Recombinant Human Keratinocyte Growth Factor Research Articles

Recombinant Human Keratinocyte Growth Factor Ameliorates Cancer Treatment-Induced Oral Mucositis on a Chip.

Oral mucositis (OM) is a severe complication of cancer therapies caused by off-target cytotoxicity. Palifermin, which is recombinant human keratinocyte growth factor (KGF), is currently the only mitigating treatment available to a subset of OM patients. This study used a previously established model of oral mucositis on a chip (OM-OC) comprised of a confluent human gingival keratinocytes (GIE) layer attached to a basement membrane-lined subepithelial layer consisting of human gingival fibroblasts (HGF) and human dermal microvascular endothelial cells (HMEC) on a stable collagen I gel. Cisplatin, radiation, and combined treatments are followed by a recovery period in the OM-OC to determine possible cellular and molecular mechanisms of OM under effects of KGF. Cancer treatments affected the keratinocyte layer, causing death and epithelial barrier loss. Both keratinocytes and subepithelial cells died rapidly, as evidenced by propidium iodide staining. In response to radiation exposure, cell death occurred in the apical epithelial layer, predominantly, within 24h. Cisplatin exposure predominantly promoted death of basal epithelial cells within 32-36h. Presence of KGF in OM-OC protected tissues from damage caused by cancer treatments in a dose-dependent manner, being more effective at 10 ng/mL. As verified by F-actin staining and the Alamar Blue assay, KGF contributed to epithelial survival and induced proliferation of GIE and HGF as well as HMEC within 120h. When the expression of eighty inflammatory cytokines is evaluated at OM induction (Day 12) and resolution (Day 18) stages in OM-OC, some cytokines are identified as potential novel therapeutic targets. In comparison with chemoradiation exposure, KGF treatment showed a trend to decrease IL-8 and TNF-a expression at Day 12 and 18, and TGF-β1 at Day 18 in OM-OC. Taken together, these findings support the utility of OM-OC as a platform to model epithelial damage and evaluate molecular mechanisms following OM treatment.

Enhancement of recombinant human keratinocyte growth factor 1 protein production in transgenic hazelnut (Corylus avellana L.) plant cell suspension cultures under RAmy3D inducible promoter

Enhancement of recombinant human keratinocyte growth factor 1 protein production in transgenic hazelnut (Corylus avellana L.) plant cell suspension cultures under RAmy3D inducible promoter

Rational Design and Production of Bioactive Analogs of Recombinant Human Keratinocyte Growth Factor (rhKGF) with Reduced Aggregation Propensity.

Recombinant human keratinocyte growth factor (rhKGF) is a highly aggregation-prone therapeutic protein. The present study aimed to reduce aggregation propensity of rhKGF by engineering the aggregation hotspots. Initially, 21 mutants were designed based on the previously-identified aggregation-prone regions (APRs) and then four of them including mutants No. 4 (L91K, I119K), 7 (V13S, L91K), 14 (L91D, I119D), and 21 (A51E) were selected based on molecular dynamics (MD) simulations for further experimental studies. The recombinantly produced rhKGF and mutants were analyzed regarding secondary structure, thermal stability, aggregation propensity, and biological activity. Far-UV CD spectroscopy showed that the mutants have similar secondary structure with rhKGF. A51E mutant showed enhanced stability and decreased monomer loss under heat stress suggesting its reduced aggregation propensity compared to rhKGF. Mutant No. 14 showed higher stability and less aggregation tendency than mutant No. 4 indicating that only mutations decreasing pI of rhKGF are effective in reducing its aggregation tendency. All of the mutants were at least as potent as rhKGF in stimulating proliferation of MCF-7 epithelial cells. Our results identified A51E as an equally potent, more stable, and less aggregation-prone analog of rhKGF which could be a promising alternative drug candidate for the commercially available rhKGF (Palifermin).

