Utility of Intravenous Curcumin Nanodelivery Systems for Improving In Vivo Pharmacokinetics and Anticancer Pharmacodynamics.

Curcumin nanoformulations for intravenous injection have been developed to offset poor absorption, biotransformation, degradation, and excessive clearance associated with parenteral delivery. This review investigates (1) whether intravenous nanoformulations improve curcumin pharmacokinetics (PK) and (2) whether improved PK yields greater therapeutic efficacy. Standard PK parameters (measured maximum concentration [Cmax], area under the curve [AUC], distribution volume [Vd], and clearance [CL]) of intravenously administered free curcumin in mice and rats were sourced from literature and compared to curcumin formulated in nanoparticles, micelles, and liposomes. The studies that also featured analysis of pharmacodynamics (PD) in murine cancer models were used to determine whether improved PK of nanoencapsulated curcumin resulted in improved PD. The distribution and clearance of free and nanoformulated curcumin were very fast, typically accounting for >80% curcumin elimination from plasma within 60 min. Case-matched analysis demonstrated that curcumin nanoencapsulation generally improved curcumin PK in terms of measured Cmax (n = 27) and AUC (n = 33), and to a lesser extent Vd and CL. However, when the data were unpaired and clustered for comparative analysis, only 5 out of the 12 analyzed nanoformulations maintained a higher relative curcumin concentration in plasma over time compared to free curcumin. Quantitative analysis of the mean plasma concentration of free curcumin versus nanoformulated curcumin did not reveal an overall marked improvement in curcumin PK. No correlation was found between PK and PD, suggesting that augmentation of the systemic presence of curcumin does not necessarily lead to greater therapeutic efficacy.

Connective Tissue Growth Factor Is Related to All-cause Mortality in Hemodialysis Patients and Is Lowered by On-line Hemodiafiltration: Results from the Convective Transport Study.

Connective tissue growth factor (CTGF) plays a key role in the pathogenesis of tissue fibrosis. The aminoterminal fragment of CTGF is a middle molecule that accumulates in chronic kidney disease. The aims of this study are to explore determinants of plasma CTGF in hemodialysis (HD) patients, investigate whether CTGF relates to all-cause mortality in HD patients, and investigate whether online-hemodiafiltration (HDF) lowers CTGF. Data from 404 patients participating in the CONvective TRAnsport STudy (CONTRAST) were analyzed. Patients were randomized to low-flux HD or HDF. Pre-dialysis CTGF was measured by sandwich ELISA at baseline, after six and 12 months. CTGF was inversely related in multivariable analysis to glomerular filtration rate (GFR) (p < 0.001) and positively to cardiovascular disease (CVD) (p = 0.006), dialysis vintage (p < 0.001), interleukin-6 (p < 0.001), beta-2-microglobulin (p = 0.045), polycystic kidney disease (p < 0.001), tubulointerstitial nephritis (p = 0.002), and renal vascular disease (p = 0.041). Patients in the highest quartile had a higher mortality risk compared to those in the lowest quartile (HR 1.7, 95% CI: 1.02-2.88, p = 0.043). HDF lowered CTGF with 4.8% between baseline and six months, whereas during HD, CTGF increased with 4.9% (p < 0.001). In conclusion, in HD patients, CTGF is related to GFR, CVD and underlying renal disease and increased the risk of all-cause mortality. HDF reduces CTGF.

Hyperthermia-triggered release of hypoxic cell radiosensitizers from temperature-sensitive liposomes improves radiotherapy efficacy in vitro.

