilutions (1-16 fold) was incubated with antibody-conjugated sheep red blood cells for 1 h. Serum from the fed mice required less dilution, consistent with inhibition of the membrane attack complex (IC₅₀ 1.92 fold vs. 6. 8 9 fold for mice fed normal chow). These data indicate that a 3.59 fold protection was achieved. They establish that ATAC is absorbed after oral administration, and, at the doses tested, is an effective inhibitor of MAC formation.

B6SJL-Tg mice develop early memory deficits due to the rapid buildup of beta amyloid protein deposits. The memory of B6SJL-Tg mice fed normal or ATAC chow was tested using a standard water maze test. It was performed in a pool of 1.5 meter diameter with opaque fluid and a 10 cm diameter hidden platform. Mice were placed in the pool for first-day visible training, followed by four days of training where the platform was hidden. The next day they were measured with the hidden platform removed to determine how quickly they returned to where the hidden platform had been placed and thus how well they remembered its location. The tracking of animal movements in the area where the platform had been located was captured by an HVS2020 plus image analyzer. Data were analyzed by two-way ANOVA. It was found that ATAC treated mice performed 2.5 fold better than the untreated mice. The data are shown in Figure 10. In summary, these in vivo data on Alzheimer disease transgenic mice show that ATAC is not only safe, but beneficial in these animals. It improves weight gain and memory retention, which correlates with its ability to inhibit formation of the membrane attack complex of complement.

Applicability of the invention to the treatment of human disease.

General considerations . The complement system has usually been interpreted as serving only the adaptive immune system. But it is also a mainstay of the innate immune system. It is called into play in all chronic degenerative diseases. If it is activated to the extent that the MAC is formed, there is danger of the pathology being exacerbated through bystander lysis. Damage can also occur by chronic activation of the alternative complement pathway. Therapeutic opportunities for intervention in a spectrum of human disease states have never been explored because there has never been previously described an orally effective complement inhibitor which is selective for blocking the MAC and alternative pathway activation. The invention described here illustrates examples of diseases where benefit in common degenerative diseases can be expected from utilization of the invention described here.

Rheumatoid arthritis . There is strong evidence that both the classical and alternative pathways of complement are pathologically activated in rheumatoid arthritis (Okroj et al. 2007). The arthritic joint contains proteins capable of activating complement as well as proteins signifying that both the classical and alternative pathways have been activated. In mouse models of rheumatoid arthritis, resistance can be achieved by deletion of C3, C5, or factor B (Okroj et al. 2007). These data indicate that ATA or ATAC should be effective in rheumatoid arthritis.

Multiple sclerosis : Multiple sclerosis is a relapsing-remitting disease characterized by inflammation of the white matter of brain. Specific antibodies have been detected which target myelin antigens indicating that it is an autoimmune disorder (Genain et al. 1999). Complement will be activated in this process indicating the appropriateness of ATAC therapy.

Malaria infection : Malaria is a prevalent disease in Africa and south East Asia, resulting in an estimated 650,000 deaths per year. The infective agent, plasmodium falciparum, transmitted by mosquitos, produces enhanced complement activation in humans and susceptible animals. IgG and C3bBb complexes have been identified on erythrocytes of infected humans indicating damage caused by activation of both the classical and alternative pathways (Silver et al. 2010). Accordingly, treatment with ATAC should have beneficial effects.

Paroxysmal nocturnal hemoglobinemia : Paroxysmal nocturnal hemoglobinemia results from a clonal deficiency in erythrocytes of the X chromosome gene PIGA. As a consequence, the glycosal phophatidylinosotol moiety necessary for anchoring membrane proteins such as CD 55 and CD 59 is non functional. Erythrocytes and platelets lack the capacity to restrict cell-surface activation of the alternative pathway. Patients are subject to fatal thrombotic and hemolytic attacks. A treatment which is partially effective is to administer at biweekly intervals the monoclonal antibody eculizumab, which blocks C5 cleavage, preventing synthesis of the membrane attack complex. However this treatment is less than satisfactory being effective in only 49% of patients (Hillmen et al. 2006). A probable reason is that it does not block C3 convertase activity. C3 convertase is unregulated due to the CD 55 deficiency (Parker 2010). ATAC, because it is orally effective and compensates for both deficiencies, should be a truly definitive treatment for paroxysmal nocturnal hemoglobinemia.