High-level production of keratinocyte growth factor 2 in Escherichia coli

Recombinant human keratinocyte growth factor 2 (KGF-2), also known as repifermin, is used in various therapeutic applications. However, KGF-2 production has not been optimized for facilitating large-scale production. Therefore, we attempted to attain high-level production of bioactive KGF-2. KGF-2 was fused with 6HFh8 (6HFh8-KGF-2) at the tobacco etch virus protease cleavage site. The 6HFh8-KGF-2 was expressed in Escherichia coli with high expression levels of approximately 33% and 20% of soluble protein in flask culture and 5 L fermentation, respectively. 6HFh8-KGF-2 was purified via nickel affinity chromatography. To maintain a stable form of KGF-2, the conditions of the cleavage reaction were optimized based on the isoelectric point. KGF-2 was purified via ion-exchange chromatography to high purity (>99%) with an optimal purification yield (91%). Circular dichroism spectroscopy demonstrated that purified KGF-2 had a secondary structure and thermal stability similar to that of commercial KGF-2. Bioactivity assays indicated that purified KGF-2 could induce MCF-7 cell proliferation in the same manner as commercial KGF-2. These results demonstrate that bioactive KGF-2 was overexpressed in E. coli and purified to high quality. Our findings indicated that bioactive KGF-2 can be produced in large quantities in E. coli.

Interactions of heparin derivatives with recombinant human keratinocyte growth factor: Structural stability and bioactivity effect study.

Heparin and heparan sulfate are important glycosaminoglycans that can regulate the activities of many vital proteins, especially the fibroblast growth factor (FGF) family. Because FGF7 (KGF) has an important role in tissue repair and maintaining the integrity of the mucosal barrier, recombinant human keratinocyte growth factor (rhKGF, palifermin) has been approved for the treatment of wound healing and oral cavity. Due to heparin plays an important role in the KGF signaling pathway, a more detailed study of the drug-drug interactions (DDIs) between rhKGF and heparin at the atomic level and investigating their synergistic effect on each other in terms of biology, especially in silico, is necessary for a better understanding of DDIs. In this study, DDIs between rhKGF and low-molecular weight heparin types (LMWH) were investigated. In this regard, scrutiny of the influence of the synergistic heparin types on the structure and biostability of rhKGF is accomplished using computational methods such as molecular docking and molecular dynamic simulations (MDs). Subsequently, the motion behavior of rhKGF in interaction with LMWHs was evaluated based on eigenvectors by using principal component analysis (PCA). Also, the binding free energies of rhKGF-LMWH complexes were calculated by the molecular mechanics/Poisson-Boltzmann surface area (MM-BPSA) method. The result showed that rhKGF-idraparinux (-6.9kcal/mol) and rhKGF-heparin (-6.0kcal/mol) complexes had significant binding affinity as well as they had a more stable binding to rhKGF than to other LMWH during 100 ns simulation. However, in order to confirm the curative effect of these drugs, clinical trials must be done.

CT-207 Protracted Diarrhea from High-Dose Melphalan in Autologous Hematopoietic Transplantation: Response to Short Course of Oral Budesonide

Approximately, 40% of patients receiving high dose melphalan (HDM) develop grade III or IV diarrhea (CTCAE 5.0) that promptly improves upon neutrophilic engraftment. Persistence of grade III diarrhea beyond 72 hours post-engraftment, though uncommon results in significant morbidity and prolongs hospitalization. Enlighten by a histopathologic finding of crypt inflammation, we utilized a short course of oral budesonide in a patient resulting in rapid resolution of diarrhea. We herein document 3 such cases and discuss their role in reducing morbidity and duration of hospitalization. All patients (age 44, 63 and 65, 2 female) received melphalan 200 mg/m2. Neutrophilic engraftment occurred on day +11 in 2 patients and day +12 in 1. Persistent grade III diarrhea 72 hours after neutrophil engraftment is defined as protracted diarrhea from melphalan. Infectious workup included stool examination for salmonella, shigella, rotavirus, cryptosporidium, and clostridium difficile we negative. Colonoscopy in one patient confirmed patchy crypt apoptosis and crypt abscess formation. Standard supportive care including antidiarrheals offered no improvement. Oral budesonide (BUD)was introduced at 5, 7- and 16-days post-engraftment (day + 17, +18 and +27 of ASCT). The dose of BUD comprised 6 mg/day in 2 while one patient received a 9 mg/day dose. Two patients had rapid resolution of diarrhea within 48 hours, while the third patient improved in 7 days. Total duration of BUD was 2, 2, and 26 days. Patients received a total cumulative dose of 12 mg, 15 mg, and 117 mg. Time to discharge from BUD initiation was 2, 2, and 7 days. A translational model of melphalan-induced gut toxicity reveals drug-host-microbe interaction that drives tissue injury and inflammation. Palifermin, a recombinant human keratinocyte growth factor is effective in reducing diarrhea of HDM. However, its cost is prohibitive. While newer melphalan preparations are available and prevention of melphalan-induced GI toxicity is being studied (Uproleselan), a short course of oral budesonide provided rapid resolution and facilitated patient discharge. Oral BUD results in rapid resolution of lower GI toxicity of HDM and potentially lowers the cost of hospitalization.

