Intranasal delivery of N-terminal modified leptin-pluronic conjugate for treatment of obesity.

Leptin is an adipocyte-secreted hormone that is delivered via a specific transport system across the blood-brain barrier (BBB) to the brain where it acts on the hypothalamus receptors to control appetite and thermogenesis. Peripheral resistance to leptin due to its impaired brain delivery prevents therapeutic use of leptin in overweight and moderately obese patients. To address this problem, we modified the N-terminal amine of leptin with Pluronic P85 (LepNP85) and administered this conjugate intranasally using the nose-to-brain (INB) route to bypass the BBB. We compared this conjugate with the native leptin, the N-terminal leptin conjugate with poly(ethylene glycol) (LepNPEG5K), and two conjugates of leptin with Pluronic P85 attached randomly to the lysine amino groups of the hormone. Compared to the random conjugates of leptin with P85, LepNP85 has shown higher affinity upon binding with the leptin receptor, and similarly to native hormone activated hypothalamus receptors after direct injection into brain. After INB delivery, LepNP85 conjugate was transported to the brain and accumulated in the hypothalamus and hippocampus to a greater extent than the native leptin and LepNPEG5K and activated leptin receptors in hypothalamus at lower dose than native leptin. Our work suggests that LepNP85 can access the brain directly after INB delivery and confirms our hypothesis that the improvement in brain accumulation of this conjugate is due to its enhanced brain absorption. In conclusion, the LepNP85 with optimized conjugation chemistry is a promising candidate for treatment of obesity.

Multifunctional theranostic Pluronic mixed micelles improve targeted photoactivity of Verteporfin in cancer cells.

Nanotechnology development provides new strategies to treat cancer by integration of different treatment modalities in a single multifunctional nanoparticle. In this scenario, we applied the multifunctional Pluronic P123/F127 mixed micelles for Verteporfin-mediated photodynamic therapy in PC3 and MCF-7 cancer cells. Micelles functionalization aimed the targeted delivery by the insertion of biotin moiety on micelle surface and fluorescence image-based through rhodamine-B dye conjugation in the polymer chains. Multifunctional Pluronics formed spherical nanoparticulated micelles that efficiently encapsulated the photosensitizer Verteporfin maintaining its favorable photophysical properties. Lyophilized formulations were stable at least for 6months and readily reconstituted in aqueous media. The multifunctional micelles were stable in protein-rich media due to the dual Pluronic mixed micelles characteristic: high drug loading capacity provided by its micellar core and high kinetic stability due its biocompatible shell. Biotin surface functionalized micelles showed higher internalization rates due biotin-mediated endocytosis, as demonstrated by competitive cellular uptake studies. Rhodamine B-tagged micelles allowed monitoring cellular uptake and intracellular distribution of the formulations. Confocal microscopy studies demonstrated a larger intracellular distribution of the formulation and photosensitizer, which could drive Verteporfin to act on multiple cell sites. Formulations were not toxic in the dark condition, but showed high Verteporfin-induced phototoxicity against both cancer cell lines at low drug and light doses. These results point Verteporfin-loaded multifunctional micelles as a promising tool to further developments in photodynamic therapy of cancer.

pH-responsive copolymers based on pluronic P123-poly(ß-amino ester): Synthesis, characterization and application of copolymer micelles.

A novel amphiphilic and pH-responsive copolymer, pluronie P123-poly(ß-amino ester) (P123-PAE), was firstly designed and synthesized using a Michael-type step polymerization. Nano-sized polymeric micelles based on P123-PAE block copolymer were prepared by self-assembly. Curcumin (Cur), a potential cancer therapy drug, was efficiently encapsulated into the P123-PAE micelles to enhance anticancer efficacy. The obtained Cur loaded P123-PAE micelles (Cur-P123-PAE) presented a spherical shape and high drug loading (18.4%). Interestingly, when the media pH decreased from 7.4 to 5.5, the particle size of the micelles shrank from 152.5nm to 122.1nm due to the protonation of PAE blocks, and the zeta potential of the P123-PAE micelles changed from weakly positive (1.5mV) to highly positive (9.0mV) over a pH range from 7.4 to 5.5. In vitro drug release studies demonstrated that the release rate of Cur was markedly influenced by pH. In vitro cytotoxicity tests showed that all the blank micelles were non-toxic. Cur-P123-PAE exhibited similar antitumor effect against MCF-7 and HepG2 cells compared to solubilized Cur solution. Using Coumarin-6 as a fluorescence probe, it was observed that Cur-P123-PAE micelles experienced longer circulation followed by accumulation at tumor tissues with stronger fluorescence intensity. The results of pharmacokinetics studies showed that the P123-PAE micelles could significantly prolong the retention time of Cur in vivo.