Alzheimer 's disease . It has long been known that beta amyloid protein deposits in brain, which are believed to be the primary cause of the disease, can be identified by the opsonizing components of complement. It was demonstrated that this was due to C1q binding to beta amyloid protein (Rogers et al. , 1992). It was also demonstrated that the membrane attack complex of complement decorated damaged neurites in the vicinity of the deposits, indicating self damage by the complement system (McGeer et al ., 1989). Taken together, these data illustrate that the opsonizing aspects of complement need to be preserved so that phagocytosis of the beta amyloid deposits can occur, while the membrane attack complex needs to be selectively blocked so that self damage to host neurons can be eliminated.

Age related macular degeneration. Opsonizing components of complement have been identified in association with drusen, which are the extracellular deposits associated with the disease. The membrane attack complex has been found near the degenerating retinal pigment epithelial cells (Anderson et al. , 2002). Genetic analyses have revealed that polymorphisms in Factor H, Complement Factor B, and C3 all significantly influence the risk of suffering from age related macular degeneration (Anderson et al. , 2010). These data illustrate that the opsonizing aspects of complement need to be preserved so that phagocytosis of drusen can occur, while the membrane attack complex needs to be selectively blocked so that self damage to retinal pigment epitheleial cells can be eliminated.

Atherosclerosis. Atherosclerosis has not generally been considered to be exacerbated by the complement system. However the mRNA for C-reactive protein, a known activator of complement, is upregulated more than ten fold in the area of atherosclerotic plaques. Plaque areas showing upregulation of C-reactive protein and the opsonization components of complement also demonstrate presence of the membrane attack complex (Yasojima et al. , 2001). This is a further example of a common human degenerative condition where the membrane attack complex is present in a sterile situation and can therefore only damage host tissue. Again, the invention described here will preserve the desirable phagocytosis stimulating aspect of complement, while eliminating the self damaging aspect of the membrane attack complex.

As those skilled in the art will know, these diseases are only examples of many that may be found where the invention described here will have therapeutic benefit.

Background Art

Numerous agents have been described which will inhibit the complement system. These include heparin, suramin, epsilon- aminocaproic acid, and tranexamic acid. However, no orally effective agents have been described that will leave the necessary opsonization of the classical complement pathway functional, but which will prevent self damage either by blocking C3 convertase activity of the alternative pathway, as well as assembly of the membrane attack complex by both pathways. The only approved agent for treating aberrant complement activation is eculizumab, a humanized monoclonal antibody which blocks C5 conversion of the alternative pathway. It has been approved for the treatment of paroxysmal nocturnal hemoglobinemia. It is effective in 49% of cases (Hillmen et al. 2006). However it does not block the earlier step of C3 convertase, which can result in ongoing hemolysis of erythrocytes (Parker 2012). Moreover, as a high MW immunoglobulin antibody, it will not cross the blood brain barrier and will not be effective in CNS disorders.

We show in this invention that components of less than 1 kDa MW of the aurin tricarboxylic acid synthetic complex (ATAC) block C3 convertase of the alternative pathway, as well as MAC assembly at the final stage of C9 addition to C5b8 of both the alternative and classical pathways. We further show that they are safe and effective following oral administration.

Complement is a key component of both the innate and adaptive immune systems. It carries out four major functions: recognition of a target for disposal, opsonization to assist phagocytosis, generation of anaphylatoxins, and direct killing of cells by insertion of the membrane attack complex (MAC) into viable cell surfaces. Although complement is an essential defense system of living organisms, it is widely regarded as a two edged sword. Its opsonizing components are beneficial, but the membrane attack complex is potentially self damaging.