STATEMENT OF RETRACTION: Recombinant human keratinocyte growth factor attenuates apoptosis in elastase induced emphysematous mice lungs

STATEMENT OF RETRACTION: Recombinant human keratinocyte growth factor attenuates apoptosis in elastase induced emphysematous mice lungs

Evaluation of clinical and histological effects of KGF-2 and NGF on corneal wound healing in an experimental alkali burn rabbit model

Endogenously produced peptide growth factors such as keratinocyte growth factor-2 (KGF-2) and nerve growth factor (NGF) play a key role in the natural corneal wound healing process. However, this self-healing ability of the corneal tissue is often impaired in cases of severe corneal damage, as in corneal alkali injuries. In the present study, we investigated the clinical and histopathological effects of topical recombinant human keratinocyte growth factor-2 and nerve growth factor treatments in a rabbit model of corneal alkali burn. After induction of an alkali burn, 24 rabbits were divided equally into three groups: control group, KGF-2 group, and NGF group. Clinical parameters including epithelial healing, opacification, neovascularization and central corneal thickness were evaluated on the first (D1), seventh (D7) and fourteenth (D14) days after injury. Corneal histology was performed using hematoxylin/eosin (H&E) and Masson's Trichrome stains. Immunohistochemical staining for matrix metalloproteinase-2 (MMP-2), MMP-9 and transforming growth factor-β (TGF-β) was performed. On D14, the percentage of epithelial defect and opacity were significantly less in the KGF-2 and NGF groups compared to the control group (p < 0.05). There was no significant difference between the groups in central corneal thickness. In the evaluation of neovascularization on D14, the NGF group was significantly less vascularized than the control group (p = 0.011). Histological examination showed a significant increase in stromal edema and inflammation in the control group compared to both treatment groups (p < 0.05). There was also a significant difference between the NGF and control groups in histological evaluation of epithelial repair and vascularization (p < 0.05). When immunoreactivity of MMP-2, MMP-9 and TGF-β was examined, there was a significant increase in the control group compared to the NGF group (p < 0.05). Taken together, both NGF and KGF-2 treatments were effective for early re-epithelialization and decrease in inflammation, opacity and neovascularization after corneal alkali burn. The inhibitory effect of NGF treatment on chemical-induced neovascularization was found to be superior to KGF-2 treatment.

Intratracheal Keratinocyte Growth Factor Enhances Surfactant Protein B Expression in Mechanically Ventilated Preterm Pigs.