Enhanced brain delivery of lamotrigine with Pluronic(®) P123-based nanocarrier.

BACKGROUND: P-glycoprotein (P-gp) mediated drug efflux across the blood-brain barrier (BBB) is an important mechanism underlying poor brain penetration of certain antiepileptic drugs (AEDs). Nanomaterials, as drug carriers, can overcome P-gp activity and improve the targeted delivery of AEDs. However, their applications in the delivery of AEDs have not been adequately investigated. The objective of this study was to develop a nano-scale delivery system to improve the solubility and brain penetration of the antiepileptic drug lamotrigine (LTG). METHODS: LTG-loaded Pluronic(®) P123 (P123) polymeric micelles (P123/LTG) were prepared by thin-film hydration, and brain penetration capability of the nanocarrier was evaluated. RESULTS: The mean encapsulating efficiency for the optimized formulation was 98.07%; drug-loading was 5.63%, and particle size was 18.73 nm. The solubility of LTG in P123/LTG can increase to 2.17 mg/mL, making it available as a solution. The in vitro release of LTG from P123LTG presented a sustained-release property. Compared with free LTG, the LTG-incorporated micelles accumulated more in the brain at 0.5, 1, and 4 hours after intravenous administration in rats. Pretreatment with systemic verapamil increased the rapid brain penetration of free LTG but not P123/LTG. Incorporating another P-gp substrate (Rhodamine 123) into P123 micelles also showed higher efficiency in penetrating the BBB in vitro and in vivo. CONCLUSION: These results indicated that P123 micelles have the potential to overcome the activity of P-gp expressed on the BBB and therefore show potential for the targeted delivery of AEDs. Future studies are necessary to further evaluate the appropriateness of the nanocarrier to enhance the efficacy of AEDs.

[Blood plasma protein adsorption capacity of perfluorocarbon emulsion stabilized by proxanol 268 (in vitro and in vivo studies)].

The adsorption abilities of the perfluorocarbon emulsion stabilized by Proxanol 268 were investigated in vitro and in vivo. In vitro, the saturation point for the blood plasma proteins was nearly reached after five minutes of incubation of the emulsion with human/rabbit blood plasma and was stable for all incubation periods studied. The decrease in volume ratio (emulsion/plasma) was accompanied by the increase in the adsorptive capacity of the emulsion with maximal values at 1/10 (3.2 and 1.5 mg of proteins per 1 ml of the emulsion, for human and rabbit blood plasma, respectively) that was unchanged at lower ratios. In vivo, in rabbits, intravenously injected with the emulsion, the proteins with molecular masses of 12, 25, 32, 44, 55, 70, and 200 kDa were adsorbed by the emulsion (as in vitro) if it was used 6 hours or less before testing. More delayed testing (6 h) revealed elimination of proteins with molecular masses of 25 and 44 kDa and an additional pool of adsorpted new ones of 27, 50, and 150 kDa. Specific adsorptive capacity of the emulsion enhanced gradually after emulsion injection and reached its maximum (3.5-5 mg of proteins per 1 ml of the emulsion) after 24 hours.

Influence of Pluronic 85 and ketoconazole on disposition and efficacy of ivermectin in sheep infected with a multiple resistant Haemonchus contortus isolate.

