English
中文Introduction
Asthma is a condition affecting the respiratory system and is characterized by difficulty in breathing, wheezing, coughing and heaviness of chest. The symptoms are the results of narrowing down of the trachea, bronchi and other components of the respiratory system due to forceful contraction of smooth muscles on one hand and thickening of tissue due to inflammatory response in the other. A number of cells are involved in the inflammatory response such as Eosinophils, T-lymphocytes, Mast cells, Dendritic cells and Macrophages (Barnes, 2006). Asthma may occur alone or associated with a constellation of symptoms as seen in chronic obstructive pulmonary disorder. Asthma is mostly reversible with treatment but the efficacy gets reduced in COPD. The primary line of treatment for Asthma or COPD is β2 agonists, anti-muscarinics, corticosteroids and antibodies (Barnes, 2006). All the drugs used for treatment act by reducing the underlying inflammation and thus assist in the opening of the airways which results in the patients breathing better. Hence it is common practice to assess the efficacy of an anti-asthmatic agent by its ability to control inflammation faster and longer than the existing agents.
Several assays are in use for assessing the anti-inflammatory response of anti-asthmatics. All the assays are done in an experimental set up in the lab (in vitro) and focus on measuring the ability of a drug to reduce oxidative burst which is basically the amount of superoxide anion (O2-) that is released during inflammation. The oxidative burst mainly occurs due to increase in the activity of Neutrophils following surface activation of their receptors such as inflammatory molecules or other pathogens. Also oxidative burst may occur by activation of proteins of signal transduction pathway by soluble factors such as PMA (Phorbol Myristyl Acetate, involved in inflammatory response. During most inflammatory processes reactive oxygen species are produced, the abundant being the superoxide ion (O2-), which is produced when electron transfer takes place between NADPH or NADH and oxygen catalyzed by the enzyme NAD(P)H oxidase (Babiora, 1978; Babiorb, 1978; Johnston, 1978).The superoxide anion is subsequently converted to hydrogen peroxide and hypochlorite (HOCL) which may occur with or without enzyme mediation. All the reactive oxygen species and byproducts are produced to ward off bacterial attack, only when they are produced more than the need do they become harmful. For a drug to be really effective, it must be rapidly absorbed and have an immediate response with respect to reducing the inflammatory response (Barnes, 2006).
Aim
The main aim of the work was to make a thorough literature survey for the availability of different assay methods employed in the analysis of anti-inflammatory properties of candidate drug molecules using oxidative burst as a reference. After weighing the benefits and disadvantages, a versatile assay was selected. The molecule under study which in this case a nebulized Budenoside (prepared from nanoparticle hydrogel technology) was assayed using the selected method.
Assays for assessing attenuation of oxidative burst
There are a number of assays which are being used for determining the anti-inflammatory properties of drugs. The most important are:
2. Assay of hydrogen peroxide: Hydrogen peroxide is one of the secondary products produced from superoxide anion spontaneously or with the action of enzymes such as superoxide-dis-mutase (Pick and Mizel, 1981). The original assay for the amount of hydrogen peroxide present involved the calculation of loss of fluorescence of the compound Scopoletin (Root et al, 1975) upon the presence of Hydrogen peroxide and the enzyme Horseradish peroxidase (HRP). This assay had some disadvantages as it required a spectrofluorometer for measuring fluorescence, temperature control of cuvette, limited sample assaying ability and lack of versatility if application. This assay was later modified by inclusion of Phenol Red (Phenol Sulponpthalein) instead of Scopoletin (Pick and Kiesari, 1980; 1981). The substrate Phenol Red gets oxidized to an unknown product which can be measured by getting absorbance values using wavelength between 600 to 610 nanometers. In most cases when the assay is done on culture plates there is interference from Phenol Red which is then converted to a yellow colour by changing the pH of the solution. The advantages of this method Phenol Red method are very similar to the Ferricytochrome C reduction assay method. Drug efficiency in this case is assessed by its ability to elicit lower absorbance values.
3. Tetrazolium Salt (MTT) reduction assay: This assay is also a very useful predictor of efficacy of anti-inflammatory drugs. In this assay MTT {3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide} is used as a substrate (Mossman, 1983; Pruett and Loftis, 1990). It is a yellow tetrazolium salt. This salt is reduced to a purple coloured Formazan compound by the enzyme Mitchondrial reductase, an enzyme present in mitochondria. The Formazan compound is insoluble and is made soluble by adding mild acidified solution of sodoum dodecyl sulphate to the solution. The absorbance of the solution is then calculated using a spectrophotometer using a wavelength between 500 to 600 nanometers. The detection step can also be done using an ELISA reader fitted with appropriate interference filters. Again the less the absorbance value, the more the efficacy of the drug in controlling inflammation. This assay is also very precise and fast. In this assay MTT is not the only tetrazolium salt used. Another compound called MTS {3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium}is also used (Cory et al, 1991). Unlike MTT, this compound requires an additional agent in the reaction called Phenazine methosulfate (PMS). The MTS reaction also produces a water soluble byproduct unlike the MTT insoluble one. Moreover, MTS+PMS reaction is more sensitive and less cytotoxic due of solubility of reaction end product. In addition to spectrophotometric evaluation, this assay can also be done on tissues (histological evaluation). Other less common tetrazolium salts used are XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide), INT (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride) and TTC (2,3,5-Triphenyl-2H-tetrazolium chloride).
