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Effect of Bleach on Bacterial Growth

Abstract Household chlorine bleach is used as disinfectant and sterilizing agent for various purposes in our daily life due to its bactericidal properties. The purpose of the study is to estimate the growth of inhibition of Escherichia coli in the presence of different dilution of Clorox solution. ToxTrakTM toxicity test was the best method used for this study and toxicity of the controls and samples was recorded using spectrophotometer at 603 nm wavelength. The initial and final absorbance values give the percent of inhibition of bacterial growth in the samples and control. Variability in the color change in the control and samples from blue to pink gives the extent of growth of inhibition of E.coli bacteria. From this study we can conclude that household chlorine bleach have bactericidal properties and can be used for various useful purposes in our daily life. As household chlorine bleach consists of low levels of chlorine it is recommended that not to mix with other bleaching or cleaning agents as it becomes more toxic and causes severe health problems to humans and animals.
Introduction In our daily life we use several cleaning agents to wash clothes, and for cleaning many surface areas in house. All the washing detergents and cleaning agents consists of low levels of chlorine that can be used as disinfect and sterilizing agent. The purpose of the experiment is to test the toxicity of household chlorine bleach (Clorox) on regular occurring bacteria Escherichia coli grown in lauryl tryptose growth medium. Household chlorine bleach consists 3-6% of sodium hypochlorite and oxygen bleach. Due to chlorine bleach’s bactericidal abilities it is used as a disinfectant and sterilizing agent ( www.wikipedia.org). Chlorine was proven to be toxic when ingested; it causes gastro intestinal damage or corrosive tissue damage due to its corrosive nature. According to EPA, exposure to chlorine even in small amounts can be harmful to living organisms (Heather, N., 2010).
This experiment focuses on a method called ToxTrakTM toxicity test to identify the inhibitory effect of chlorine on E.coli based on color change. The test tubes are added with reagents contained in a reagent pillow and an accelerator solution to increase the rate of the reaction, so that we need not wait for long hours. A visible color change depicts the activity of the test compound on E.coli. The difference in the initial and final absorbance recorded using a spectrophotometer derives the inhibition percentage (Toxtrak Toxicity test, lab handout). This test identifies the potentiality of compound chlorine as a disinfectant or toxicant to microorganisms and it is estimated that chlorine bleach would inhibit the growth of E.coli.
Materials and Methods Clorox was brought from Wal-Mart (Troy, Al), toxtrak testing kit (accelerator solution, reagent pillows and test tubes), lauryl tryptose broth was brought from HACH chemical company (Loveland, CO). One day old E.coli in a lauryl tryptose broth, pipettes, deionized water and spectrophotometer are the required materials. The test tubes were labeled. The blank (only deionized water) and control was prepared, the control has all the reagents except the sample chlorine. Different concentrations (100%, 10%, 1%) of chlorine were prepared by serial dilution. Equal amounts of reagent pillows (2), E.Coli sample (1ml), and accelerator solution (4drops) were added in the sample test tubes. The blank was kept in the spectrophotometer at 603 nm wavelength, absorbance was made to zero and the readings of control and samples were taken. The reading of the control has to be taken until it reaches 0.06 abs. After 30 minutes, final readings were taken and the initial and final absorbances of the samples and control were used to calculate the percentage of inhibition (Toxtrak Toxicity test, lab handout).
Results A significant change in color was observed in a very short span of time which indicates active inhibition of bacteria for chlorine. The color was changed from blue to pink. The percentage inhibition was calculated by finding the ratio between the sample and control. For this initially it is required to find the difference between initial and final concentration of each sample.
Discussion The color of control and samples were changed from blue to pink. Reduction of redox-active dye, resazurin by the bacterial respiration was the basis for the toxtrack method. The color of control and samples were changed from blue to pink when resazurin was reduced. The result shows decrease in the growth of E.coli from sample 1 to 3. But reading of sample 2 shows a negative inhibition than expected inhibition may due to error in preparation of sample 2. Control shows 0% inhibition growth due to absence of E.coli bacteria. Variability in the color change from blue to pink gives the extent of growth of inhibition of E.coli bacteria.
Conclusion The purpose of this study is to estimate the percent of inhibition of bacterial growth upon using household chlorine bleach. Toxtrack toxicity test was used for this study as it is inexpensive and takes less time. From this study we can conclude that there is a significant reduction in the growth of E.coli by using along with Clorox solution. This study shows that household chlorine bleach (Clorox) has bactericidal properties and can be used as disinfectant and sterilizing agent for various purposes in our daily life. It is recommended that not to mix household chlorine bleach with other bleaching or cleaning agents as it cause serious health problems to human health and animals (Chlorine Bleach and Mold Clean Up, www.spore-tech.com).