Non-specific mechanisms involving ATP-binding cassette drug efflux transporters may play an important role in xenobiotic clearance in ovine gastro-intestinal nematodes. By using transporter inhibitors, the aim of this trial was to assess the possibility of increasing drug bioavailability in the host in an attempt to improve treatment efficacy. Thirty-six lambs were infected with 5000 multiple-drug resistant Haemonchus contortus third stage larvae and separated into six groups (n=6): ivermectin alone (IVM; 0.2 mg/kg body-weight, BW), ketoconazole alone (KET; 10 mg/kg BW), Pluronic 85 alone (P85; 4 mg/kg BW), IVM+KET, IVM+P85 or untreated control. Ivermectin was administered once on day 28 post-infection for all appropriate groups, whereas KET and P85 were administered as five separate doses on day 26-30 post-infection inclusive. The resultant data showed that concomitant administration of KET or P85 with IVM induced increases in plasma and tissue concentrations of IVM in treated animals, resulting in a two-fold increase in the area under the time-concentration curve (p<0.05). Faecal egg counts and worm burdens of the IVM+KET and IVM+P85 groups were lower than in the untreated, KET and P85 alone control animals. Worm burdens were reduced by between 16% and 51% with IVM+KET and IVM+P85 respectively compared to untreated control animals. The co-administration of P85 with IVM increased the efficacy by 34%, compared with IVM alone, in terms of worm count reduction of the multi-resistant isolate of H. contortus.

Protein conjugation with amphiphilic block copolymers for enhanced cellular delivery.

Modification of a model protein, horseradish peroxidase (HRP), with amphiphilic block copolymer poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (Pluronic), was previously shown to enhance the transport of this protein across the blood-brain barrier in vivo and brain microvessel endothelial cells in vitro. This work develops procedures for synthesis and characterization of HRP with Pluronic copolymers, having different lengths of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) blocks. Four monoamine Pluronic derivatives (L81, P85, L121, P123) were synthesized and successfully conjugated to a model protein, HRP, via biodegradable or nondegradable linkers (dithiobis(succinimidyl propionate) (DSP), dimethyl 3,3'-dithiobispropionimidate (DTBP), and disuccinimidyl propionate (DSS)). The conjugation was confirmed by HRP amino group titration, matrix-assisted laser desorption/ionization-time of flight spectroscopy, and cation-exchange chromatography. HRP conjugates containing an average of one to two Pluronic moieties and retaining in most cases over 70% of the activity were synthesized. Increased cellular uptake of these conjugates was demonstrated using the Mardin-Derby canine kidney cell line and primary bovine brain microvessel endothelial cells. The optimal modifications included Pluronic L81 and P85. These copolymers have shorter PPO chains compared to Pluronic P123 and L121, which were less efficient. There was little if any dependence of the uptake on the length of the hydrophilic PEO block for the optimal modifications. The proposed modifications may be used to increase cellular uptake of other proteins.

Complexation and release of doxorubicin from its complexes with pluronic P85-b-poly(acrylic acid) block copolymers.

Poly(acrylic acid) (PAA) was attached on both termini of Pluronic P85 copolymer (EO27PO39EO27) via atom transfer radical polymerization (ATRP) to produce a novel block copolymer, PAA-b-P85-b-PAA (P85PAA). The P85PAA-DOX complex formation and drug loading were strongly dependent on the PAA segment length and pH, where the protonation of carboxyl groups in the PAA segment at pH < 7.2 reduced the binding sites of DOX onto P85PAA chains, resulting in a diminished DOX uptake at low pH. The composition of copolymer-DOX complexes at pH 7.2 was close to the stoichiometric 1:1 DOX:carboxyl molar ratio, confirming the dominance of electrostatic interactions between cationic DOX molecules and carboxyl groups. The stability study of the copolymer-DOX complex suggested that non-polyelectrolyte interactions may also participate in the complexation of drug and P85PAA block copolymer. DOX loading at pH 5.0 decreased to 60% of the total binding capacity, indicating that protonation of carboxyl groups reduced the DOX binding to P85PAA block copolymer. DOX release from the complex is a pH-responsive process, where the protonation of carboxyl groups at mildly acidic condition resulted in a faster dissociation of copolymer-DOX complex, leading to an accelerated release of DOX at pH 5.0. Thus, complexation of DOX with P85PAA yielded a drug delivery system affording a pH-triggered release of DOX in an acidic environment of pH 5.0.