Hypoxia is a characteristic feature of solid tumors and an important cause of resistance to radiotherapy. Hypoxic cell radiosensitizers have been shown to increase radiotherapy efficacy, but dose-limiting side effects prevent their widespread use in the clinic. We propose the encapsulation of hypoxic cell radiosensitizers in temperature-sensitive liposomes (TSL) to target the radiosensitizers specifically to tumors and to avoid unwanted accumulation in healthy tissues. The main objective of the present study is to develop and characterize TSL loaded with the radiosensitizer pimonidazole (PMZ) and to evaluate the in vitro efficacy of free PMZ and PMZ encapsulated in TSL in combination with hyperthermia and radiotherapy. PMZ was actively loaded into TSL at different drug/lipid ratios, and the physicochemical characteristics and the stability of the resulting TSL-PMZ were evaluated. PMZ release was determined at 37 °C and 42 °C in HEPES buffer saline and fetal bovine serum. The concentration-dependent radiosensitizing effect of PMZ was investigated by exposing FaDu cells to different PMZ concentrations under hypoxic conditions followed by exposure to ionizing irradiation. The efficacy of TSL-PMZ in combination with hyperthermia and radiotherapy was determined in vitro, assessing cell survival and DNA damage by means of the clonogenic assay and histone H2AX phosphorylation, respectively. All TSL-PMZ formulations showed high encapsulation efficiencies and were stable for 30 d upon storage at 4 °C and 20 °C. Fast PMZ release was observed at 42 °C, regardless of the drug/lipid ratio. Increasing the PMZ concentration significantly enhanced the effect of ionizing irradiation. Pre-heated TSL-PMZ in combination with radiotherapy caused a 14.3-fold increase in cell death as compared to radiotherapy treatment alone. In conclusion, our results indicate that TSL-PMZ in combination with hyperthermia can assist in improving the efficacy of radiotherapy under hypoxic conditions.

Reversibly core-crosslinked PEG-P(HPMA) micelles: Platinum coordination chemistry for competitive-ligand-regulated drug delivery.

HYPOTHESIS: The presence of pendant thioether groups on poly(ethylene glycol)-poly(N(2-hydroxypropyl) methacrylamide) (PEG-P(HPMA)) block copolymers allows for platinum-mediated coordinative micellar core-crosslinking, resulting in enhanced micellar stability and stimulus-responsive drug delivery. EXPERIMENTS: A new PEG-P(HPMA) based block copolymer with pendant 4-(methylthio)benzoyl (MTB) groups along the P(HPMA) block was synthesized by free radical polymerization of a novel HPMA-MTB monomer using a PEG based macro-initiator. As crosslinker the metal-organic linker [ethylenediamineplatinum(II)]2+ was used, herein called Lx, which is a coordinative linker molecule that has been used for the conjugation of drug molecules to a number of synthetic or natural carrier systems such as hyperbranched polymers and antibodies. FINDINGS: The introduction of Lx in the micellar core results in a smaller size, a lower critical micelle concentration and a better retention of the hydrophobic drug curcumin thanks to coordination bonds between the central platinum atom of Lx and thioether groups on different polymer chains. The drug release from Lx crosslinked micelles is significantly accelerated under conditions mimicking the intracellular environment due to competitive coordination and subsequent micellar de-crosslinking. Because of their straightforward preparation and favorable drug release characteristics, core-crosslinked Lx PEG-P(HPMA) micelles hold promise as a versatile nanomedicine platform.

Folate-dactolisib conjugates for targeting tubular cells in polycystic kidneys.

The aim of the present study was to develop folic acid (FA) conjugates which can deliver the kinase inhibitor dactolisib to the kidneys via folate receptor-mediated uptake in tubular epithelial cells. Dactolisib is a dual inhibitor of phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) and is considered an attractive agent for treatment of polycystic kidney disease. The ethylenediamine platinum(II) linker, herein called Lx, was employed to couple dactolisib via coordination chemistry to thiol-containing FA-spacer adducts to yield FA-Lx-dactolisib conjugates. The dye lissamine was coupled via similar linker chemistry to folate to yield fluorescent FA-Lx-lissamine conjugates. Three different spacers (PEG5-Cys, PEG27-Cys or an Asp-Arg-Asp-Asp-Cys peptide spacer) were used to compare the influence of hydrophilicity and charged groups in the spacer on interaction with target cells and in vivo organ distribution of the final conjugates. The purity and identity of the final products were confirmed by UPLC and LC-MS analysis, respectively. FA-Lx-dactolisib conjugates were stable in serum and culture medium, while dactolisib was released from the conjugates in the presence of glutathione. All three type of conjugates were internalized efficiently by HK-2 cells and uptake could be blocked by an excess of folic acid in the medium, demonstrating FR mediated uptake. FA-Lx-dactolisib conjugates showed nanomolar inhibition of the PI3K pathway (Akt phosphorylation) and mTOR pathway (S6 phosphorylation) in cultured kidney epithelial cells (HK-2 cells). After intraperitoneal administration, all three types conjugates accumulated extensively in kidneys of iKsp-Pkd1del mice with polycystic kidney disease. In conclusion, folate conjugates were successfully prepared by platinum(II) coordination chemistry and accumulated in a target-specific manner in kidney cells and polycystic kidneys. The folate conjugate of dactolisib thus may have potential for targeted therapy of polycystic kidney disease.