Bevacizumab Pharmacology and Applications

Introduction Avastin® (Bevacizumab solution for injection; Genentech, San-Francisco, Canada), is a humanised (93% human, 7% murine sequence) IgG 1 monoclonal antibody prepared by recombinant DNA technology. It is a Vascular Endothelial Growth Factor (VEGF) specific angiogenesis inhibitor. It belongs to the class of drug considered the fourth modality for cancer treatment. In 2004, it became the first angiogenesis inhibitor drug approved solely for cancer chemotherapy; firstly for the treatment of colorectal cancer, and later approved for other malignant conditions (1, 43).
Chemistry/composition Typically, Bevacizumab is a monomer with molecular weight of 149KDa. It has 3 major fragments namely: Fv (Fragment variable of murine origin), Fab (Fragment antigen binding) and FC (Fragment crystallisable of human origin) which performs effector function. Each V (variable) domain contains 3 short stretches of peptide and hypervariable sequences (HV1, HV2 and HV3) known as the complementarity determining regions (CDR) – antigen binding region (30). The heavy chains show C-terminal heterogeneity (lysine variants) and also contain one N-linked glycosylation on asparagine at position 303. Two inter-chain covalent disulfide bonds couple the two heavy chains. Each light chain is covalently joined through a disulfide bond at cysteine 214 to a heavy chain at cysteine 226 (6).
Avastin comes as a lyophilised solid for reconstitution prior to use. The solid is a clear to slightly opalescent, colourless to brown, sterile; pH 6.2 solution given by intravenous infusion. It is supplied in 100mg and 400mg preservative-free single use vials to deliver 4ml or 16ml of Avastin (25mg/ml). The 100mg product is formulated in 240mg α, α-tetrahalose dehydrate (acceptable non-compendial specifications), 23.2mg sodium phosphate (monobasic, monohydrate), 4.8mg sodium phosphate (dibasic, anhydrous), 1.6mg polysorbate 20, and water for injection USP. Its 400mg product contains the same amount of ingredients in the order of 4-folds. It can be stored up to 24 months at 2-8OC and given IV, mostly on days 0,28,35 and 42 or every 14 days (6).
Pharmacology Tumor Angiogenesis
Angiogenesis is the formation of new blood vessels sprouting from the pre-existing vasculature. It occurs in normal and pathological conditions, including those associated with cancer (2,9). The association of angiogenesis and cancer was initially discovered about fifty decades ago (11-13). Folkman et al., in 1971, first proved that angiogenesis was one of the major steps in tumor progression and metastasis (4,14) shown in Fig.2. Gullino showed that cells in pre-malignant tissues acquired angiogenic capacity which is dominant when malignancy is attained (15), which was confirmed through genetic studies, that acquisition of an angiogenic phenotype, was one of the hallmarks of cancer (3, 16-18).
VEGF A is the major angiogenic factor and regulator of tumor neovascularisation in humans. It enhances endothelial cell proliferation and blood vessel formation. Over-expression of VEGF in most tumors worsens the prognosis (8, 20). Avastin® explores the most efficacious of the three possible mode of blocking VEGF’s activity (neutralise VEGF); others either block production (Iressa) or block receptors for VEGF and other angiogenic stimulators -Sutent (7).
Fig. 2. Tumour angiogenesis. Angiogenesis is initiated by the production of angiogenic factors from tumour cells, such as vascular endothelial growth factor (VEGF). Upon binding to its cognate receptors on endothelial cells, VEGF triggers endothelial cell proliferation and migration. Degradation and invasion of extracellular matrix (ECM) then follow. Endothelial cells assemble into a tubular structure. The process is completed by loop formation and vessel wall maturation. (EC = endothelial cell) – Ref-[7].
Pharmacodynamics:
Bevacizumab binds with high affinity, to all human VEGF-A with its Fab fragment, which interacts selectively with ligand VEGF (complement fixation), prior to VEGF’s connection to the natural endothelial receptor, which leads to antibody-dependent cellular cytotoxicity (ADCC). Normal ligand-receptor interaction is blocked; also receptor phosphorylation and downstream pathways are activated. Thus, vascularisation is regressed; tumor vasculature formation and tumor growth are also inhibited (23). It binds and inhibits the biological activity of humanVEGF during in vitro and in vivo assay systems (64). Preclinical in vivo tumor growth studies of Bevacizumab administration to xenograft and metastatic models of cancer in mice showed reduction of microvascular tumor growth and inhibition of metastatic disease progression (24, 25).
Pharmacokinetics:
In contrast to small molecule drugs, the typical metabolic enzymes and transporter proteins such as cytochrome P450 and multi-drug resistance efflux pumps are not involved in the disposition of monoclonal antibodies (mAbs) (62). Pharmacokinetic studies of Bevacizumab were conducted in mice, rats and rabbits given by intraperitoneal injection; absorption was complete which was slower for subcutaneous injection. Its distribution was assessed in rabbit model because recombinant mAb (rhuMAb VEGF) was discovered to bind to rabbit VEGF but not mouse or rat VEGF recombinant MAb (24).