Distribution kinetics of a micelle-forming block copolymer Pluronic P85.

Pluronic block copolymers, micelle-forming polymeric surfactants, are currently being evaluated in chemotherapy clinical trials in combination with doxorubicin to treat multidrug-resistant (MDR) tumors. This study examines the pharmacokinetics and biodistribution of Pluronic P85 (P85), a potent inhibitor of P-glycoprotein (Pgp). P85 was radioactively labeled and administered intravenously (i.v.) to mice. The concentration of the copolymer was varied to examine the effects of micelle formation on the distribution kinetics. The main pharmacokinetic parameters (the area under the curve, half-life, clearance, mean residence time, and volume of distribution) were determined. The results suggest that half-life of P85 varies from 60 to 90 h, depending on its aggregation state. Formation of micelles decreased the uptake of the block copolymer in the liver. However, it had no effect on the total clearance, suggesting that the elimination of P85 was controlled by the renal elimination of P85 unimers and not by the rate of micelle disposition or disintegration. The total clearance value suggests that a significant portion of P85 is reabsorbed back into the blood, probably through the kidney's tubular membranes.

Altered organ accumulation of oligonucleotides using polyethyleneimine grafted with poly(ethylene oxide) or pluronic as carriers.

Passive targeting provides a simple strategy based on natural properties of the carriers to deliver DNA molecules to desired compartments. Polyethylenimine (PEI) is a potent non-viral system that has been known to deliver efficiently both plasmids and oligonucleotides (ODNs) in vitro. However, in vivo systemic administration of DNA/PEI complexes has encountered significant difficulties because these complexes are toxic and have low biodistribution in target tissues. This study evaluates PEI grafted with poly(ethylene oxide) (PEO(8K)-g-PEI(2K)) and PEI grafted with non-ionic amphiphilic block copolymer, Pluronic P85 (P85-g-PEI(2K)) as carriers for systemic delivery of ODNs. Following i.v. injection an antisense ODN formulated with PEO(8K)-g-PEI(2K) accumulated mainly in kidneys, while the same ODN formulated with P85-g-PEI(2K) was found almost exclusively in the liver. Furthermore, in the case of the animals injected with the P85-g-PEI(2K)-based complexes most of the ODN was found in hepatocytes, while only a minor portion of ODN was found in the lymphocyte/monocyte populations. The results of this study suggest that formulating ODN with PEO(8K)-g-PEI(2K) and P85-g-PEI(2K) carriers allows targeting of the ODN to the liver or kidneys, respectively. The variation in the tissue distribution of ODN observed with the two carriers is probably due to the different hydrophilic-lipophilic balance of the polyether chains grafted to PEI in these molecules. Therefore, polyether-grafted PEI carriers provide a simple way to enhance ODN accumulation in a desired compartment without the need of a specific targeting moiety.

Pharmacokinetics of RheothRx injection in healthy male volunteers.

The objectives of this study were to evaluate the safety and tolerability of RheothRx (poloxamer 188) injection administered as an intravenous (i.v.) infusion to healthy male volunteers and to determine the pharmacokinetic profile of poloxamer 188. Thirty-six healthy male volunteers were enrolled in a randomized, double-blind, placebo-controlled, dose-escalation trial for RheothRx injection. The volunteers were randomized to three treatment groups (12 per treatment group, with eight receiving active therapy and four receiving placebo). In each treatment group, volunteers received RheothRx injection or placebo as an i.v. infusion on two occasions at least 3 weeks apart to make a total of six doses being studied (10, 30, and 45 mg/kg/h for 72 h, 60 mg/kg/h for 43.3 to 72 h, 60 and 90 mg/kg/h for 24 h). Serial plasma samples were collected during and up to 36 h after the end of the infusions; urine was collected over intervals from the start of the infusion until 36 h after the infusions were terminated. Plasma and urine samples were assayed for poloxamer 188 by gel-permeation chromatography. Pharmacokinetic parameter values were calculated by noncompartmental and compartmental methods. Poloxamer 188 was eliminated primarily by renal excretion. Estimates of clearance, elimination rate constant, and apparent volume of distribution at steady state values were independent of infusion rate. Poloxamer 188 displayed no apparent infusion rate dependence in its pharmacokinetics.