Bronchopulmonary dysplasia (BPD) is a devastating disease of prematurity that is associated with mechanical ventilation and hyperoxia. We used preterm pigs delivered at gestational day 102 as a translational model for 26–28-week infants to test the hypothesis administering recombinant human keratinocyte growth factor (rhKGF) at initiation of mechanical ventilation will stimulate type II cell proliferation and surfactant production, mitigate ventilator induced lung injury, and reduce epithelial to mesenchymal transition considered as a precursor to BPD. Newborn preterm pigs were intubated and randomized to receive intratracheal rhKGF (20 μg/kg; n = 6) or saline (0.5 ml 0.9% saline; control; n = 6) before initiating 24 h of ventilation followed by extubation to nasal oxygen for 12 h before euthanasia and collection of lungs for histopathology and immunohistochemistry to assess expression of surfactant protein B and markers of epithelial to mesenchymal transition. rhKGF pigs required less oxygen during mechanical ventilation, had higher tidal volumes at similar peak pressures indicative of improved lung compliance, and survival was higher after extubation (83% vs. 16%). rhKGF increased surfactant protein B expression (p < 0.05) and reduced TGF-1β (p < 0.05), that inhibits surfactant production and is a prominent marker for epithelial to mesenchymal transition. Our findings suggest intratracheal administration of rhKGF at initiation of mechanical ventilation enhances surfactant production, reduces ventilator induced lung injury, and attenuates epithelial-mesenchymal transition while improving pulmonary functions. rhKGF is a potential therapeutic strategy to mitigate pulmonary responses of preterm infants that require mechanical ventilation and thereby reduce the incidence and severity of bronchopulmonary dysplasia.

Toxicity Evaluation of Long-Term Topical Application of Recombinant Human Keratinocyte Growth Factor-2 Eye Drops on Macaca Fascicularis.

Recombinant human keratinocyte growth factor-2 (rhKGF-2), an effective agent for the regeneration of epithelial tissue, was found to have great potential for use in treatments of corneal diseases that involve corneal epithelial defects. Furthermore, the safety of long-term and high-dose external use of KGF-2 eye drops in rabbits has been well established previously. The aim of this study is to determine the safe dose range and target organs for toxicity of rhKGF-2 eye drops in Macaca fascicularis (M. fascicularis). The M. fascicularis animals were administered with different doses of rhKGF-2 eye drops (125, 500, and 2000 μg/ml) for four consecutive weeks, followed by a 2 week recovery period. No significant differences in weight, electrocardiogram characteristics, blood and urine indexes, pathology, and bone marrow cells were detected among the animals in different groups. The corneas of some animals in the middle- and high-dose groups showed fluorescence when stained with sodium fluorescein, and then the staining disappeared on days 28 and 42. Anti-rhKGF-2 antibodies were detected in a small number of animals in the high-dose group, and their level decreased after rhKGF-2 withdrawal. No neutralizing antibodies were detected. The result demonstrated that there was no obvious adverse reaction when topical application of rhKGF-2 eye drops at the dosage of 125 or 500 μg/ml on the M. fascicularis. This study is of great significance for the future clinical transformation of rhKGF-2 eye drops.

RhKGF-2 Attenuates Smoke Inhalation Lung Injury of Rats via Activating PI3K/Akt/Nrf2 and Repressing FoxO1-NLRP3 Inflammasome.

Smoke inhalation injury is an acute pathological change caused by thermal stimulation or toxic substance absorption through respiratory epithelial cells. This study aims to probe the protective effect and mechanism of recombinant human keratinocyte growth factor 2 (rhKGF-2) against smoke inhalation-induced lung injury (SILI) in rats. The SILI was induced in rats using a smoke exposure model, which were then treated with rhKGF-2. The rat blood was collected for blood-gas analysis, and the levels of inflammatory factors and oxidative stress markers in the plasma were measured. The rat lung tissues were collected. The pathological changes and cell apoptosis were determined by hematoxylin-eosin (HE) staining and TdT-mediated dUTP nick end labeling (TUNEL) assay, and the PI3K/Akt/Nrf2/HO-1/NQO1, and FoxO1-NLRP3 inflammasome expression were verified by western blot (WB). Both of the human alveolar epithelial cell (HPAEpiC) and primary rat alveolar epithelial cell were exposed to lipopolysaccharide (LPS) for making in-vitro alveolar epithelial cell injury model. After treatment with rhKGF-2, GSK2126458 (PI3K inhibitor) and AS1842856 (FoxO1 inhibitor), the cell viability, apoptosis, inflammation, oxidative stress, reactive oxygen species (ROS), PI3K/Akt/Nrf2, HO-1/NQO1, and FoxO1-NLRP3 in HPAEpiC and primary rat alveolar epithelial cell were examined. The data suggested that rhKGF-2 reduced LPS-induced HPAEpiC cell and primary rat alveolar epithelial cell apoptosis and the expression of inflammatory factors and oxidative stress factors. Moreover, rhKGF-2 improved the blood gas and alleviated SILI-induced lung histopathological injury in vivo via repressing inflammation, NLRP3 inflammasome activation and oxidative stress. Mechanistically, rhKGF-2 activated PI3K/Akt pathway, enhanced Nrf2/HO-1/NQO1 expression, and attenuated FoxO1-NLRP3 inflammasome both in vitro and in vivo. However, pharmaceutical inhibition of PI3K/Akt pathway attenuated rhKGF-2-mediated protective effects against SILI, while suppressing FoxO1 promoted rhKGF-2-mediated protective effects. Taken together, this study demonstrated that rhKGF-2 mitigated SILI by regulating the PI3K/Akt/Nrf2 pathway and the FoxO1-NLRP3 axis, which provides new reference in treating SILI.

