Study on biopharmaceutics classification and oral bioavailability of a novel multikinase inhibitor NCE for cancer therapy.

Specific biopharmaceutics classification investigation and study on phamacokinetic profile of a novel drug candidate (2-methylcarbamoyl-4-{4-[3- (trifluoromethyl) benzamido] phenoxy} pyridinium 4-methylbenzenesulfonate monohydrate, NCE) were carried out. Equilibrium solubility and intrinsic dissolution rate (IDR) of NCE were estimated in different phosphate buffers. Effective intestinal permeability (P(eff)) of NCE was determined using single-pass intestinal perfusion technique in rat duodenum, jejunum and ileum at three concentrations. Theophylline (high permeability) and ranitidine (low permeability) were also applied to access the permeability of NCE as reference compounds. The bioavailability after intragastrical and intravenous administration was measured in beagle dogs. The solubility of NCE in tested phosphate buffers was quite low with the maximum solubility of 81.73 µg/mL at pH 1.0. The intrinsic dissolution ratio of NCE was 1 × 10⁻4 mg·min⁻¹·cm⁻². The P(eff) value of NCE in all intestinal segments was more proximate to the high-permeability reference theophylline. Therefore, NCE was classified as class II drug according to Biopharmaceutics Classification System due to its low solubility and high intestinal permeability. In addition, concentration-dependent permeability was not observed in all the segments, indicating that there might be passive transportation for NCE. The absolute oral bioavailability of NCE in beagle dogs was 26.75%. Therefore, dissolution promotion will be crucial for oral formulation development and intravenous administration route will also be suggested for further NCE formulation development. All the data would provide a reference for biopharmaceutics classification research of other novel drug candidates.

AAV-mediated human PEDF inhibits tumor growth and metastasis in murine colorectal peritoneal carcinomatosis model.

BACKGROUND: Angiogenesis plays an important role in tumor growth and metastasis, therefore antiangiogenic therapy was widely investigated as a promising approach for cancer therapy. Recently, pigment epithelium-derived factor (PEDF) has been shown to be the most potent inhibitor of angiogenesis. Adeno-associated virus (AAV) vectors have been intensively studied due to their wide tropisms, nonpathogenicity, and long-term transgene expression in vivo. The objective of this work was to evaluate the ability of AAV-mediated human PEDF (hPEDF) as a potent tumor suppressor and a potential candidate for cancer gene therapy. METHODS: Recombinant AAV2 encoding hPEDF (rAAV2-hPEDF) was constructed and produced, and then was assigned for in vitro and in vivo experiments. Conditioned medium from cells infected with rAAV2-hPEDF was used for cell proliferation and tube formation tests of human umbilical vein endothelial cells (HUVECs). Subsequently, colorectal peritoneal carcinomatosis (CRPC) mouse model was established and treated with rAAV2-hPEDF. Therapeutic efficacy of rAAV2-hPEDF were investigated, including tumor growth and metastasis, survival time, microvessel density (MVD) and apoptosis index of tumor tissues, and hPEDF levels in serum and ascites. RESULTS: rAAV2-hPEDF was successfully constructed, and transmission electron microscope (TEM) showed that rAAV2-hPEDF particles were non-enveloped icosahedral shape with a diameter of approximately 20 nm. rAAV2-hPEDF-infected cells expressed hPEDF protein, and the conditioned medium from infected cells inhibited proliferation and tube-formation of HUVECs in vitro. Furthermore, in CRPC mouse model, rAAV2-hPEDF significantly suppressed tumor growth and metastasis, and prolonged survival time of treated mice. Immunofluorescence studies indicated that rAAV2-hPEDF could inhibit angiogenesis and induce apoptosis in tumor tissues. Besides, hPEDF levels in serum and ascites of rAAV2-hPEDF-treated mice were significant higher than those in rAAV2-null or normal saline (NS) groups. CONCLUSIONS: Thus, our results suggest that rAAV2-hPEDF may be a potential candidate as an antiangiogenic therapy agent.

A novel vaccine delivery system: biodegradable nanoparticles in thermosensitive hydrogel.