The complement system as it is understood today is illustrated in Figure 1. It consists of two main pathways: the classical and the alternative. The pathways have differing opsonizing mechanisms, but they have in common assembly of the terminal components to form the membrane attack complex (C5b-9). The classical pathway commences with the C1q component of the C1 complex recognizing a target that needs to be phagocytosed. Subsequent steps involve dissociation of the C1 complex, cleavage of C2, C4, and C3 to provide amplification as well as covalent attachment of the activated complement components to the target. By this means the target is disposed of by phagocytes that have receptors for the activated complement components so attached.

Both pathways result in C5 being cleaved into C5a and C5b. The released C5b fragment can then insert itself into the membranes of nearby cells. C6, C7, C8 and C9 (n) can then become sequentially attached to the membranes. The addition of C9 renders the complex functional by opening holes in the membranes, thus leading to death of the cells. Its physiological purpose is to kill foreign pathogens, but in the case of sterile lesions, it can destroy host cells by the phenomenon known as bystander lysis.

The complement system therefore operates in two parts. The first part is opsonization, which prepares targeted tissue for phagocytosis. The second part is assembly of the membrane attack complex, which has the purpose of killing cells. The former is essential, but the latter is not. For example, approximately 0.12% of Japanese are homozygous for the nonsense CGA-TGA (arginine 95stop) mutation in exon 4 of C9 (Kira et al. , 1999). These individuals cannot make a functioning membrane attack complex. This means that there are more than 150,000 Japanese leading healthy lives despite this deficiency. The Japanese experience indicates that selective inhibition of membrane attack complex formation on a long term basis is a viable therapeutic strategy.

The membrane attack complex exacerbates the pathology in all diseases where there is persistent overactivity of the complement system. In addition, pathology can be exacerbated in diseases in which there is alternative pathway C3 convertase over activity. Such diseases include, but are not limited to, rheumatoid arthritis, paroxysmal nocturnal hemoglobinemia, multiple sclerosis, malaria infection, Alzheimer disease, age related macular degeneration, and atherosclerosis. The purpose of this invention is to provide a method for successfully treating such conditions. We screened a large library of organic compounds for any that might have promise of being a selective inhibitor of these pathways. Commercially supplied 'aurin tricarboxylic acid' was the only material to pass the initial screening test. We found that the product contained only a small amount of aurin tricarboxylic acid. It consisted mostly of a complex of high molecular weight materials. We fractionated the crude material and investigated the properties of components of less than 1kDa MW. The desired properties were identified in true aurin tricarboxylic acid (ATA, MW422), aurin quadracarboxylic acid (AQA, MW572), aurin hexacarboxylic acid (AHA, MW858), and their combination which we term the low molecular weight aurin tricarboxylic acid complex (ATAC).

Related Publications

Patent Documents

McGeer et al. United States Patent application 13/195,216 filed August 1, 2011.

Bernstein et al. United States Patent 4,007,290 issued Feb. 8, 1977.

Other Publications

Anderson DH, Mullins RF, Hageman GS, Johnson LV . 2002. A role for local inflammation in the formation of drusen in the aging eye. Am. J. Ophthalmol. 134(3): 411-431 .

Anderson DH, Radeke MJ, Gallo NB, Chapin EA, Johnson PT, Curlettie CR, Hancox LS, Hu J, Ebright JN, Malek G, Hauser MA, Rickman CB, Bok D, Hageman GS, Johnson LV . 2010. The pivotal role of the complement system in aging and age-related macular degeneration: hypothesis revisited. Prog. Ret. Eye Res. 29: 95-112.

Cushman M, Kanamathareddy S . 1990. Synthesis of the covalent hydrate of the incorrectly assumed structure of aurintricarboxylic acid. Tetrahedron 46(5): 1491-1498 .

Cushman M, Kananathareddy S, De Clercq E, Scols D, Goldman ME, Bowen JA . 1991. Synthesis and anti-HIV activities of low molecular weight aurintricarboxylic acid fragments and related compounds. J. Med. Chem 34: 337-342.