Synthesis and characterization of amino acid substituted sunitinib analogues for the treatment of AML.

Acute myeloid leukemia (AML) is the most common type of leukemia in adults. Sunitinib, a multikinase inhibitor, was the first Fms-like tyrosine kinase 3 (FLT3) inhibitor clinically used against AML. Off-target effects are a major concern for multikinase inhibitors. As targeted delivery may reduce such undesired side effects, our goal was to develop novel amino acid substituted derivatives of sunitinib which are potent candidates to be used conjugated with antibodies and peptides. In the current paper we present the synthesis, physicochemical and in vitro characterization of sixty two Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutant kinase inhibitors, bearing amino acid moieties, fit to be conjugated with peptide-based delivery systems via their carboxyl group. We determined the solubility, pKa, CHI and LogP values of the compounds along with their inhibition potential against FLT3-ITD mutant kinase and on MV4-11 cell line. The ester derivatives of the compounds inhibit the growth of the MV4-11 leukemia cell line at submicromolar concentration.

Adsorption of phospholipids at oil/water interfaces during emulsification is controlled by stress relaxation and diffusion.

Adsorption of phosphatidylcholines at oil/water interfaces strongly deviates from spread monolayers at air/water surfaces. Understanding its nature and consequences could vastly improve applications in medical nanoemulsions and biotechnologies. Adsorption kinetics at interfaces of water with different oil phases were measured by profile analysis tensiometry. Adsorption kinetics for 2 different phospholipids, DPPC and POPC, as well as 2 organic phases, squalene and squalane, show that formation of interfacial monolayers is initially dominated by stress-relaxation in the first minutes. Diffusion only gradually contributes to a decrease in interfacial tension at later stages of time and higher film pressures. The results can be applied for the optimization of emulsification protocols using mechanical treatments. Emulsions using phospholipids with unsaturated fatty acids are dominated much more strongly by stress-relaxation and cover interfaces very fast compared to those with saturated fatty acids. In contrast, phospholipid layers consisting of saturated fatty acids converge faster towards the equilibrium than those with unsaturated fatty acids.

Influence of cholesterol inclusion on the doxorubicin release characteristics of lysolipid-based thermosensitive liposomes.

Fast hyperthermia (i.e. 39-42 °C) triggered doxorubicin release from lysolipid-containing thermosensitive liposomes (LTSL) in the tumor vasculature has been demonstrated to result in considerable enhancement of bioavailable drug levels in heated tumor tissue in preclinical tumor models. However, there is also significant leakage of doxorubicin already at 37 °C in the bloodstream, making these LTSL less efficient and increasing the risk for systemic toxicity. In conventional liposomes, cholesterol is incorporated in the bilayer to increase the stability of the liposomes. Here, we investigate the effect of cholesterol inclusion on the doxorubicin release characteristics of LTSL at 37 °C and hyperthermic temperatures. For this purpose, three LTSL formulations with 0, 5 and 10 mol% cholesterol were prepared. Inclusion of cholesterol reduced the undesired doxorubicin leakage at 37 °C in Hepes-buffered saline (HBS) as well as in fetal bovine serum (FBS). The incorporation of cholesterol in the LTSL bilayers did not influence the hyperthermia-triggered release property of the LTSL. These results were supported by DSC measurements. Therefore, in conclusion, our data indicate that cholesterol inclusion in LTSL offers a simple solution to the problem of significant leakage of doxorubicin from LTSL already at 37 °C in the bloodstream.

Complement activation in vitro and reactogenicity of low-molecular weight dextran-coated SPIONs in the pig CARPA model: Correlation with physicochemical features and clinical information.

