Design, modeling, expression, and chemoselective PEGylation of a new nanosize cysteine analog of erythropoietin

Reza Ahangari Cohan1, Armin Madadkar-Sobhani2,3, Hossein Khanahmad1, Farzin Roohvand4, Mohammad Reza Aghasadeghi4, Mohammad Hossein Hedayati5, Zahra Barghi5, Mehdi Shafiee Ardestani4, Davoud Nouri Inanlou1, Dariush Norouzian11Research and Development Department, Production and Research Complex, Pasteur Institute of Iran, Tehran, Iran; 2Department of Bioinformatics, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran; 3Department of Life Sciences, Barcelona Supercomputing Center, Barcelona, Spain; 4Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran; 5Quality Control Department, Production and Research Complex, Pasteur Institute of Iran, Tehran, IranBackground: Recombinant human erythropoietin (rhEPO) is considered to be one of the most pivotal pharmaceutical drugs in the market because of its clinical application in the treatment of anemia-associated disorders worldwide. However, like other therapeutic proteins, it does not have suitable pharmacokinetic properties for it to be administrated at least two to three times per week. Chemoselective cysteine PEGylation, employing molecular dynamics and graphics in in silico studies, can be considered to overcome such a problem.Methods: A special kind of EPO analog was elicited based on a literature review, homology modeling, molecular dynamic simulation, and factors affecting the PEGylation reaction. Then, cDNA of the selected analog was generated by site-directed mutagenesis and subsequently cloned into the expression vector. The construct was transfected to Chinese hamster ovary/dhfr- cells, and highly expressed clones were selected via methotrexate amplification. Ion-immobilized affinity and size exclusion (SE) chromatography techniques were used to purify the expressed analog. Thereafter, chemoselective PEGylation was performed and a nanosize PEGylated EPO was obtained through dialysis. The in vitro biologic assay and in vivo pharmacokinetic parameters were studied. Finally, E31C analog Fourier transform infrared, analytical SE-high-performance liquid chromatography, zeta potential, and size before and after PEGylation were characterized.Results: The findings indicate that a novel nanosize EPO31-PEG has a five-fold longer terminal half-life in rats with similar biologic activity compared with unmodified rhEPO in proliferation cell assay. The results also show that EPO31-PEG size and charge versus unmodified protein was increased in a nanospectrum, and this may be one criterion of EPO biologic potency enhancement.Discussion: This kind of novel engineered nanosize PEGylated EPO has remarkable advantages over rhEPO.Keywords: nanoPEGylated EPO, cysteine PEGylation, pharmacokinetic propert

Assessment of sensitivity, specificity and predictive value of Widal test in Typhoid fever patients in Hamadan

Serological Widal test is a fast, reliable and easy to perform and this test still is a suitable diagnostic method for diagnosing of typhoidal disease in many clinical laboratories. However, there are some doubtful reports regarding diagnostic value and credibility of this test, which was a motivation for present research. In this study, a total of 378 patients in two groups including 182 typhoidal patients with positive culture for Salmonella Typhi and para-Typhi A, B, C and 196 non-typhoidal febrile patients with negative culture for Salmonella, were studied in Hamadan city during 1994-97. All patients had at least one Widal test. In first group, 52.7% of patients have a titer of ?1:40 using anti-O (A, B, D) and 42.3% of patients also have a titer of ?1:80 using anti-O (A, B, D). In second group, 7.2% of patients have a titer of ?1:40 and 5.6% of them have a titer of ?1:80. In first group, 66.4% of patients had positive blood culture for S.typhi, however, 31.3% of them had positive antibody for OD titer. The results of this study indicated that sensitivity of Widal test for titer of ?1:80 was 86%, specificity 64% and PPV 42% and NPV 95%, therefore the negative Widal test did not have significant effect on the clinical diagnosis of typhoid disease