In this work, a novel vaccine delivery system, biodegradable nanoparticles (NPs) in thermosensitive hydrogel, was investigated. Human basic fibroblast growth factor (bFGF)-loaded NPs (bFGF-NPs) were prepared, and then bFGF-NPs were incorporated into thermosensitive hydrogel to form bFGF-NPs in a hydrogel composite (bFGF-NPs/hydrogel). bFGF-NPs/hydrogel was an injectable sol at ambient temperature, but was converted into a non-flowing gel at body temperature. The in vitro release profile showed that bFGF could be released from bFGF-NPs or bFGF-NPs/hydrogel at an extended period, but the release rate of bFGF-NPs/hydrogel was much lower. In vivo experiments suggested that immunogenicity of bFGF improved significantly after being incorporated into the NPs/hydrogel composite, and strong humoral immunity was maintained for longer than 12 weeks. Furthermore, an in vivo protective anti-tumor immunity assay indicated that immunization with bFGF-NPs/hydrogel could induce significant suppression of the growth and metastases of tumors. Thus, the NPs/hydrogel composite may have great potential application as a novel vaccine delivery system.

Biodegradable thermosensitive injectable PEG-PCL-PEG hydrogel for bFGF antigen delivery to improve humoral immunity.

In this contribution, a biodegradable and injectable thermosensitive poly(ethylene glycol)-poly(epsilon-caprolactone)-poly(ethylene glycol) (PEG-PCL-PEG, PECE) hydrogel system was successfully prepared for basic fibroblastic growth factor (bFGF) antigen delivery. bFGF encapsulated PECE hydrogel system (bFGF-hydrogel) is an injectable free-flowing sol at ambient temperature, and forms a non-flowing gel at physiological temperature acting as antigen depot. Furthermore, the cytotoxicity results showed that the PECE hydrogel could be regarded as a safe carrier, and bFGF could be released from the hydrogel system in an extended period in vitro. Otherwise, the immunogenicity of bFGF was improved significantly after encapsulated into the hydrogel. Strong humoral immunity created by bFGF-hydrogel was maintained for more than 14 weeks. Therefore, the prepared bFGF loaded PECE hydrogel might have great potential as a novel vaccine adjuvant for protein antigen.

In vitro drug release behavior from a novel thermosensitive composite hydrogel based on Pluronic f127 and poly(ethylene glycol)-poly(epsilon-caprolactone)-poly(ethylene glycol) copolymer.

BACKGROUND: Most conventional methods for delivering chemotherapeutic agents fail to achieve therapeutic concentrations of drugs, despite reaching toxic systemic levels. Novel controlled drug delivery systems are designed to deliver drugs at predetermined rates for predefined periods at the target organ and overcome the shortcomings of conventional drug formulations therefore could diminish the side effects and improve the life quality of the patients. Thus, a suitable controlled drug delivery system is extremely important for chemotherapy. RESULTS: A novel biodegradable thermosensitive composite hydrogel, based on poly(ethylene glycol)-poly(epsilon-caprolactone)-poly(ethylene glycol) (PEG-PCL-PEG, PECE) and Pluronic F127 copolymer, was successfully prepared in this work, which underwent thermosensitive sol-gel-sol transition. And it was flowing sol at ambient temperature but became non-flowing gel at body temperature. By varying the composition, sol-gel-sol transition and in vitro drug release behavior of the composite hydrogel could be adjusted. Cytotoxicity of the composite hydrogel was conducted by cell viability assay using human HEK293 cells. The 293 cell viability of composite hydrogel copolymers were yet higher than 71.4%, even when the input copolymers were 500 microg per well. Vitamin B12 (VB12), honokiol (HK), and bovine serum albumin (BSA) were used as model drugs to investigate the in vitro release behavior of hydrophilic small molecular drug, hydrophobic small molecular drug, and protein drug from the composite hydrogel respectively. All the above-mentioned drugs in this work could be released slowly from composite hydrogel in an extended period. Chemical composition of composite hydrogel, initial drug loading, and hydrogel concentration substantially affected the drug release behavior. The higher Pluronic F127 content, lower initial drug loading amount, or lower hydrogel concentration resulted in higher cumulative release rate. CONCLUSION: The results showed that composite hydrogel prepared in this paper were biocompatible with low cell cytotoxicity, and the drugs in this work could be released slowly from composite hydrogel in an extended period, which suggested that the composite hydrogel might have great potential applications in biomedical fields.