The unique magnetic properties of superparamagnetic iron oxide nanoparticles (SPIONs) have led to their increasing use in drug delivery and imaging applications. Some polymer-coated SPIONs, however, share with many other nanoparticles the potential of causing hypersensitivity reactions (HSRs) known as complement (C) activation-related pseudoallergy (CARPA). In order to explore the roles of iron core composition and particle surface coating in SPION-induced CARPA, we measured C activation by 6 different SPIONs in a human serum that is known to react to nanoparticles (NPs) with strong C activation. Remarkably, only the carboxymethyldextran-coated (ferucarbotran, Resosvist®) and dextran-coated (ferumoxtran-10, Sinerem®) SPIONs caused significant C activation, while the citric acid, phosphatidylcholine, starch and chitosan-coated SPIONs had no such effect. Focusing on Resovist and Sinerem, we found Sinerem to be a stronger activator of C than Resovist, although the individual variation in 15 different human sera was substantial. Further analysis of C activation by Sinerem indicated biphasic dose dependence and significant production of C split product Bb but not C4d, attesting to alternative pathway C activation only at low doses. Consistent with the strong C activation by Sinerem and previous reports of HSRs in man, injection of Sinerem in a pig led to dose-dependent CARPA, while Resovist was reaction-free. Using nanoparticle tracking analysis, it was further determined that Sinerem, more than Resovist, displayed multimodal size distribution and significant fraction of aggregates - factors which are known to promote C activation and CARPA. Taken together, our findings offer physicochemical insight into how key compositional factors and nanoparticle size distribution affect SPION-induced CARPA, a knowledge that could lead to the development of SPIONs with improved safety profiles.

Instability Mechanisms of Water-in-Oil Nanoemulsions with Phospholipids: Temporal and Morphological Structures.

Many food preparations, pharmaceuticals, and cosmetics use water-in-oil (W/O) emulsions stabilized by phospholipids. Moreover, recent technological developments try to produce liposomes or lipid coated capsules from W/O emulsions, but are faced with colloidal instabilities. To explore these instability mechanisms, emulsification by sonication was applied in three cycles, and the sample stability was studied for 3 h after each cycle. Clearly identifiable temporal structures of instability provide evidence about the emulsion morphology: an initial regime of about 10 min is shown to be governed by coalescence after which Ostwald ripening dominates. Transport via molecular diffusion in Ostwald ripening is commonly based on the mutual solubility of the two phases and is therefore prohibited in emulsions composed of immiscible phases. However, in the case of water in oil emulsified by phospholipids, these form water-loaded reverse micelles in oil, which enable Ostwald ripening despite the low solubility of water in oil, as is shown for squalene. As is proved for the phospholipid dipalmitoylphosphatidylcholine (DPPC), concentrations below the critical aggregation concentration (CAC) form monolayers at the interfaces and smaller droplet sizes. In contrast, phospholipid concentrations above the CAC create complex multilayers at the interface with larger droplet sizes. The key factors for stable W/O emulsions in classical or innovative applications are first, the minimization of the phospholipids' capacity to form reversed micelles, and second, the adaption of the initial phospholipid concentration to the water content to enable an optimized coverage of phospholipids at the interfaces for the intended drop size.

PulmoStent: In Vitro to In Vivo Evaluation of a Tissue Engineered Endobronchial Stent.

Currently, there is no optimal treatment available for end stage tumour patients with airway stenosis. The PulmoStent concept aims on overcoming current hurdles in airway stenting by combining a nitinol stent with a nutrient-permeable membrane, which prevents tumour ingrowth. Respiratory epithelial cells can be seeded onto the cover to restore mucociliary clearance. In this study, a novel hand-braided dog bone stent was developed, covered with a polycarbonate urethane nonwoven and mechanically tested. Design and manufacturing of stent and cover were improved in an iterative process according to predefined requirements for permeability and mechanical properties and finally tested in a proof of concept animal study in sheep for up to 24 weeks. In each animal two stents were implanted, one of which was cell-seeded by endoscopic spraying in situ. We demonstrated the suitability of this membrane for our concept by glucose transport testing and in vitro culture of respiratory epithelial cells. In the animal study, no migration occurred in any of the twelve stents. There was only mild granulation tissue formation and tissue reaction; no severe mucus plugging was observed. Thus, the PulmoStent concept might be a step forward for palliative treatment of airway stenosis with a biohybrid stent device.

Selection and fabrication of a non-woven polycarbonate urethane cover for a tissue engineered airway stent.