The in vitro characterisation and biodistribution of some non-ionic surfactant coated liposomes in the rabbit.

The degree of adsorption of some novel silicone glycol copolymers onto polystyrene microspheres was studied and compared with the sorption onto small unilamellar vesicles (SUVs) composed of egg phosphatidylcholine (EPC) and prepared by the detergent dialysis technique. These non-ionic surfactants are 'comb' polymers of the ABn type where A is a silicone chain with n pendant polyglycol chains (B). Photon correlation spectroscopy was used to measure the adsorbed layer thickness (delta h) following polymer sorption from aqueous solutions. delta h on latex particles was a function of the length of the polymer hydrophilic chains. Upon incubation with SUVs, delta h of the different polymers was similar (3 nm) and significantly less (two sample t-test, p < 0.01) than the corresponding delta h on the polystyrene latex which could be attributed to the penetration of the polymers into the outer phospholipid bilayer. The glycol chains of the silicone polymers are assumed to be in a helical and planar position. Efflux of 5(6)-carboxyfluorescein from EPC liposomes was increased by the presence of these polymers. The highest retention (49% at 5 h) was obtained with SUVs coated with the silicone polymer possessing the highest glycol content and the longest ethylene oxide chains. Sterically stabilised vesicles were also formed by coating dipalmitoyl phosphatidyl-choline (DPPC)/cholesterol (Chol) (molar ratio 1:1) with two of these silicone glycol copolymers and Poloxamer 338. The liposomes were labelled with 67gallium-desferrioxamine (67Ga-DF). Incubation of radiolabelled Poloxamer 338-coated vesicles in saline or serum at 37 degrees C for 24 h resulted in less stable liposomes compared to the more stable non-coated or silicone coated vesicles. Following intravenous (i.v.) administration in rabbits, free 67Ga-DF rapidly disappeared from the circulation (half-life = 41.4 min) and accumulated in the bladder. Two populations of vesicles were prepared (136 +/- 2.9 nm and 100 +/- 1.4 nm). 24 h after i.v. injection of the different formulations of the 100 nm liposomes in rabbits, 20-27% of the activity was retained in blood. The silicone polymer with the highest glycol content and the longest ethylene oxide chains showed the longest half-life (21.4 h). Using gamma scintigraphy, the liver/spleen uptake of the 136 nm non-coated vesicles was 57% which was significantly reduced to 37% upon coating the liposomes with the silicone glycol copolymers. At 30 min post i.v. injection, approximately 10% of the activity was associated with the heart/lung region irrespective of liposome size or polymer coating.(ABSTRACT TRUNCATED AT 400 WORDS)

[Evaluation of pluronic F127 as a base for gradual release of anticancer drug].

The property of pluronic F127 (PLF) as a base for an anticancer agent, mitomycin C (MMC) was evaluated. A weighed amount of PLF and MMC were slowly added to cold water and mixed completely. The solution was warmed to 37 degrees C and MMC containing PLF gel was formed. The cumulative amount of MMC which was released from the gel during a 3-hour period was calculated. 5.5 percent of MMC was released from PLF-MMC gel which contained 20 or 25% PLF and 3.8% MMC from PLF-MMC gel containing 30% PLF. Intraperitoneal (i.p.) administration of large amount of PLF gel alone in rabbits or mouse revealed severe hepatorenal toxicity, and acute i.p. administered LD50 of PLF was supposed to be between 1.7 g and 5.0 g/kg body weight in mouse. Further evaluation of gradual releasing property of PLF is needed.