Thermoreversible gel-sol behavior of biodegradable PCL-PEG-PCL triblock copolymer in aqueous solutions.

A series of biodegradable PCL-PEG-PCL block copolymers were successfully synthesized by ring-opening polymerization of epsilon-caprolactone initiated by poly(ethylene glycol) (PEG), which were characterized by (1)H NMR, (13)C NMR, and FTIR. Their aqueous solution displayed special gel-sol transition behavior with temperature increasing from 4 to 100 degrees C, when the polymer concentration was above corresponding critical gel concentration (CGC). The gel-sol phase diagram was recorded using test tube inverting method and DSC method, which depended not only on chemical composition of copolymers, but also on heating history of copolymer's aqueous solution. As a result, the gel-sol transition temperature could be adjusted, which might be very useful for its application in biomedical fields such as injectable drug delivery system. And the typical shell-core structure of PCL-PEG-PCL micelles was introduced. The micelle-packing and partial crystallization might be the key gelation machanism for this gel-sol transition behavior of PCL-PEG-PCL aqueous solution.

Preparation and characterization of honokiol nanoparticles.

In this paper, honokiol nanoparticles were prepared by emulsion solvent evaporation method. The prepared honokiol nanoparticles were characterized by particle size distribution, morphology, zeta potential and crystallography. Results showed that the obtained honokiol nanoparticles at size of 33 nm might be amorphous, and could be well dispersed in water. Due to the great dispersibility in water, the obtained honokiol nanoparticles might have great potential in medical field.

Enhanced efficacy of combination therapy with adeno­associated virus-delivered pigment epithelium-derived factor and cisplatin in a mouse model of Lewis lung carcinoma.

Pigment epithelium-derived factor (PEDF) is a potent inhibitor of angiogenesis, and the antitumor effect of adeno-associated virus (AAV)-mediated PEDF expression has been demonstrated in a range of animal models. The combined treatment of low-dose chemotherapy and gene therapy inhibits the growth of solid tumors more effectively than current traditional therapies or gene therapy alone. In the present study, the effect of treatment with an AAV2 vector harboring the human PEDF (hPEDF) gene in combination with low-dose cisplatin on the growth of Lewis lung carcinoma (LLC) in mice was assessed. LLC cells were infected with AAV-enhanced green fluorescent protein (EGFP) in the presence or absence of cisplatin, and then the effect of cisplatin on AAV-mediated gene expression was evaluated by image and flow cytometric analysis. Tumor growth, survival time, vascular endothelial growth factor (VEGF) expression, microvessel density (MVD) and apoptotic index were analyzed in C57BL/6 mice treated with AAV-hPEDF, cisplatin or cisplatin plus AAV-hPEDF. The results of the present study provide evidence that cisplatin treatment is able to enhance AAV-mediated gene expression in LLC cells. In addition, the combined treatment of cisplatin plus AAV­hPEDF markedly prolonged the survival time of the mice and inhibited tumor growth, resulting in significant suppression of tumor angiogenesis and induction of tumor apoptosis in vivo, and also protected against cisplatin-related toxicity. These findings suggest that combination of AAV-hPEDF and cisplatin has potential as a novel therapeutic strategy for lung cancer.

Prevention of postsurgical cauterization-induced peritoneal adhesions by biodegradable and thermosensitive micelles.

Postsurgical peritoneal adhesion is a major concern in clinical practice which causes significant morbidity and mortality. In this study, we investigated the efficacy of biodegradable and injectable thermosensitive poly(ethylene glycol)-poly(epsilon-caprolactone)-poly(ethylene glycol) (PEG-PCL-PEG) micelles in preventing postsurgical cauterization-induced peritoneal adhesion. The biodegradable PEG-PCL-PEG copolymer could form nano-sized micelles in water, which instantly turned into a non-flowing gel at body temperature due to micellar aggregation. Moreover, a novel sidewall and cecum cauterization rat model was developed and the micelles were assigned for adhesion prevention tests. The PEG-PCL-PEG micelles could be administered by an ordinary syringe and provided unrestricted coverage of the cauterized peritoneum. The micelles instantly formed a gel in situ at body temperature and the formed gel could adhere to the cauterized sites as a durable barrier during critical time of adhesion formation. All rats from the control group (n = 10) developed score 5 adhesion, whereas, eight out of ten rats in the micelle-treated group showed no adhesion at all. Besides, cauterization-induced adhesion formation, adhesiveness and degradation of micelles, remesothelization of peritoneum, and restoration of cauterized tissue were investigated in detail. Our results thus indicated that, it was feasible to use biodegradable and injectable thermosensitive PEG-PCL-PEG micelles for prevention of peritoneal adhesions after surgery.