A Prospective Cohort Study Comparing Long-Term Outcomes with and without Palifermin in Patients Receiving Hematopoietic Cell Transplantation for Hematologic Malignancies

The incidence of debilitating oral mucositis (OM) can be as high as 99% after myeloablative conditioning regimens preparing patients with hematologic malignancies for hematopoietic cell transplantation (HCT). Palifermin (KGF) is a recombinant human keratinocyte growth factor that reduces the incidence and duration of severe OM. The long-term safety of KGF has not been well established, however. In this long-term prospective matched-cohort study, patients who received KGF (cases) and underwent autologous or allogeneic HCT for hematologic malignancies between 2006 and 2013 were matched 1:1 to patients who did not receive KGF (controls). The primary outcome was overall survival (OS). Other outcomes were disease relapse, new malignancies, pancreatitis, renal failure requiring dialysis, pulmonary complications, cataract surgery, and acute and chronic graft-versus-host disease (GVHD). The analysis population comprised 2191 matched pairs with a wide range of diseases and donor types that received diverse conditioning and GVHD preventive regimens, representing contemporary practice patterns. The median duration of follow-up was 8 years (range, 1 to 12.5 years). In multivariate analyses, the probabilities of OS (relative risk [RR], 1.01; 95% confidence interval [CI], 0.91 to 1.12), relapse (RR, 1.06; 95% CI, 0.94 to 1.18), new malignancies (RR, 0.89; 95% CI, 0.67 to 1.18), and cataract surgery (RR, 1.05; 95% CI, 0.74 to 1.50) were not statistically significantly different between cases and controls. In univariate analyses, no increased risks were observed for renal failure requiring dialysis, pancreatitis, acute GVHD, chronic GVHD, interstitial pneumonitis/acute respiratory distress syndrome/idiopathic pneumonia syndrome, or bronchiolitis obliterans/cryptogenic organizing pneumonia/bronchiolitis obliterans organizing pneumonia among cases compared with controls. This long-term prospective safety cohort study demonstrates that the KGF group had no increased risk of overall mortality, relapse, new malignancies, or any other key outcome. The broad inclusion criteria allow the results to be generalized to contemporary practice for patients with a wide range of diseases and receiving a wide range of HCT conditioning regimens and graft sources from diverse donor types.

Potential aggregation hot spots in recombinant human keratinocyte growth factor: a computational study