Cushman M, Wang P, Stowell JG, Schols D, De Clercq E . 1992. Structural investigation and anti-HIV activities of high molecular weight ATA polymers. J. Org. Chem. 57 : 7 2 41-7248 .

Genain CP, Cannelloa B, Hauser SI et al. 1999. Identification of autoantibodies associated with myelin damage in multiple sclerosis. Nat. Med. 5, 170-175.

Gonzalez RG, Blackburn BJ, Schleich T . 1979. Fractionation and structural elucidation of the active components of aurintricarboxylic acid, a potent inhibitor of protein nucleic acid interactions. Biochimica et Biophysica Acta 562 : 534-545.

Heisig GB, Lauer M . 1941. Ammonium salt of aurin tricarboxylic acid. Organic Syntheses 1: 54 .

Hillmen, P. , Young, N.S., Schubert, J., et al. 2006. The complement inhibitor eculizumab in paroxysmal nocturnal hemoglobinuria. N. Engl. J. Med. 355, 1233-1243.

Kira S, Ihara K, Takada H, Gondo K, Hara T . 1998. Nonsense mutation in exon 4 of human complement C9 gene is the major cause of Japanese complement C9 deficiency. Human Gen. 102(6): 605-610 .

Lee, M. , Guo, J. P., Schwab, C., McGeer, E. G., and McGeer, P. L. (2012)Selective inhibition of the membrane attack complex of complement by low molecular weight components of the aurin tricarboxylic acid synthetic complex.Neurobiol. Aging. doi: http://dx.doi.org/10.1016/j.neurobiolaging.2011.12.005.

McGeer PL, Akiyama H, Itagaki S, McGeer EG . 1989. Activation of the classical complement pathway in brain tissue of Alzheimer patients. Neuroscience Letters 107: 341-346

Okraj, M., Heinegard, d., Holmdahl, R., and Blom, A.M. (2007) Rheumatoid arthritis and the complement system. Ann. Med. 39, 517-530.

Owens MR , Holme S . 1996. Aurin tricarboxylic acid inhibits adhesion of platelets to subendothelium. Thrombosis Res. 81 : 177-185 .

Parker CJ. 2012 Paroxysmal nocturnal hemoglobinuria 19(3):141-148

Rogers J, Cooper NR, Webster S, Schultz J, McGeer PL, Styren SD, Civin WH, Brachova L, Bradt B, Ward P, Lieberburg I. Complement activation by b-amyloid in Alzheimer disease. 1992. Proc Natl Acad Sci USA 89:10016-10020, 1992

Silver KL, Higgins SJ, McDonald CR, and Kain KC 2010. Complement driven immune responses to malaria: fuelling severe malarial diseases. Cellular Microbial. 8, 1036-1045.

Yasojima K, Schwab C, McGeer EG, McGeer PL . 2001. Generation of C-reactive protein and complement components in atherosclerotic plaques. American J. Pathol. 158(3): 1039-1051.

Technical Problem: For many years it has been known that complement activation which exceeds the limitations of endogenous controlling factors, can result in self damage to viable host tissue. The organs and cells damaged may be brain and spinal cord cells, especially neurons and oligodendroglia; retinal cells, especially pigment epithelial cells; heart and arterial muscle cells; joint cells; and kidney cells. Heretofore there has been no satisfactory method of treating the various conditions which generate such unwanted complement activation in these organs and tissues.

Technical Solution:

The solution to this general problem is to administer ATA, AQA, AHA and their derivatives, alone or in combination, in sufficient doses to block selectively formation of C3 convertase and the membrane attack complex of complement in these tissues. The active ingredients may be administered orally, intravenously, subcutaneously, or by direct injection into an affected area such as an inflamed joint or muscle.

Advantageous Effects. th effects will be to prevent further self damage by the unwanted complement activation with amelioration of the pathology.

Description of Drawings

Figure 1. Shows a standard schematic representation of the classical complement pathway as activated in Alzheimer disease, and the alternative complement pathway as activated in age related macular degeneration. Notice that assembly of the membrane attack complex is common to both the classical and alternative pathways.

