Abstract
In this study, we validated a double-sandwich enzyme-linked immunosorbent assay (ELISA) to investigate the pharmacokinetics of a recombinant human acidic fibroblast growth factor (rh-aFGF) hydrogel in rat skin and serum. A total of 130 Sprague-Dawley rats were divided into a control group, rh-aFGF hydrogel group, and a positive-control group (commercial rh-aFGF-Ai). We first determined the dilution ratio of skin homogenate and then validated the quantitative range, specificity, precision, and accuracy of our double-sandwich ELISA method, as well as the stability of our rh-aFGF hydrogel. For our pharmacokinetic study, skin and serum samples were collected at 0.5, 1, 2, 4, 6, and 10 h after rh-aFGF administration, and the concentration of rh-aFGF was measured by ELISA. The results showed that a 10-fold dilution of the skin tissue homogenate circumvented non-specific interference with endogenous proteins. The quantitative scope of the rh-aFGF calibration curve ranged from 62.5 to 4,000 pg/mL. The precision and accuracy of rh-aFGF quality-control samples were below 20%. Furthermore, bFGF, FGF21, KGF-2, and insulin did not interfere with the detection of aFGF, confirming that our method was specific. Rh-aFGF was stable under normal storage conditions. The maximum concentration (Cmax) and time to peak (Tmax) of the rh-aFGF hydrogel were 909.2 pg/cm2 and 0.5 h, respectively. The relative bioavailability (F) of the rh-aFGF hydrogel was 120% compared with that of rh-aFGF-Ai. The serum concentration of rh-aFGF was too low to be detected. Taken together, the pharmacokinetics of this rh-aFGF hydrogel provide further support for clinical research on rh-aFGF, and our double-sandwich ELISA method may be useful for pharmacokinetic studies of other protein-based drugs.
Highlights
With recent advances in biotechnology, an increasing number of protein-based drugs have been applied in clinical settings and have become the first choice of treatment for many diseases (Lewis and Richard, 2015)
Considering that skin tissue contains endogenous Acidic fibroblast growth factor (aFGF) and other proteins that interfere with the determination of the rhaFGF concentration, we first studied the dilution ratio of the tissue
The relative error (RE) of high, middle, and low concentrations of recombinant human acidic fibroblast growth factor (rh-aFGF) samples calculated using the standard curve prepared with 10× diluted skin homogenate were 93.5%, 89.8%, and 88.0%, respectively (Figure 1B)
Summary
With recent advances in biotechnology, an increasing number of protein-based drugs have been applied in clinical settings and have become the first choice of treatment for many diseases (Lewis and Richard, 2015). When proteinbased drugs are administered topically, their low concentrations and interference of endogenous proteins make analysis of their pharmacokinetics difficult (Sun et al, 2017). Ma et al performed a series of clinical trials that showed that topical application of rh-aFGF may represent a novel clinical treatment for deep burns and scalds (Ma et al, 2007). Previous research in our laboratory has shown that an rh-aFGF hydrogel significantly promotes the healing process of a full skin scald in a rat model of diabetes mellitus (Hui et al, 2018). It is necessary to study the pharmacokinetics of topical administration of rh-aFGF hydrogels to shed light on their toxicology and guarantee safety upon application
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