The recombinant human keratinocyte growth factor (rhKGF) is a highly aggregation-prone therapeutic protein. The high aggregation liability of rhKGF is manifested by loss of the monomeric state, and accumulation of the aggregated species even at moderate temperatures. Here, we analyzed the rhKGF for its vulnerability toward aggregation by detection of aggregation-prone regions (APRs) using several sequence-based computational tools including TANGO, ZipperDB, AGGRESCAN, Zyggregator, Camsol, PASTA, SALSA, WALTZ, SODA, Amylpred, AMYPDB, and structure-based tools including SolubiS, CamSol structurally corrected, Aggrescan3D and spatial aggregation propensity (SAP) algorithm. The sequence-based prediction of APRs in rhKGF indicated that they are mainly located at positions 10–30, 40–60, 61–66, 88–120, and 130–140. Mapping on the rhKGF structure revealed that most of these residues including F16-R25, I43, E45, R47-I56, F61, Y62, N66, L88-E91, E108-F110, A112, N114, T131, and H133-T140 are surface-exposed in the native state which can promote aggregation without major unfolding event, or the conformational change may occur in the oligomers. The other regions are buried in the native state and their contribution to non-native aggregation is mediated by a preceding unfolding event. The structure-based prediction of APRs using the SAP tool limited the number of identified APRs to the dynamically-exposed hydrophobic residues including V12, A50, V51, L88, I89, L90, I118, L135, and I139 mediating the native-state aggregation. Our analysis of APRs in rhKGF identified the regions determining the intrinsic aggregation propensity of the rhKGF which are the candidate positions for engineering the rhKGF to reduce its aggregation tendency. Communicated by Ramaswamy H. Sarma

Fabrication and Evaluation of Transdermal Microneedles for a Recombinant Human Keratinocyte Growth Factor.

Microneedle transdermal patches are a combination of hypodermic needles and transdermal patches used to overcome the individual limitations of both injections and patches. The objective of this study was to design a minimally invasive, biodegradable polymeric recombinant human keratinocyte growth factor (rHuKGF) microneedle array and evaluate the prepared biodegradable microneedles using in vitro techniques. Biodegradable polymeric microneedle arrays were fabricated out of poly lactic-co-glycolic acid (PLGA) using the micromolding technique under aseptic conditions, and the morphology of the microneedles was characterized using light microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to rule out drug-polymer interactions. Standard procedures were used to analyze the prepared microneedle arrays for in vitro drug release and to perform a microneedle insertion test. Enzyme-linked immunosorbent assay was used to quantify rHuKGF. The PLGA polymer was safe for use in the fabrication of rHuKGF microneedles as there was no interaction between the drug and the polymer. The fabricated rHuKGF microneedle arrays had fully formed microneedles with a height of 600 µm and a base of 300 µm. The drug from the microneedle patch was released in vitro within 30 minutes. The strength of the microneedles in the patch was good, as they were able to reach a depth of 381±3.56 µm into parafilm without any structural change or fracture. Microneedle transdermal patches were successfully prepared for rHuKGF, and their evaluation suggested excellent quality and uniformity of patch characteristics. This can have potential applications in the therapeutic arena, offering advantages in terms of reduced dosing frequency, improved patient compliance, and bioavailability.

In silico mutagenesis in recombinant human keratinocyte growth factor: Improvement of stability and activity in addition to decrement immunogenicity

The recombinant human keratinocyte growth factor (rhKGF) is clinically applied to decrease the incidence and duration of cancer therapeutic agents. Particularly, it is extensively used for oral mucositis after chemotherapy-induced damage of different human cancers. However, the usage of rhKGF in treatment is limited owing to its short half-life, poor stability, immunogenicity, tendency to aggregate, and side effects. Therefore, there is a need to enhance the stability and to reduce immunogenicity of rhKGF for therapeutic applications. In this study, the stability, activity, and immunogenicity of rhKGF were improved using computational methods. The several mutations were generated based on sequence alignment, amino acids physic-chemical properties, and the structure simulation. The 3D structure of rhKGF and proposed mutants were predicted by Modeller v9.15 program, and then were evaluated using PROSESS, PROCHECK, and ProSA web tools. Afterwards, the effect of these mutants on rhKGF structure, stability, activity, and its interaction with fibroblast growth factor receptor2-IIb (FGFR2-IIb) was analyzed through utilizing GROMACS molecular dynamics simulations and docking tools, respectively. Also, binding free energies were calculated by the Molecular Mechanics/Poisson–Boltzmann Surface Area (MM/PBSA) method. We found that F63Y, R121K, and combine1 (K38R, F63Y, K72E, N105S) mutants lead to reduction of the number of T-cell epitopes. However, all of the selected mutants, except for R121K, could considerably increase stability and affinity of the rhKGF to FGFR2-IIb, in silico. In conclusion, this study, for the first time, offered that the combine1 and F63Y mutants could highly improve the stability and activity of rhKGF and even reduce immunogenicity without having any significant effect on the biological functions of rhKGF.