One of the major problems in end-stage bronchotracheal cancer is stenosis of the upper airways, either due to luminal ingrowth of the tumor or mucus plugging. Airway stents that suppress tumor ingrowth and sustain mucociliary transport can alleviate these problems in end-stage bronchial cancer. We evaluated different types of polymeric covers for a tissue engineered airway stent. The distinguishing feature of this stent concept is that respiratory epithelial cells can grow on the luminal surface of the stent which facilitates mucociliary clearance. To facilitate growth of epithelial cells at the air-liquid interface of the stent, we developed a polyurethane cover that allows transport of nutrients to the cells. Nonwoven polycarbonate urethane (PCU) covers were prepared by a spraying process and evaluated for their porosity and glucose permeability. Respiratory epithelial cells harvested from sheep trachea were cultured onto the selected PCU cover and remained viable at the air-liquid interface when cultured for 21days. Lastly, we evaluated the radial force of a PCU-covered nitinol stent, and showed the PCU covers did not adversely affect the mechanical properties of the stents for their intended application in the smaller bronchi. These in vitro data corroborate the design of a novel airway stent for palliative treatment of bronchotracheal stenosis by combination of stent-technology with tissue-engineered epithelial cells.

Gene based therapies for kidney regeneration.

In this review we provide an overview of the expanding molecular toolbox that is available for gene based therapies and how these therapies can be used for a large variety of kidney diseases. Gene based therapies range from restoring gene function in genetic kidney diseases to steering complex molecular pathways in chronic kidney disorders, and can provide a treatment or cure for diseases that otherwise may not be targeted. This approach involves the delivery of recombinant DNA sequences harboring therapeutic genes to improve cell function and thereby promote kidney regeneration. Depending on the therapy, the recombinant DNA will express a gene that directly plays a role in the function of the cell (gene addition), that regulates the expression of an endogenous gene (gene regulation), or that even changes the DNA sequence of endogenous genes (gene editing). Some interventions involve permanent changes in the genome whereas others are only temporary and leave no trace. Efficient and safe delivery are important steps for all gene based therapies and also depend on the mode of action of the therapeutic gene. Here we provide examples on how the different methods can be used to treat various diseases, which technologies are now emerging (such as gene repair through CRISPR/Cas9) and what the opportunities, perspectives, potential and the limitations of these therapies are for the treatment of kidney diseases.

Inhibition of Octreotide Acylation Inside PLGA Microspheres by Derivatization of the Amines of the Peptide with a Self-Immolative Protecting Group.

Acylation of biopharmaceuticals such as peptides has been identified as a major obstacle for the successful development of PLGA controlled release formulations. The purpose of this study was to develop a method to inhibit peptide acylation in poly(d,l-lactide-co-glycolide) (PLGA) formulations by reversibly and temporarily blocking the amine groups of a model peptide (octreotide) with a self-immolative protecting group (SIP), O-4-nitrophenyl-O'-4-acetoxybenzyl carbonate. The octreotide with two self-immolative protecting groups (OctdiSIP) on the N-terminus and lysine side chain was synthesized by reaction of the peptide with O-4-nitrophenyl-O'-4-acetoxybenzyl carbonate, purified by preparative RP-HPLC and characterized by mass spectrometry. Degradation studies of OctdiSIP in aqueous solutions of different pH values showed that protected octreotide was stable at low pH (pH 5) whereas the protecting group was eliminated at physiological pH, especially in the presence of an esterase, to generate native octreotide. OctdiSIP encapsulated in PLGA microspheres, prepared using a double emulsion solvent evaporation method, showed substantial inhibition of acylation as compared to the unprotected octreotide: 52.5% of unprotected octreotide was acylated after 50 days incubation of microspheres in PBS pH 7.4 at 37 °C, whereas OctdiSIP showed only 5.0% acylation in the same time frame. In conclusion, the incorporation of self-immolative protection groups provides a viable approach for inhibition of acylation of peptides in PLGA delivery systems.

Elevated Urinary Connective Tissue Growth Factor in Diabetic Nephropathy Is Caused by Local Production and Tubular Dysfunction.

Connective tissue growth factor (CTGF; CCN2) plays a role in the development of diabetic nephropathy (DN). Urinary CTGF (uCTGF) is elevated in DN patients and has been proposed as a biomarker for disease progression, but it is unknown which pathophysiological factors contribute to elevated uCTGF. We studied renal handling of CTGF by infusion of recombinant CTGF in diabetic mice. In addition, uCTGF was measured in type 1 DN patients and compared with glomerular and tubular dysfunction and damage markers. In diabetic mice, uCTGF was increased and fractional excretion (FE) of recombinant CTGF was substantially elevated indicating reduced tubular reabsorption. FE of recombinant CTGF correlated with excretion of endogenous CTGF. CTGF mRNA was mainly localized in glomeruli and medullary tubules. Comparison of FE of endogenous and recombinant CTGF indicated that 60% of uCTGF had a direct renal source, while 40% originated from plasma CTGF. In DN patients, uCTGF was independently associated with markers of proximal and distal tubular dysfunction and damage. In conclusion, uCTGF in DN is elevated as a result of both increased local production and reduced reabsorption due to tubular dysfunction. We submit that uCTGF is a biomarker reflecting both glomerular and tubulointerstitial hallmarks of diabetic kidney disease.