Figure 2. Shows the putative structure and mass of the three components of the aurin tricaboxylic acid synthetic complex (ATAC) of less than 1 KDa with c orresponding mass-spec analyses of the separated components. (a) ATA, MW 422 (5,5'-((3-carboxy-4-oxocyclohexa-2,5-dienn-1-ylidene)methylene)bis(2-hydroxybenzoic acid) ( b ) AQA, MW 572 (putative structure 5,5-((3-carboxy-5-((3carboxy-4-oxocyclohexa-2,5-dien-1-ylidene)methyl)-4-hydroxyphenyl)methylene)bis(2hydroxybenzoic acid)) ( c ) AHA, MW858 (putative structure, 5.5'-((3-carboxy-5-((3-carboxy-4-oxocyclohexa-2,5-dien-1-ylidene)methyl)-4-hydroxybenzyl)-4-hydroxyphenyl)methylene)bis(2-hydroxybenzoic acid)). ES- means negative scan mode, giving values of -1 to the true mass. ES+ mean positive scan mode giving values of +1 to the true mass.

Figure 3. Shows the CH50 analyses of human and rat serum . Notice the almost identical IC₅₀ values of each component . They were (nM) for ATA 544, for AQA 576, for AHA 559 and for ATAC 580. The IC₅₀ for ATAC in rat serum was 268 nM.

Figure 4. Shows Western blot analyses demonstrating that ATA, AQA, AHA, and ATAC act selectively by blocking the addition of C9 to C5b678 thus preventing formation of the membrane attack complex. Normal human serum was pre treated with aliquots of aqueous solutions of ATA, AQA, AHA and ATAC prior to adding sheep red blood cells sensitized to human complement. The reaction mixtures were incubated at 37ºC for 1 h. Aliquots were loaded on 10% polyacrylamide gels and subjected to SDS-PAGE. Proteins were transferred to membranes and developed with appropriate primary antibodies to complement proteins (Table 1): (a) Western blots of membranes developed with antibodies to C1q, C3, C4 and C5. Lane 1, untreated serum; lane 2, serum with red blood cells added; lane 3 serum with red blood cells protected with ATAC. Notice that in untreated serum, bands for C1q, C3, C4, and C5 were readily detected. In lanes 2 and 3, the activated products C3d, C4d, and C5a were detected indicating opsonization had taken place. In lane 2, the MAC was detected, but not in lane 3, indicating that ATAC was blocking MAC formation. To analyze which step in MAC formation was involved, western blot membranes were treated with antibodies to C6, C7, C8, and C9 for (b) ATAC, (c) ATA, (d) AQA and (e) AHA. The results are identical. In each panel, lane 1 is serum, lane 2 is unprotected red blood cells, lane 3 is red blood cells protected with either ATA, AQA, AHA, or ATAC , and lane 4 is the same as lane 3 but with C9 protein supplementation. It shows that C6, C7, C8 and C9 are readily detected in untreated serum. Lane 2 shows that, in unprotected red blood cells that have become hemolysed by complement attack, only C5b-9, the fully formed membrane attack complex, is detected. Lane 3, in which the cells have been protected either by ATA, AQA, AHA or ATAC, the membrane attack complex does not fully form but becomes arrested at the C8 stage. The C6 antibody detects C5b6, C5b67, and C5b678. The C7 antibody detects C5b67 and C5b678, while the C8 antibody detects C5b678. Lane 4 provides confirmation that the blockade occurs only at the C9 stage. It can be seen that C5b-9 is now detected upon probing with C6, C7, C8 and C9, thus establishing that the ATAC block was at the C9 stage. A very faint C9 band is still visible in the blots indicating that not all the added C9 was consumed in the process.