Rabbit as an Animal Model for Pharmacokinetics Studies of Enteric Capsule Contains Recombinant Human Keratinocyte Growth Factor Loaded Chitosan Nanoparticles.

BackgroundRecombinant human keratinocyte growth factor (rHuKGF) has gained considerable attention by researchers as epithelial cells proliferating agent. Moreover, intravenous truncated rHuKGF (palifermin) has been approved by Food and Drug Administration (FDA) to treat and prevent chemotherapy-induced oral mucositis and small intestine ulceration. The labile structure and short circulation time of rHuKGF in-vivo are the main obstacles that reduce the oral bioactivity and dosage of such proteins at the target site.ObjectiveFormulation of methacrylic acid-methyl methacrylate copolymer-coated capsules filled with chitosan nanoparticles loaded with rHuKGF for oral delivery.MethodsWe report on chitosan nanoparticles (CNPs) with diameter < 200 nm, prepared by ionic gelation, loaded with rHuKGF and filled in methacrylic acid-methyl methacrylate copolymer-coated capsules for oral delivery. The pharmacokinetic parameters were determined based on the serum levels of rHuKGF, following a single intravenous (IV) or oral dosages using a rabbit model. Furthermore, fluorescent microscope imaging was conducted to investigate the cellular uptake of the rhodamine-labelled rHuKGF-loaded nanoparticles. The proliferation effect of the formulation on FHs 74 Int cells was studied as well by MTT assay.ResultsThe mucoadhesive and absorption enhancement properties of chitosan and the protective effect of methacrylic acid-methyl methacrylate copolymer against rHuKGF release at the stomach, low pH, were combined to promote and ensure rHuKGF intestinal delivery and increase serum levels of rHuKGF. In addition, in-vitro studies revealed the protein bioactivity since rHuKGF-loaded CNPs significantly increased the proliferation of FHs 74 Int cells.ConclusionThe study revealed that oral administration of rHuKGF–loaded CNPs in methacrylic acid-methyl methacrylate copolymer-coated capsules is practically alternative to the IV administration since the absolute bioavailability of the orally administered rHuKGF–loaded CNPs, using the rabbit as animal model, was 69%. Fluorescent microscope imaging revealed that rhodamine-labelled rHuKGF-loaded CNPs were taken up by FHs 74 Int cells, after 6 hours’ incubation time, followed by increase in the proliferation rate.

Repair Effects of KGF on Ischemia-Reperfusion–Induced Flap Injury via Activating Nrf2 Signaling

BackgroundIschemia-reperfusion (IR) injury is a main cause to and the mechanism of necrosis after flap transplantation. Researches were hardly conducted on the role and possible mechanism of keratinocyte growth factor (KGF) in association with IR flap injury. Materials and methodsA CoCl2-stimulated hypoxia cell model was established to investigate the effects of KGF on cell viability, apoptosis, cell cycle, and reactive oxygen species level. The experiments were performed by cell counting kit-8 and flow cytometry as required. Meanwhile, the expressions of cell cycle-related and nuclear factor E2–related factor 2 (Nrf2) signaling–related genes were determined using quantitative real-time PCR and Western blot. The right dorsolateral areas of Institute of Cancer Research mice were marked as flaps, the pedicle of which formed an IR process through clamping and loosening. Tissue morphologies were observed using hematoxylin and eosin staining 24 h after the surgery. The effects of KGF on cell apoptosis and associated genes expressions were studied by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling, immunohistochemistry, and Western blot. ResultsHaCAT cells treated with 40 μM CoCl2 could not only reduce cell viability, promote cell apoptosis, arrest G1 phase of cell cycle and increase the activity of reactive oxygen species but also downregulate the expressions of c-myc, c-fos, transforming growth factor-α, Nrf2, heme oxygenase-1, and gamma-glutamyl cysteine synthetase. Additional recombinant human KGF, on one hand, could protect the cells from hypoxia injury. On the other hand, recombinant human KGF could significantly inhibit cell apoptosis, increase KGF activity, and increase the Nrf2, heme oxygenase-1, and gamma-glutamyl cysteine synthetase proteins levels in IR flap tissues. ConclusionsKGF played an important role in protecting mice flaps from IR injury, and the possible mechanism was involved in activating the Nrf2 signaling.