Local therapeutic efficacy with reduced systemic side effects by rapamycin-loaded subcapsular microspheres.

Kidney injury triggers fibrosis, the final common pathway of chronic kidney disease (CKD). The increase of CKD prevalence worldwide urgently calls for new therapies. Available systemic treatment such as rapamycin are associated with serious side effects. To study the potential of local antifibrotic therapy, we administered rapamycin-loaded microspheres under the kidney capsule of ureter-obstructed rats and assessed the local antifibrotic effects and systemic side effects of rapamycin. After 7 days, microsphere depots were easily identifiable under the kidney capsule. Both systemic and local rapamycin treatment reduced intrarenal mTOR activity, myofibroblast accumulation, expression of fibrotic genes, and T-lymphocyte infiltration. Upon local treatment, inhibition of mTOR activity and reduction of myofibroblast accumulation were limited to the immediate vicinity of the subcapsular pocket, while reduction of T-cell infiltration was widespread. In contrast to systemically administered rapamycin, local treatment did not induce off target effects such as weight loss. Thus subcapsular delivery of rapamycin-loaded microspheres successfully inhibited local fibrotic response in UUO with less systemic effects. Therapeutic effect of released rapamycin was most prominent in close vicinity to the implanted microspheres.

Features of complement activation-related pseudoallergy to liposomes with different surface charge and PEGylation: comparison of the porcine and rat responses.

Pigs are known to provide a sensitive model for studying complement (C) activation-related pseudoallergy (CARPA), a hypersensitivity reaction to liposomal and many other nanomedicines that limits their clinical use. The utility of rats as a CARPA model has, however, not been analyzed to date in detail. The present study compared the two models by inducing CARPA with i.v. bolus injections of two reactogenic liposomes that differed from each other in surface properties: one was AmBisome, a strong anionic, free-surface small unilamellar liposome (SUV), while the other was neutral, polyethylene glycol (PEG)-grafted SUV wherein the 2 kDa-PEG was anchored to the membrane via cholesterol (Chol-PEG). Both in pigs and rats AmBisome caused significant consumption of C3, indicating C activation, along with paralleling massive changes in blood pressure, white blood cell, platelet counts and in plasma thromboxane B2 levels, indicating CARPA. These processes were similar in the two species in terms of kinetics, but significantly differed in the doses that caused major hemodynamic changes (~0.01 and ~22 mg phospholipid (PL)/kg in pigs and rats, respectively). Pigs responded to AmBisome with pulmonary hypertension and systemic hypotension, and the reaction was not tachyphylactic. The major response of rats was systemic hypotension, leukopenia followed by leukocytosis, and thrombocytopenia. Chol-PEG liposomes caused severe reaction in pigs at 0.1 mg/kg, while the reaction they caused in rats was mild even at 300 mg PL/kg. Importantly, the reaction to Chol-PEG in pigs was partly tachyphylactic. These observations highlight fundamental differences in the immune mechanisms of porcine and rat CARPA, and also show a major impact of liposome surface characteristics, determining the presence or absence of tachyphylaxis. The data suggest that rats are 2-3 orders of magnitude less sensitive to liposomal CARPA than pigs; however, the causes of these differences, the PEG-dependent tachyphylaxis and the massive reactivity of Chol-PEG liposomes remain unclear.

Release behavior and intra-articular biocompatibility of celecoxib-loaded acetyl-capped PCLA-PEG-PCLA thermogels.