Figure 5 . Shows western blots of membranes developed with antibodies to properdin, C3/C3b, Factor B/Bb and Factor D, demonstrating the effect of inhibiting classical pathway activation with C1 inhibitor or C4b antibody, and showing inhibition of C3 convertase by ATA . (a) Normal serum demonstrates detectable bands for properdin, C3, Factor B and Factor D (lane1). Upon activation with zymosan in the presence of C1 inhibitor, bands corresponding to PC3b, PC3bBb and PC3bBbC3b appear on blots developed with properdin and C3b antibodies, and PC3bBb and PC3bBb and PC3bBbC3b on the one developed with Factor Bb antibody (lane 2). These data demonstrate that properdin is required for C3b binding to initiate the alternative pathway, and that C3 and C5 convertases are activated. The addition of ATA results in bands appearing only for PC3b and PC3bB, indicating a block at the stage of Factor D cleavage of bound Factor B (lane 3). Lane 4 where properdin is added, and lane 5 where Factor D is added, both show reappearance of weak bands for PC3bBb and PC3bBbC3b, indicating partial recovery of alternative pathway activation. No bands for Factor D were detected on the erythrocyte membranes, indicating that this protease did not become bound but remained in solution. Three independent experiments were performed and these are representative. (b) Western blots of the residual serum developed with the antibody to C5/C5a. A band for C5 was readily detected in normal serum (lane 1). Treatment with zymosan and C1 inhibitor resulted in disappearance of the C5 band and appearance of the activation product C5a (lane 2). The addition of ATA and C1 inhibitor (lane 3) prevented cleavage of C5, which was partially antagonized by treatment with properdin (1 microgm/ml, lane 4) and Factor D (0.1microgm/ml, lane 5). (c) Treatment of the residual membranes with antibodies to C5/C5b, C6, C7, C8 and C9. Lane 1 of normal serum shows that each complement protein was detected in normal serum. Lane 2 of membranes following serum treatment with zymosan and C1 inhibitor resulted in disappearance of each of the protein bands and appearance of the MAC formation components C5b6, C5b67, C5b678, and the fully formed C5b-9. Lane 3 in which ATA was added shows that complete blockade appeared with no activation bands appearing on the membranes. Lanes 4 and 5, where the serum was supplemented with properdin and Factor D respectively, showed partial activation of the complement system with weaker bands for C5b6, C5b67, and C5b678 appearing, but there was still blockade at the C5b-9 stage indicating that ATA was also blocking the addition of C9 to C5b-8.

Figure 6 . Is a diagram showing the binding of ATA to Factor D and C9, but not to properdin, factor B, C2, C3, C4, C5, C6, C7, or C8. These proteins were applied to microwell plates in concentrations of 1-32 ng/ml, following which ATA at 100 micrograms/ml was added.

Figure 7. Is a schematic diagram of the alternative complement pathway illustrating blockade by ATA at the C3 convertase and C9 addition to C5b-8 stages.

Figure 8 . Shows a comparison of CH50 results in human serum of ATAC and the methyl derivatives of ATAC. The methyl derivatives were less effective than ATA with an estimated IC₅₀ of 2.52 microM.

Figure 9. Shows the effects of orally administered ATAC on complement activation of mouse serum. Serum from six B6SJL-Tg mice fed normal chow was combined and compared with the combined serum from six B6SJL-Tg mice fed ATAC supplemented chow. The sera were subjected to 1-16 fold dilutions. The solutions (25 microliters) were incubated with 100 microliters of antibody-conjugated sheep red blood cells (5×10⁶ cells) for 1 h. The mixtures were centrifuged, and the relative amount of hemoglobin released into 100 microliters of supernatant recorded by the absorbance at 405 nanometers. Serum from mice fed normal chow required more dilution than ATAC-fed mice for hemolysis to occur. The IC₅₀s were 6.89 and 1.92 fold respectively corresponding to a 3.59 fold protection.

Figure 10 . Shows memory retention of ATAC fed B6SJL-Tg mice compared with normal chow fed B6SJL-Tg mice as assessed by the rate of searching in the vicinity of the hidden platform after its removal on day 6 of testing. ATAC fed mice showed a significantly greater time searching in the correct area of the missing platform than mice fed normal chow, indicating a better retention of memory.

