Absorption, metabolism and excretion of once-weekly somapacitan, a long-acting growth hormone derivative, after single subcutaneous dosing in human subjects.

Somapacitan is a reversible albumin-binding growth hormone (GH) derivative in clinical development for once-weekly administration in patients with adult GH deficiency (AGHD) and children with GH deficiency (GHD). To date, the use of somapacitan in AGHD or severe AGHD has been approved in the USA and Japan, respectively. This study (ClinicalTrials.gov, NCT02962440) investigated the absorption, metabolism and excretion, as well as the pharmacokinetics (PK), of tritium-labelled somapacitan ([3H]-somapacitan). Seven healthy males received a single subcutaneous dose of 6 mg somapacitan containing [3H]-somapacitan 20 MBq. Blood, serum, plasma, urine, faeces, and expired air were collected for radioactivity assessment. Metabolites were identified and quantified in plasma and urine collected. The PK of plasma components were determined, and the radioactive peaks of the most abundant plasma metabolites and urine metabolites were selected for analysis. Twenty-eight days after dosing, 94.0% of the administered dose was recovered as [3H]-somapacitan-related material, most of which was excreted in urine (80.9%); 12.9% was excreted in faeces, and an insignificant amount (0.2%) was exhaled in expired air. PK properties of [3H]-somapacitan-related material appeared to be consistent across plasma, serum and blood. Three abundant plasma metabolites (P1, M1 and M1B) and two abundant urine metabolites (M4 and M5) were identified. The total exposure of intact somapacitan accounted for 59% of the total exposure of all somapacitan-related material, P1 accounted for 21% and M1 plus M1B accounted for 12%. M4 and M5 were the most abundant urine metabolites and accounted for 37% and 8% of the dosed [3H]-somapacitan radioactivity, respectively. No intact somapacitan was found in excreta. Two subjects had six adverse events (AEs); all were mild in severity and unlikely to be related to trial product. The majority of dosed [3H]-somapacitan (94%) was recovered as excreted metabolites. Urine was the major route for excretion of somapacitan metabolites, followed by faeces, and exhalation in expired air was negligible. The low molecular weights of identified urine metabolites demonstrate that somapacitan was extensively degraded to small residual fragments that were excreted (fully biodegradable). The extensive metabolic degradation and full elimination of metabolites in excreta were the major clearance pathways of somapacitan and the key elements in its biological fate. A single dose of 6 mg somapacitan (containing [3H]-somapacitan) in healthy male subjects was well tolerated with no unexpected safety issues identified.

Bioinspired Microenvironment Responsive Nanoprodrug as an Efficient Hydrophobic Drug Self-Delivery System for Cancer Therapy.

Artemisinin compounds have shown satisfactory safety records in anti-malarial clinical practice over decades and have revealed value as inexpensive anti-tumor adjuvant chemotherapeutic drugs. However, the rational design and precise preparation of nanomedicines based on the artemisinin drugs are still limited due to their non-aromatic and fragile chemical structure. Herein, a bioinspired coordination-driven self-assembly strategy was developed to manufacture the artemisinin-based nanoprodrug with a significantly increased drug loading efficacy (∼70 wt %) and decreased preparation complexity compared to conventional nanodrugs. The nanoprodrug has suitable size distribution and robust colloidal stability for cancer targeting in vivo. The nanoprodrug was able to quickly disassemble in the tumor microenvironment with weak acidity and a high glutathione concentration, which guarantees a better tumor inhibitory effect than direct administration and fewer side effects on normal tissues in vivo. This work highlights a new strategy to harness a robust, simplified, organic solvent-free, and highly repeatable route for nanoprodrug manufacturing, which may offer opportunities to develop cost-effective, safe, and clinically available nanomedicines.

Capabilities of selenoneine to cross the in vitro blood-brain barrier model.

The naturally occurring selenoneine (SeN), the selenium analogue of the sulfur-containing antioxidant ergothioneine, can be found in high abundance in several marine fish species. However, data on biological properties of SeN and its relevance for human health are still scarce. This study aims to investigate the transfer and presystemic metabolism of SeN in a well-established in vitro model of the blood-brain barrier (BBB). Therefore, SeN and the reference Se species selenite and Se-methylselenocysteine (MeSeCys) were applied to primary porcine brain capillary endothelial cells (PBCECs). Se content of culture media and cell lysates was measured via ICP-MS/MS. Speciation analysis was conducted by HPLC-ICP-MS. Barrier integrity was shown to be unaffected during transfer experiments. SeN demonstrated the lowest transfer rates and permeability coefficient (6.7 × 10-7 cm s-1) in comparison to selenite and MeSeCys. No side-directed accumulation was observed after both-sided application of SeN. However, concentration-dependent transfer of SeN indicated possible presence of transporters on both sides of the barrier. Speciation analysis demonstrated no methylation of SeN by the PBCECs. Several derivatives of SeN detected in the media of the BBB model were also found in cell-free media containing SeN and hence not considered to be true metabolites of the PBCECs. In concluding, SeN is likely to have a slow transfer rate to the brain and not being metabolized by the brain endothelial cells. Since this study demonstrates that SeN may reach the brain tissue, further studies are needed to investigate possible health-promoting effects of SeN in humans.

Optimal Monitoring of Weekly IGF-I Levels During Growth Hormone Therapy With Once-Weekly Somapacitan.