Synthesis and characterization of injectable, thermosensitive, and biocompatible acellular bone matrix/poly(ethylene glycol)-poly (ε-caprolactone)-poly(ethylene glycol) hydrogel composite.

In orthopedic tissue engineering, the extensively applied acellular bone matrix (ABM) can seldom be prefabricated just right to mold the cavity of the diverse defects, might induce severe inflammation on account of the migration of small granules and usually bring the patients great pain in the treatment. In this study, a new injectable thermosensitive ABM/PECE composite with good biocompatibility was designed and prepared by adding the ABM granules into the triblock copolymer poly(ethylene eglycol)-poly(ε-caprolactone)-poly(ethylene eglycol) (PEG-PCL-PEG, PECE). The PECE was synthesized by ring-opening copolymerization and characterized by ¹H NMR. The ABM was prepared by acellular treatment of natural bone and ground to fine granules. The obtained ABM/PECE composite showed the most important absorption bands of ABM and PECE copolymer in FT-IR spectroscopy and underwent sol-gel phage transition from solution to nonflowing hydrogel at 37°C. SEM results indicated that the ABM/PECE composite with different ABM contents all presented similar porous 3D structure. ABM/PECE composite presented mild cytotoxicity to rat MSCs in vitro and good biocompatibility in the BALB/c mice subcutis up to 4 weeks. In conclusion, all the results confirmed that the injectable thermosensitive ABM/PECE composite was a promising candidate for orthopedic tissue engineering in a minimally-invasive way.

Prognostic significance of tumor-associated macrophages in solid tumor: a meta-analysis of the literature.

PURPOSE: Tumor associated macrophages (TAMs) are considered with the capacity to have both negative and positive effects on tumor growth. The prognostic value of TAM for survival in patients with solid tumor remains controversial. EXPERIMENTAL DESIGN: We conducted a meta-analysis of 55 studies (n = 8,692 patients) that evaluated the correlation between TAM (detected by immunohistochemistry) and clinical staging, overall survival (OS) and disease free survival (DFS). The impact of M1 and M2 type TAM (n = 5) on survival was also examined. RESULTS: High density of TAM was significantly associated with late clinical staging in patients with breast cancer [risk ratio (RR)  = 1.20 (95% confidence interval (CI), 1.14-1.28)] and bladder cancer [RR = 3.30 (95%CI, 1.56-6.96)] and with early clinical staging in patients with ovarian cancer [RR = 0.52 (95%CI, 0.35-0.77)]. Negative effects of TAM on OS was shown in patients with gastric cancer [RR = 1.64 (95%CI, 1.24-2.16)], breast cancer [RR = 8.62 (95%CI, 3.10-23.95)], bladder cancer [RR = 5.00 (95%CI, 1.98-12.63)], ovarian cancer [RR = 2.55 (95%CI, 1.60-4.06)], oral cancer [RR = 2.03 (95%CI, 1.47-2.80)] and thyroid cancer [RR = 2.72 (95%CI, 1.26-5.86)],and positive effects was displayed in patients with colorectal cancer [RR = 0.64 (95%CI, 0.43-0.96)]. No significant effect was showed between TAM and DFS. There was also no significant effect of two phenotypes of TAM on survival. CONCLUSIONS: Although some modest bias cannot be excluded, high density of TAM seems to be associated with worse OS in patients with gastric cancer, urogenital cancer and head and neck cancer, with better OS in patients with colorectal cancer.

Novel vaccine adjuvant LPS-Hydrogel for truncated basic fibroblast growth factor to induce antitumor immunity.