In this study, we investigated the in vitro and in vivo properties and performance of a celecoxib-loaded hydrogel based on a fully acetyl-capped PCLA-PEG-PCLA triblock copolymer. Blends of different compositions of celocoxib, a drug used for pain management in osteoarthritis, and the acetyl-capped PCLA-PEG-PCLA triblock copolymer were mixed with buffer to yield temperature-responsive gelling systems. These systems containing up to 50 mg celecoxib/g gel, were sols at room temperature and converted into immobile gels at 37 °C. In vitro, release of celecoxib started after a ∼10-day lag phase followed by a sustained release of ∼90 days. The release was proven to be mediated by polymer dissolution from the gels. In vivo (subcutaneous injection in rats) experiments showed an initial celecoxib release of ∼30% during the first 3 days followed by a sustained release of celecoxib for 4-8 weeks. The absence of a lag phase and the faster release seen in vivo were likely due to the enhanced celecoxib solubility in biological fluids and active degradation of the gel by macrophages. Finally, intra-articular biocompatibility of the 50 mg/g celecoxib-loaded gel was demonstrated using µCT-scanning and histology, where no cartilage or bone changes were observed following injection into the knee joints of healthy rats. In conclusion, this study shows that celecoxib-loaded acetyl-capped PCLA-PEG-PCLA hydrogels form a safe drug delivery platform for sustained intra-articular release.

Computer modeling assisted design of monodisperse PLGA microspheres with controlled porosity affords zero order release of an encapsulated macromolecule for 3 months.

PURPOSE: The aim of this study was the development of poly(D,L-lactide-co-glycolide) (PLGA) microspheres with controlled porosity, to obtain microspheres that afford continuous release of a macromolecular model compound (blue dextran). METHODS: PLGA microspheres with a size of around 40 µm and narrow size distribution (span value of 0.3) were prepared with a double emulsion membrane emulsification method. Gene expression programming (GEP) analysis was applied to design and formulate a batch of microspheres with controlled porosity that shows continuous release of blue dextran. RESULTS: Low porous microspheres with a high loading efficiency were formed at high polymer concentrations (30% w/w in the oil phase) and were characterized with a burst release <10% and a three-phasic release profile of blue dextran. Increasing porosity (10% w/w polymer concentrations), a sustained release of blue dextran was obtained albeit with up to 40% of burst release. The desired formulation, calculated by GEP, resulted in microspheres with 72% loading efficiency and intermediate porosity. Blue dextran was indeed released continuously in almost a zero order manner over a period of 3 months after an initial small burst release of 9%. CONCLUSIONS: By fine-tuning the porosity, the release profile of PLGA microspheres for macromolecules can be predicted and changed from a three-phasic to a continuous release.

Targeted inhibition of renal Rho kinase reduces macrophage infiltration and lymphangiogenesis in acute renal allograft rejection.

The Rho kinase pathway plays an important role in epithelial dedifferentiation and inflammatory cell infiltration. Recent studies suggest that inflammation promotes lymphangiogenesis, which has been associated with renal allograft rejection. We investigated whether targeted inhibition of the Rho kinase pathway in proximal tubular cells reduces inflammation and lymphangiogenesis in acute renal allograft rejection. The Rho kinase inhibitor Y27632 was coupled to lysozyme (Y27632-lysozyme), providing a kidney-specific conjugate that can release its drug in proximal tubular cells. Isogenic (Fisher-Fisher, n=18), or allogenic (Fisher-Lewis, n=24) kidney transplantations were performed, with the contralateral kidney remaining in situ. To elicit acute rejection, no immunosuppressive treatment was given. Animals were treated daily with Y27632-lysozyme (10 mg/kg/day i.v.) or vehicle (saline i.v.) until sacrifice (1 or 4 days post-transplantation). After allogenic transplantation, interstitial macrophage accumulation was strongly reduced by Y27632-lysozyme at day 4 after transplantation. Interstitial lymphangiogenesis, which was induced in allografts as compared to control kidney, was also reduced by renal Rho kinase inhibition at day 4 after transplantation. The increase of vimentin and procollagen-1alpha1 gene expression in renal allografts from day 1 to day 4 after transplantation was significantly reduced by Y27632-lysozyme. Y27632-lysozyme did not affect systolic blood pressure in isogenic or allogenic transplantation groups. In cultured tubular epithelial cells (NRK-52E), Rho kinase inhibition dose-dependently reduced IL-1ß-induced MCP-1 gene expression. Renal inhibition of Rho kinase causes a marked reduction in renal inflammation and renal lymphangiogenesis during acute transplant rejection, suggesting that this treatment regimen is a valuable future treatment in renal transplantation.