4. Reduction of water soluble tetrazolium salt (WST-1) assay: The Ferricytochrome C assay has issues of background absorbance because the unreduced product also displays absorbance at 550 nanometer wavelength thus reducing the sensitivity of the assay. To obviate this problem, the water soluble tertazolium salt was used as substrate (Tan and Berridge, 2000). The water soluble tetrazolium salt is highly permeable through the cell membrane and is readily reduced to a water soluble formazan product by superoxide anion. The reduction of WT1 to a Formazan derivative is confirmed by the lack of reduction upon addition of the enzyme Superoxide-dis-mutase. The easiness and low background issue makes this assay very precise and suitable for usage in a microplate reader or a spectrophotometer thus enabling assaying of larger number of samples. The less the formazan product, the greater the efficiency of a drug in controlling inflammation. This assay is almost two fold more sensitive than Ferricytochrome C method. Last but not least, the water soluble tetrazolium sat has few inhibitors compared to the Ferricytochrome C method which has numerous inhibitors thus increasing the possibility of deactivation.
5. Chemiluminesence assay: This assay also measures the reactive oxygen species that arise during inflammation. In this assay luminal to isoluminol is used as the substrate. In this assay the enzyme NADPH or NADH catalyzes the reaction between the substrate and Superoxide anion. This releases light energy which can be measured using a photographic plate or a microtiter plate. This assay is very sensitive than the Ferricytochrome C method as it can measure NADPH enzyme activity in few cells. The levels of NADPH can be assessed both within and outside the cell. The technique requires special luminometers but is very precise, rapid, user-friendly, cheap and simple. Like some assays this assay also requires a peroxidase to move the reaction faster.
6. ELISA Assay: The term ELISA stands for Enzyme Linked Immunosorbent Assay. In this procedure any of the above mentioned substrates can be used. However, the assaying procedure is different as there is no spectrophotometer or microscope or microplate reader. Instead an ELISA reader is used (Pick and Mizel, 1981). The advantages of using this reader is that it is very sensitive. This assay can be modified in such as way that any of the inflammatory proteins are detected with an antibody against them. The antibody is linked to an enzyme, mainly HRP (Horseradish peroxidase) or AP (Alkaline phosphatase). When appropriate substrates these enzymes carry reduction which results in the development of a colored product that can then be quantified using the ELISA reader.
Modified MTT method: In the present study a modified MTT technique was used to evaluate the efficacy of a nebulized Budesonide preparation prepared using nanoparticle hydrogel technology (Nanagel). Commercially available Budesonide preparation for asthma treatment is delivered as an oral inhalant and is marketed under the trade name Pulmicort. The main aim of the study was to evaluate the efficacy of the new Budesonide preparation the water soluble tetrazolium salt (WST-1) assay. Since it was unavailable, the procedure was carried out using a modified MTT assay. All the experiments were done using HaCaT (Immortalized human keratinocyte) cell line. It was found that this line was best suited for the assay experiments. The cytokine TNF-α and the bacterial fMLP (N-formyl-methionine-leucine-phenylalanine) were used for generating oxidative burst. In the modified MTT method, additional controls were added such as MTT alone, MTT with enzyme Superoxide-dis-mutase, MTT with oxidative burst inducer (fMLP or TNF-α) and all the three together. This kind of an arrangement helps in deriving appropriate conclusion from the assay and accounts for background issues. Following this assay it was found that Budesonide delivery using Nanagel was three times more efficient than the commercial preparation Pulmicort even within a span of twenty-four hours.
Discussion and Conclusion
Thus there are many assays for the evaluation of anti-inflammatory propertied of candidate drug molecules base on their ability to curb oxidative burst. But each method has its advantages and disadvantages but the water soluble tetrazolium assay (WST-1) is considered the best followed by the MTT method which also yields very good results in terms of simplicity, precise nature and economy. More studies have to be for the development of highly simple and cost effective assays which permit large scale screening of thousands of compounds at once without compromising accuracy and reliability. Also it confirmed that Budesonide preparation using nanoparticle technology is superior to commercially available Pulmicort. The three time difference in efficiency may be attributed to the faster absorption and delivery of Nanagel compared to Pulmicort. However, more studies need to be done with increased concentrations of Pulmicort to arrive at a plausible conclusion. Budesonide seems to hold good promise as a fast acting inhalant but the data comparing it to Pulmicort must be reproduced using the efficient water soluble tertazolium assay (WST-1) method. Most corticosteroids have a slow absorption and hence upon inhalation create soreness and hoarseness of voice due to vocal cord atrophy. But many of the side effects of corticosteroids can be prevented by using a rapidly absorbable inhalant. In this regard Nanagel proved its worth as a highly efficient anti-asthmatic and anti-inflammatory drug.
