1. Lipoprotein atherogenesis – background

One of the main processes that turn plasma lipoproteins into atherogenic products is lipid peroxidation. This has been extensively studied for more than 40 years, but although this reaction can be easily imitated and reproduced in vitro, the cause of it in vivo remains uncertain.

CTL's discovery of AtheroAbzymes provided the answser to this. Antibodies extracted from diseased human aorta were found to cause lipid peroxidation: incubation of these antibodies with normal plasma low density lipoproteins, LDL, demonstrated that they were not only able to bind to the intact LDL, but also to cause their peroxidation.

The other factor which makes lipoproteins atherogenic, originally observed in the 70s, is their formation of immune complexes. This was partially explained by the fact that when lipoproteins become oxidised the immune system starts to recognise the modified lipids as “foreign” and the production of the appropriate antibodies is triggered.

Kinetic analysis of this oxidation demonstrated that this reaction had an apparent enzymatic profile, graph 1. The Km was 0.69 + 0.012 mg LDL cholesterol, and their Vm was 0.83 + 0.105 µM of malonaldehyde per hour, i.e. in a range of physiological concentration of the substrate and in a “real-time” range of bio-catalysis.

Graph 1. Rate of LDL peroxidation by AtheroAbzymes as a function of substrate concentration, 1mg of lesion IgG, pH 5.6

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2. Cross-reactivity of AtheroAbzymes

Why does the immune system make such damaging antibodies against its own molecules, lipoproteins? One suggestion is that perhaps there is a case of broken down mimicry of parasitic micro-organisms (bacteria). This could originally have helped the parasite to avoid being attacked by the host’s immune system. However, at a certain stage, the tolerance of the system failed and production of antibodies against these mimicry bacteria epitopes begins. The subsequent result of this is the attack and damage of its own host molecules.

To verify this hypothesis, CTL conducted experiments with Chlamydia pneumoniae, the bacteria which has most commonly been reported in the last 15 years to be associated with the development of atherosclerosis.

Control	+AtheroAbzymes

Photo 1. Lysis of Chlamydia pneumoniae by AtheroAbzymes, MIF (×3,500)

It was found that the addition of IgG extracted from human atheroma to a suspension of Chlamydia pneumoniae bacteria caused their direct bacteriolysis. This was observed both in microfluorescent, MIF, (photos 1) and electron microscopy experiments (photo 2).

Control	+ AtheroAbzymes IgG

Photo 2. Lysis of Chlamydia pneumoniae elementary bodies by atheroma IgG, electron microscopy (×220,000)

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3. Clinical validation of AtheroAbzymes

Correlation with the development of atherosclerosis

CTL has validated the role of lipid oxidising antibodies in a number of clinical trials.

In patients with Coronary Heart Disease, CHD, using angiography data, it was shown that the level of the activity of these antibodies correlated with the degree of coronary artery stenosis. In patients with Cerebral Ischaemic Disease, it was demonstrated that the development of the stenosis of cerebral arteries, measured by ultrasound examination, also correlated with the level of the activity of AtheroAbzymes. >back to top.

Anti-AtheroAbzyme^®/TM therapy of CHD

Although the correlations described above may indicate a link between AtheroAbzyme activity and development of CHD, in order to invesstigate a possible causative role of these antibodies, a trial was initiated to evaluate any clinical effect of their inhibition.

Graph 2. Correlation of the frequency of detection of AtheroAbzymes and their activity with clinical manifestation of CHD

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Two anti-microbial drugswere selected on the basis of their additional ability to inhibit AtheroAbzymes (Doxycycline and Azithromycin). The anti-AtheroAbzyme activity of the latter was about 10 fold higher in vitro than the former (table 1).

AtheroAbzyme-positive patients with CHD were given daily administration of 500 mg of Azithromycin (on top of their traditional treatment with nitrates, β-blockers, aspirin, etc.). After two weeks of treatment, significant reduction of AtheroAbzyme activity was observed (graph 3). The treatment was continued for two months until AtheroAbzyme activity became undetectable in the serum of all patients.

At the end of this period, the clinical condition of the patients, as estimated by using the Rose G. - Blackburn H. Questionnaire, was significantly improved.

In contrast, administration of Doxycycline to AtheroAbzyme-positive CHD patients (on top of their traditional treatment with nitrates, β-blockers, aspirin, etc.) for two months did not cause any changes in AtheroAbzyme activity. After this treatment, there were no changes in the clinical condition of the patients.

Table 1. Results of in vitro screening of anti-microbial drugs with anti-AtheroAbzyme activity and their clinical effect on patients with CHD

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Graph 3. Effect of Azithromycin treatment on AtheroAbzyme^® activity in the serum of the patients with CHD

The total level of anti-Chlamydia antibodies in patients’ serum, which is linked with the intensity of the Chlamydia infectious process, albeit indirectly, was reduced after two months of the treatment either by Azithromycin or Doxycycline. This indicates that both drugs were similarly effective as anti-microbial agents. However, this quality alone was not enough to affect the clinical condition of the patients. In other words, inhibition of AtheroAbzyme activity appeared to be essential in the treatment of CHD (table 1).

The effectiveness of anti-AtheroAbzyme therapy was further evaluated after a one year follow-up of the treated patients. It was shown that Azithromycin treatment for two months resulted in inhibition of lipid oxidising anti-Chlamydia abzymes for 3-6 months with clinical benefit for one year.

Anti-AtheroAbzyme treatment alleviated symptoms of angina (clinical scores based on Rose G., Blackburn H. Questionnaire), and for 7 of the patients this resulted in their lasting remission of their condition when no apparent symptoms were registered during the observation period. This treatment also resulted in a reduction of the number of both fatal and non-fatal myocardial infarctions (table 2).

Table 2. One year follow-up of anti-AtheroAbzyme treatment of CHD

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4. Conclusion
CTL’s data indicate that Chlamydia infection may only be a trigger in the development of atherosclerosis. However, the excessive and prolonged production of originally useful bacteriolytic anti-Chlamydia antibodies, which cross-react with and convert plasma lipoproteins into atherogenic products, could be a significant pathogenic factor responsible for the development and progression of atherosclerosis.

Even two months of antibiotic treatment does not clear the Chlamydia infection and has no effect on the severity of CHD, whereas inhibition of the AtheroAbzyme activity gives clinical improvement. Therefore, for AtheroAbzymes may be considered not just as a new marker for the aggressive atherogenic process but also as a novel drug target in the treatment of the disease.

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