In silico analysis of different signal peptides for the secretory production of recombinant human keratinocyte growth factor in Escherichia coli

BackgroundThe recombinant human truncated Keratinocyte growth factor (Palifermin) is the only FDA approved medicine for the treatment of oral mucositis. The Keratinocyte growth factor is a fairly unstable protein due to its high aggregation propensity and therefore its expression as a secretory protein may results in the production of a protein with more stability, higher solubility, better folding, enhanced biological activity, N-terminal authenticity and simplified downstream processing. ObjectiveThe aim of this study was in silico evaluation of 31 different secretory signal peptides to determine the best theoretical candidates for the secretory production of recombinant truncated human KGF in E. coli. MethodsThirty different prokaryotic signal peptides experimentally shown to be capable of recombinant protein secretion in E.coli, along with the native KGF signal peptide were selected for further investigations. The signal peptide sequences were retrieved from the UniProt database. The ability of SPs to act as a secretory leader peptide for rhKGF and the location of cleavage sites were predicted by SignalP 4.1. Physicochemical properties of the signal peptides, which may influence protein secretion, were analyzed by ProtParam and PROSOII. Furthermore, the mRNA secondary structure and Gibbs free energy profile of the selected SPs were analyzed in the fusion state with the rhKGF using Visual Gene Developer package. Results and ConclusionComputational analysis of the physicochemical properties affecting protein secretion identified Sec-B dependent OmpC, Bla, and StaI and SRP dependent TolB signal peptides as the best theoretical candidates for the secretory production of recombinant truncated human KGF in E.coli.

Bioactivity Determination of a Therapeutic Recombinant Human Keratinocyte Growth Factor by a Validated Cell-based Bioassay.

The therapeutic recombinant human keratinocyte growth factor 1 (rhKGF-1) was approved by the FDA for oral mucositis resulting from hematopoietic stem cell transplantation for hematological malignancies in 2004. However, no recommended bioassay for rhKGF-1 bioactivity has been recorded in the U.S. Pharmacopoeia. In this study, we developed an rhKGF-1-dependent bioassay for determining rhKGF-1 bioactivity based on HEK293 and HaCat cell lines that stably expressed the luciferase reporter driven by the serum response element (SRE) and human fibroblast growth factor receptor (FGFR2) IIIb. A good responsiveness to rhKGF-1 and rhKGF-2 shared by target HEK293/HaCat cell lines was demonstrated. Our stringent validation was completely focused on specificity, linearity, accuracy, precision, and robustness according to the International Council for Harmonization (ICH) Q2 (R1) guidelines, AAPS/FDA Bioanalytical Workshop and the Chinese Pharmacopoeia. We confirmed the reliability of the method in determining rhKGF bioactivity. The validated method is highly timesaving, sensitive, and simple, and is especially valuable for providing information for quality control during the manufacture, research, and development of therapeutic rhKGF.

Toxicology study of long-term administration of rhKGF-2 eye drops on rabbit corneas

Keratinocyte growth factor −2 promotes corneal repair. Its mechanism of action involves regulating regeneration and migration of corneal cells, as well as activating corneal limbal stem cells. However, KGF-2 being a carcinogenic growth factor and its potential adverse effect in over dosage long-term treatment had not yet been reported. In this study, we used New Zealand white rabbits to study possible toxic effects of ocular administration of recombinant human keratinocyte growth factor-2 eye drops. Animals in the medium- and high-dose groups had some ocular irritant reactions during the course of drug administration; however this reaction was harmless to the cornea and it ended up when administration was stopped. Serum biochemistries were largely unaffected by treatment. Pathological examinations were unremarkable. We found that over-dosed administration of these eye drops caused some ocular irritation, but this irritant reaction was harmless to the eye, and it reversed after the drug was stopped. There were no apparent systemic effects of the drug.