The need to enhance the immunogenicity of tumor-associated antigens and modulate the resulting immune responses has prompted the development of new adjuvants. We prepared a novel adjuvant, lipopolysaccharides (LPS) loaded thermosensitive hydrogel (LPS-Hydrogel), for truncated basic fibroblast growth factor (tbFGF) peptide to enhance immunological responses and improve therapeutic effects in cancer. When co-formulated with tbFGF, LPS-Hydrogel formed antigen-adjuvant complexes, which enhanced antibody and cell-mediated responses in mice, thus promoting a more balanced antibody-mediated and cytotoxic T lymphocyte (CTL)-mediated immune response to inhibit tumor growth and metastases in vivo. Furthermore, the secretion of IFN-γ and IL-4 was detected, confirming activation of the two immune responses in vivo. There were no significant systemic toxicities observed with tbFGF-LPS-Hydrogel treatment. These results suggested that the thermosensitive and biodegradable LPS-Hydrogel was a novel adjuvant and carrier for peptide vaccines in cancer immunotherapy.

Thermosensitive PEG-PCL-PEG hydrogel controlled drug delivery system: sol-gel-sol transition and in vitro drug release study.

In this article, biodegradable and low molecular weight poly(ethylene glycol)-poly(epsilon-caprolactone)-poly(ethylene glycol) (PEG-PCL-PEG, PECE) triblock copolymers were successfully synthesized. Aqueous solution of the obtained PECE copolymers underwent sol-gel-sol transition as temperature increased which was flowing sol at room temperature and then turned into nonflowing gel at body temperature. Sol-gel-sol phase transition behaviors of aqueous PECE solutions were studied using rheometry and test tube-inverting method, which were affected by many factors, including the heating/cooling procedure and different additives in copolymers aqueous solution. In vitro drug release behavior was studied using bovine serum albumin (BSA) and Vitamin B(12) (VB(12)) as model drugs, and the PECE hydrogel could protect BSA from acidic degradation for 1 week at least. Therefore, PECE hydrogel is believed to be promising for injectable in situ gel-forming controlled drug delivery system due to their great thermosensitivity and biodegradability.

Acute toxicity evaluation of in situ gel-forming controlled drug delivery system based on biodegradable poly(epsilon-caprolactone)-poly(ethylene glycol)-poly(epsilon-caprolactone) copolymer.

In this paper, biodegradable poly(epsilon-caprolactone)-poly(ethylene glycol)-poly(epsilon-caprolactone) (PCL-PEG-PCL) triblock copolymer was synthesized, and was characterized by FTIR, 1H-NMR and GPC. The PCL-PEG-PCL/dimethyl sulfoxide (DMSO) solution displayed in situ gelling behavior when subcutaneously injected into the body. Toxicity tests and a histopathological study were performed in BALB/c mice. We focused mainly on acute organ toxicity of BALB/c mice by subcutaneous injection. In the acute toxicity test, the dose of subcutaneous injection was 5 g/kg body weight (b.w.), and the mice were observed continuously for 14 days. For the histopathological study, samples including heart, lung, liver, kidneys, spleen, stomach and intestine were histochemically prepared and stained with hematoxylin-eosin for histopathological examination. No mortality or significant signs of toxicity were observed during the whole observation period, and there is no significant lesion to be shown in histopathological study of major organs in the mice. Therefore, the maximal tolerance dose of dimethyl sulfoxide (DMSO) solution of PCL-PEG-PCL copolymer by subcutaneous injection was calculated to be higher than 5 g/kg b.w. Therefore, the PCL-PEG-PCL/DMSO system was thought to be non-toxic after subcutaneous injection, and it might be a candidate for an in situ gelling controlled drug delivery system.

Acute toxicity evaluation of biodegradable in situ gel-forming controlled drug delivery system based on thermosensitive PEG-PCL-PEG hydrogel.