Table 1 . Lists the antibodies used to detect complement proteins in Western blots.

Best Mode The preferred mode for delivering ATA, AQA, AHA, its ammonium or other salts, its methyl or other derivatives, or mixtures thereof, to a person or animal in need thereof, is by the oral route. Capsules or pills with any combination of the above active ingredients may be formulated with acceptable conventional pharmaceutical carriers to prolong release, enhance effectiveness, and decrease metabolism of the active ingredients. These may include, but are not limited to, lactose, stearic acid, propylene glycol, cellulose or other ingredients that are well known to those skilled in the art. The dosage of the active ingredients may vary from 50 mg to 10 grams per day depending on the needs of each particular subject.

Mode for Invention: In addition to the preferred mode,alternative modes of delivery include intravenous, subcutaneous, and direct injection into a joint or muscle. These modes would become desirable in situations where oral administration was not possible, or where high concentrations were necessary or desirable at some localized site.

Industrial Applicability

This invention should open a major new treatment field for several chronic degenerative conditions of the brain and peripheral organs for which there is currently no satisfactory treatment. These diseases include, but are not limited to, paroxysmal nocturnal hemoglobinemia, rheumatoid arthritis, multiple sclerosis, malaria infection, Alzheimer disease, age related macular degeneration, and atherosclerosis.

Sequence List Text

Claims (18)

1. A method of medical treatment by selectively inhibiting the membrane attack complex of complement in a human or other mammalian species in need thereof by administering orally or parenterally an effective amount of components of the aurin tricarboxylic acid complex of less than 1 kilodalton in molecular weight.
2. A method as claimed in claim 1 where the selective inhibitor of the membrane attack complex is aurin tricarboxylic acid.
3. A method as claimed in claim 1 where the selective inhibitor of the membrane attack complex is aurin quadracarboxylic acid.
4. A method as claimed in claim 1 where the selective inhibitor of the membrane attack complex is aurin hexacarboxylic acid.
5. A method as claimed in claim 1 where selective inhibitors of the membrane attack complex are esters of the aurin tricarboxylic acid complex of less than 1 kilodalton molecular weight.
6. A method as claimed in claim 1 where the condition in which the selective inhibitor of the membrane attack complex is needed is age related macular degeneration.
7. A method as claimed in claim 1 where the condition in which the selective inhibitor of the membrane attack complex is needed is Alzheimer 's disease.
8. A method as claimed in claim 1 where the condition in which the selective inhibitor of the membrane attack complex is needed is atherosclerosis.
9. A method as claimed in claim 1 in all conditions where it can unequivocally be established that in such conditions the membrane attack complex of complement is assembled on host cells and is causing self damage.
10. A method of medical treatment by selectively inhibiting the C3 convertase step of the alternative complement pathway in a human or other mammalian species in need thereof by administering orally or parenterally an effective amount of components of the aurin tricarboxylic acid complex of less than 1 kilodalton in molecular weight.
11. A method as claimed in claim 10 where the selective inhibitor of C3 convertase is aurin tricarboxylic acid.
12. A method as claimed in claim 10 where the selective inhibitor of C3 convertase is aurin quadracarboxylic acid.
13. A method as claimed in claim 10 where the selective inhibitor of C3 convertase is aurin hexacarboxylic acid.
14. A method as claimed in claim 10 where the condition in which the selective inhibitor of C3 convertase is needed is rheumatoid arthritis.
15. A method as claimed in claim 10 where the condition in which the selective inhibitor C3 convertase is needed is paroxysmal nocturnal hemoglobinemia.
16. A method as claimed in claim 10 where the condition in which the selective inhibitor of C3 convertase is needed is malaria infection.
17. A method as claimed in claim 10 where the condition in which the selective inhibitor of C3 convertase is needed is multiple sclerosis.
18. A method as claimed in claim 10 in all conditions where it can unequivocally be established that in that condition C3 convertase is assembled on host cells and is causing self damage.