This model showed that most of rhuMAb was retained in the plasma, with more spreading to the heart, testes, bladder and kidney in comparison with other organs (which justifies its treatment of cancer of such organs); suggesting that Endogenous antibodies and Avastin is similarly cleared and regulated by Brambell receptors (54).Its Pharmacokinetics was thus characterized as a 2-compartment model from earlier studies done. Patients who received doses ranging from 0.1 to 10mg/kg intravenously over 4-24 weeks had an average clearance of 239mL/day, with an average Vd (volume of distribution) of 3260mL in the central compartment (6). Mechanism of metabolism and elimination has not yet been described in published data. However, results from a phase II trial showed that human clearance and half-life for Avastin was about 2.79 mL/kg/day and 21days respectively (6).
Clinical applications:
It is used for the management of metastaic colorectal cancer, with intravenous interferon alfa or 5-fluorouracil-based chemotherapy for first- or second-line treatment (31); non-squamous non-small cell lung cancer, with carboplatin and paclitaxel for first line treatment of unresectable, locally advanced, recurrent or metastatic disease (32, 34); first line therapy for metastatic HER2-negative breast cancer (33,35); glioblastoma, as a single agent (intraarterially / intracranially) for patients with progressive disease following prior therapy; and macular degeneration (48,56).
Side effects/Adverse effects:
Commonly reported effects are hypertension, proteinuria and haemorrhage. Less frequent adverse events include arterial and venous thromboembolic events (ATE, VTE), congestive heart failure, wound healing complications and gastrointestinal perforations. Generally, these adverse events are not dose dependent in any indication (with the exception for hypertension and grade 1 proteinuria). These Adverse events require habitual monitoring (hypertension, proteinuria); avoid overdosage (hypertension,VTE); temporary dose stoppage (hypertension, proteinuria, VTEs, wound healing) to absolute stoppage of regimen (for all life threatening events) – 38, 40-43.
Development/Formulation:
The first approved mAbs for therapy – orthoclone (OKT3) which was of murine origin, stimulated the formation of neutralising human anti-mouse antibodies which reduced the drug’s activity (via immunogenicity). Murine MAbs were engineered to the chimeric type (mouse CDR, human Fc) but still had the same limitations typified by rituximab; however, with a longer in vivo half-life (57,62). Bevacizumab development (humanized Mabs) was meant to reduce the xenogenic portion, thereby limiting immunogenicity (23). Two methods of achieving absolute biocompatibility of mAbs with humans (fully humanized mAbs), are phage display library (Adalimumab/Humira) and use of transgenic XenoMouse® – Panitumumab / Vectibix (63).
Bevacizumab is produced by recombinant DNA technology in Chinese hamster Ovary (CHO) cells, with gentamicin (antibiotic) as part of the expression system. Its manufacture is based on a CHO master and working cell bank system, which have been thoroughly characterised and tested to exclude harmful contaminants and endogenous viruses in accordance to ICH guidelines. Manufacture consists of series of steps which include fermentation, harvest and purification. Chromatographic and viral inactivation / removal procedures were combined to purify the product (45).
Structurally, it is large, complex, lipophilic and prone to degradation by acids and enzymes as well as temperature extremes and solvents (58,69). The development of the product was intended to achieve a stable liquid intravenous formulation (as infusion), the most prominent means of delivery of antibody therapeutics, which is able to reach diffuse and inaccessible tumor sites (49,64). Also, liquid formulations are cheaper, easily developed and easier to prepare for administration.
Its presentation as a liquid formulation was limited by aggregation and deamidation reaction (water-accessible regions) which could affect the product’s integrity / efficacy (65, 68, 75) by inducing charge heterogeneity detectable by isoelectric focusing or high-performance cation-exchange chromatography (66, 67). At low concentration, it adheres to container walls and exhibits aggregation at high concentrations. Concentration allowed will not be adequate for clinical response in chronic therapy. Also, it is difficult for concentrations > 50mg/ml to pass through the needle guage due to its viscosity – solution dimerisation (70,71). It justifies its administration by infusion. Other conditions that encouraged this situation were pH 6.5-7.5, IM NaCl (72,73).
This challenge was partly managed by post-translational modification.i.e. N- linked glycosylation at asparagine 303 during manufacture of Avastin (45,55,71); even though, the antigen specificity of some therapeutics could be affected by substituting some amino acids of the Fv fragment (59-61).Also, solution formulation was lyophilised to reduce the impact of water (deamidation, adherence / aggregation and loss of potency) on Bevacizumab because it is stable to freeze drying in the absence of excipients that act as cryoprotectants (76-78). Its freeze drying with residual water content (1-8%) allows for optimal stabilization in the dry state and upon reconstitution, as confirmed by a recent study (22). Avastin was formulated with buffer (sodium phosphate – mono

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