In this work, a biodegradable poly(ethylene glycol)-poly(epsilon-caprolactone)-poly (ethylene glycol) (PEG-PCL-PEG, PECE) triblock copolymer was successfully synthesized. The aqueous solution of such PECE copolymer displayed special sol-gel-sol transition as temperature increase, which is a flowing sol at low-temperature and turns into a nonflowing gel at body temperature. The cytotoxicity of PECE copolymer was evaluated by cell viability assay using HEK 293 cells. In vivo gel formation and degradation test based on intraperitoneal and subcutaneous administration was conducted, respectively. The acute toxicity test and histopathological study were performed in BALB/c mice by intrapleural, intraperitoneal, or subcutaneous administration of PECE hydrogel (30 Wt %), respectively. The dose of intrapleural, intraperitoneal, or subcutaneous administration was up to 10 g/kg body weight (b.w.), 25 g/kg b.w., and 25 g/kg b.w., respectively, and the mice were observed continuously for 14 days. For histopathologic study, samples including heart, liver, lung, kidneys, spleen, stomach, intestine, and tissue of injection site were prepared for histochemical analysis and were stained with hematoxylin-eosin. No mortality or significant signs of acute toxicity was observed during the whole observation period and there is no significant lesion to be shown in histopathologic study of major organs. Therefore, the maximum tolerance dose of PECE hydrogel by intrapleural, intraperitoneal, or subcutaneous administration was calculated to be higher than 10 g/kg b.w., 25 g/kg b.w., and 25 g/kg b.w., respectively. The results indicated that the prepared PECE hydrogel was nontoxic after intrapleural, intraperitoneal, or subcutaneous administration, and it could be a safe candidate for in situ gel-forming controlled drug delivery system.

Biodegradable in situ gel-forming controlled drug delivery system based on thermosensitive PCL-PEG-PCL hydrogel: part 1--Synthesis, characterization, and acute toxicity evaluation.

In this work, biodegradable PCL-PEG-PCL (PCEC) triblock copolymers were successfully synthesized at one-step. Aqueous solution of PCEC copolymer displayed thermosensitive sol-gel-sol transition behavior, which is flowing sol at low temperature and turns into non-flowing gel at body temperature. The cytotoxicity of PCEC copolymer was evaluated by cell viability assay using HEK293 and WISH cells. In vivo gel-formation, degradation test, acute toxicity tests, and histopathological study of PCEC hydrogels were performed in BALB/c mice by subcutaneous administration. In acute toxicity test, the mice were observed continuously for 21 days. For histopathologic study, samples including heart, liver, spleen, lung, kidneys, and tissue of injection site were histochemical prepared and stained with hematoxylin-eosin. No mortality or significant signs of acute toxicity was observed during the whole observation period and there is no significant lesion to be shown in histopathologic study of major organs and tissue of injection site. The maximum tolerance dose (MTD) of PCEC hydrogel (20 wt%) by subcutaneous administration was calculated to be higher than 25 g/kg b.w. The results indicated that the obtained PCEC hydrogel was non-toxic after subcutaneous administration, and could be a safe candidate for in situ gel-forming controlled drug delivery system.

Preparation and characterization of magnetic poly(epsilon-caprolactone)-poly(ethylene glycol)-poly(epsilon-caprolactone) microspheres.

In this article, nano-magnetite particles (ferrofluid, Fe3O4) were prepared by chemical co-deposition method. A series of biodegradable triblock poly(epsilon-caprolactone)-poly(ethylene glycol)-poly(epsilon-caprolactone) (PCL-PEG-PCL, PCEC) copolymers were synthesized by ring-opening polymerization method from epsilon-caprolactone (epsilon-CL) initiated by poly(ethylene glycol) diol (PEG) using stannous octoate as catalyst. And the magnetic PCEC composite microspheres were prepared by solvent diffusion method. The properties of the ferrofluid, PCEC copolymer, and magnetic PCEC microspheres were studied in detail by SEM, VSM, XRD, Malvern Laser Particle Sizer, 1H-NMR, GPC, and TG/DTG. Effects of macromolecular weight and concentration of polymer, and the time for ultrasound dispersion on properties of magnetic microspheres were also investigated. The obtained magnetic PCEC microspheres might have great potential application in targeted drug delivery system or cell separation.

Synthesis, characterization, and hydrolytic degradation behavior of a novel biodegradable pH-sensitive hydrogel based on polycaprolactone, methacrylic acid, and poly(ethylene glycol).

In this work, a new kind of biodegradable pH-sensitive hydrogel was successfully synthesized by UV-initiated free radical polymerization. The obtained hydrogel was characterized by (1)H NMR and FTIR. Swelling behavior in different aqueous media and pH responsivity of the hydrogels were studied in detail as well. With increase in pH from 1.2 to 7.2, swelling ratio of the hydrogel increased. The morphology was observed by scanning electron microscopy, and the hydrolytic degradation behavior was also investigated in this work.