Proprietary Methodologies

The comparison of properties of original pharmaceutical products and corresponding generic copies is of growing scientific interest, due to the large number of molecules that have been coming off patent in recent years.

Current legislation of most pharmaceutical markets requires only a proof of bioequivalence for registration of such generics, but no in vivo clinical testing of therapeutic efficacy and safety, as the generic product contains the same active substance.

However, various in vivo comparisons of original pharmaceuticals and respective generics have shown differences in therapeutic efficacy, as well as drug safety [1, 2]. One possible explanation may be the different impurities composition in the respective original and generic formulations analyzed. There is no test method for the fast comparison of physiological activity between original pharmaceutical products and respective generics. To address this problem, IDrug has developed a new approach of comparative estimation of physiological activity of original and generic pharmaceutical products. This method has been patented [3].

The approach is based on the investigation of the influence of pharmaceutical products on the membrane structure of blood cells (erythrocytes, lymphocytes and platelets) using ESR-spectroscopy and ESR marks (or probes). The blood cells are isolated from human blood samples (Fig. 1). The alteration of the respective cell membrane structure is studied depending on active substance concentration in the solution and contact time of the solution with these cells. The alteration of the membrane structure is characterized by changes of its rigidity. These changes are compared between the various samples, which concentration corresponds to the maximum 24 hour dose of the respective pharmaceutical product. Changes of cell rigidity over time are measured as well.

Three blood cells types – erythrocytes, lymphocytes and platelets – were chosen for this analysis. Important processes such as oxygen transport, blood clotting and immune system reactions depend on these cell types. It is known that the influence of external factors on cell membranes result in structure alterations of lipid bi-layer, as well as conformational state of membrane proteins. These changes are accompanied by the alteration of physic-chemical properties (microviscosity, rigidity, polarity, permeability) and eventually result in the alteration of functional properties of the respective cell [4, 5] (Fig. 2).

The “rigidity” of a cell plays a fundamental role in the regulation of its internal processes. The deviation of the cell membrane rigidity value from its normal state (control – 0) in any direction (“+” if rigidity value is higher than in control state, and “-“ if it is less than in control state) alternates cell membrane processes and results in functional disorders of the cell.

The increase of membrane rigidity above a certain critical value alternates structure and dynamic bi-layer properties such that correct functioning of protein molecules becomes impossible. The transport function becomes broken for erythrocytes, immunological functionality for lymphocytes [6] and aggregative functionality for platelets [4, 5]. Cell shape alternates.

Our experiments have shown that blood viscosity increases when erythrocytes membrane rigidity increases. It is known that probability of clots formation increases when blood viscosity increases. Decrease of erythrocytes membrane rigidity may cause cell death. Platelets membrane rigidity decrease may increase the probability of clots formation [4]. At the same time it is possible to suppose that an increase in platelet membrane rigidity may increase the probability of bleeding.

Our research with inductors of clot formation (adenosynedephosphate (ADP), factor of platelets activator (FAT), adrenaline, thrombin, etc.) has shown that they cause a decrease of platelet membrane rigidity and therefore increase the probability of clot formation. As our experiments have shown in the case of lymphocytes, membrane rigidity increase results in an alternation of cell number and cell form, as well as a decrease of immune system activity. The influence of lymphocytes membrane rigidity on the immune system state is not studied yet. Absence of cell membrane rigidity changes confirms that there was no deviation from the normal state.

The use of different formulations of pharmaceutical products (such as Generic or Biosimilar copies of an originator product) may cause blood cell membrane alternations, resulting in cellular function changes and ultimately leading to different haematological side effect profiles of the various formulations. Thus it is important to study the alternations in blood cell membrane rigidity of alternative formulations of a pharmaceutical product, to validate if a switch of one product to the other might induce a higher or lower risk of haematological side effects.