Design of hFGF1 Variant(s) with Increased Stability and Enhanced Bioactivity

Fibroblast growth factors (FGFs) are involved in various cellular processes such as cell growth,proliferation, differentiation, migration, angiogenesis, wound healing and embryonic development. Human acidic fibroblast growth factor (hFGF1) binds non-selectively to all the four FGF-receptors and is therefore considered as a powerful mitogen with broadest specificity. However, pharmacological applications of hFGF1 are restricted due to the low thermal stability of the growth factor. hFGF1 has low thermodynamic stability under physiological temperatures which leads to impairment of cellular signaling process thereby preventing its potential mitogenic properties. hFGF1 has a heparin binding pocket at the C-terminus which comprises of positively charges residues. The interaction between the positively charged amino acids lead to electrostatic repulsions, thereby rendering instability. To overcome this instability, hFGF1 binds to the glycosaminoglycan, heparin which decreases the repulsion (s) between the positively charged residues. However, binding of heparin poses a challenge for the use of hFGF1 in wound healing. Thrombin converts fibrinogen to fibrin and works as first line of defense by blocking the loss of blood. Intriguingly, thrombin also binds to heparin. Studies on wtFGF1 have demonstrated the presence of secondary thrombin cleavage site in hFGF1. Thus, thrombin is known to cleave hFGF1 at Arg 136 and render it biologically inactive. Usually, it is considered that dependency of hFGF1 to heparin increases the plausibility of thrombin-induced degradation of the growth factor. To tackle these downfalls, I have designed and constructed several point mutations in hFGF1 to improve the thermal stability and cell proliferation ability and to subside the heparin binding affinity of the growth factor. In this dissertation, I studied single, double, triple, quadruple, and penta variants of Q54P, S61L, H107S, K126N, and R136E and examined the thermal stability, bioactivity, and heparin dependency of the protein. These studies indicate that site - directed mutagenesis in hFGF1 can impact the inherent stability of the growth factor and role of heparin in hFGF1-FGFR receptor interaction and activation

DEVELOPMENT AND VALIDATION OF STABILITY INDICATING ASSAY FOR GRISEOFULVIN BY RP-HPLC IN TABLET DOSAGE FORM

Objective: To development and validation of a stability indicating reverse phase HPLC (RP-HPLC) method for the analysis of griseofulvin, an antifungal drug, and its assay method, in tablet dosage forms.Methods: The proposed RP-HPLC method utilizes Shiseido C18, 250 mm × 4.6 mm i.d., 5μ column (at ambient temperature), isocratic run [using Methanol and Water (70:30) as mobile phase], at a flow rate of 1.0 ml/minute, and UV detection at 291 nm for analysis of griseofulvin. This method was selected after applying different chromatographic conditions, the chromatographic variables like flow rate, the composition of mobile phase and nature of stationary phase were studied.Results: The reported method is linear over the range of 0.1-1.2μg/ml with a coefficient of correlation (r2) value 0.9998, slope 274.9 and intercepts 19093. The precision study revealed that the percentage relative standard deviation was within the acceptable limit and the mean recovery was found to be between 98%-102%. Griseofulvin was exposed to acidic, alkaline, oxidative, thermal and photolytic stress conditions and the sample was taken at different time intervals. The stressed samples were analyzed by the proposed method. The proposed method can be used for routine analysis stability testing and assay of griseofulvin in quality control laboratories.Conclusion: An economical, accurate, sensitive and precise HPLC method with ultraviolet detection was developed and fully validated for quality control analysis of griseofulvin in tablets. The proposed method is very rapid, where the total analytical run time is 5.16 minute.Keywords: RP-HPLC, Method validation, Griseofulvin, Estimatio