CONTEXT: Somapacitan is a long-acting growth hormone (GH) in development for once-weekly treatment of GH deficiency (GHD). Optimal monitoring of insulin-like growth factor-I (IGF-I) levels must account for weekly IGF-I fluctuations following somapacitan administration. OBJECTIVE: To develop and assess the reliability of linear models for predicting mean and peak IGF-I levels from samples taken on different days after dosing. DESIGN: A pharmacokinetic/pharmacodynamic model was used to simulate IGF-I data in adults and children following weekly somapacitan treatment of GHD. SETTING AND PATIENTS: 39 200 IGF-I profiles were simulated with reference to data from 26 adults and 23 children with GHD. INTERVENTION(S): The simulated dose range was 0.02 to 0.12 mg/kg for adults and 0.02 to 0.16 mg/kg for children. Simulated data with >4 average standard deviation score were excluded. MAIN OUTCOME MEASURE(S): Linear models for predicting mean and peak IGF-I levels based on IGF-I samples from different days after somapacitan dose. RESULTS: Robust linear relationships were found between IGF-I sampled on any day after somapacitan dose and the weekly mean (R2 > 0.94) and peak (R2 > 0.84). Prediction uncertainties were generally low when predicting mean from samples taken on any day (residual standard deviation [RSD] ≤ 0.36) and peak from samples taken on day 1 to 4 (RSD ≤ 0.34). IGF-I monitoring on day 4 and day 2 after dose provided the most accurate estimate of IGF-I mean (RSD < 0.2) and peak (RSD < 0.1), respectively. CONCLUSIONS: Linear models provided a simple and reliable tool to aid optimal monitoring of IGF-I by predicting mean and peak IGF-I levels based on an IGF-I sample following dosing of somapacitan. A short visual summary of our work is available (1).

Selenoneine is a major selenium species in beluga skin and red blood cells of Inuit from Nunavik.

Nunavimmiut (Inuit of Nunavik, Northern Quebec, Canada) exhibit a high selenium (Se) status because of their frequent consumption of marine mammal foods. Indirect evidence from our previous studies had suggested that selenoneine - a novel selenocompound - may be accumulating in the blood of Nunavimmiut. We used a liquid-chromatography/inductively coupled tandem mass spectrometry (LC-ICP-MS/MS) method to measure concentrations of selenoneine and its methylated metabolite Se-methylselenoneine in archived red blood cells (RBC) obtained from 210 Nunavimmiut living in communities along the Hudson Strait, where marine mammal hunting and consumption are most frequent in Nunavik. This method was adapted to quantify selenoneine and its methylated metabolite in beluga mattaaq, an Inuit delicacy consisting of the skin with the underlying layer of fat and the major dietary source of Se for Nunavimmiut. Total selenium concentration was also measured in RBC and beluga mattaaq samples by isotope dilution ICP-MS/MS. The median selenoneine concentration in RBC was 413 µg Se/L (range = 3.20-3230 µg Se/L), representing 54% (median) of total Se content (range = 1.6-91%). Quantification of selenoneine in five beluga mattaaq samples (skin layer) from Nunavik revealed a median concentration of 1.8 µg Se/g wet wt (range = 1.2-7.4 µg Se/g), constituting 54% (median) of the total Se content (range = 44-74%). Se-methylselenoneine was also detected in Inuit RBC but not in beluga mattaaq, suggesting that selenoneine undergoes methylation in humans. Selenoneine may protect Nunavimmiut from methylmecury toxicity by increasing its demethylation in RBC and in turn decreasing its distribution to target organs.

Hexahistidine-metal assemblies: A promising drug delivery system.

It is of considerable interest to construct an ideal drug delivery system (i.e., high drug payload, minimal cytotoxicity, rapid endocytosis, and lysosomal escape) under mild conditions for disease treatment, tissue engineering, bioimaging, etc. Inspired by the coordinative interactions between histidine and metal ions, we present the facile synthesis of hexahistidine (His6)-metal assembly (HmA) particles under mild conditions for the first time. The HmA particles presented a high loading capacity, a wide variety of loadable drugs, minimal cytotoxicity, quick internalization, the ability to bypass the lysosomes, and rapid intracellular drug release. In addition, HmA encapsulation largely improved the antitumor ability of camptothecin (CPT) relative to free CPT. By capitalizing on these promising features in drug delivery, HmA will have great potential in various biomedical fields. STATEMENT OF SIGNIFICANCE: It is of considerable interest to construct an ideal drug delivery system (i.e., high drug payload, minimal cytotoxicity, rapid endocytosis, and lysosomal escape) under mild conditions. Inspired by the coordinative interactions between histidine and metal ions, we present for the first time the facile synthesis of Hexahistidine (His6)-metal assembly (HmA) particles under mild conditions. The HmA particles exhibited a high loading capacity, a wide variety of loadable drugs, minimal cytotoxicity, quick internalization, the ability to bypass the lysosomes, and rapid intracellular drug release. By capitalizing on these promising features in drug delivery, HmA will have great potential in various biomedical fields.

Poly(propylene imine) dendrimers with histidine-maltose shell as novel type of nanoparticles for synapse and memory protection.

Poly(propylene imine) dendrimers have been shown to be promising 3-dimensional polymers for the use in the pharmaceutical and biomedical applications. Our aims of this study were first, to synthesize a novel type of dendrimer with poly(propylene imine) core and maltose-histidine shell (G4HisMal) assessing if maltose-histidine shell can improve the biocompatibility and the ability to cross the blood-brain barrier, and second, to investigate the potential of G4HisMal to protect Alzheimer disease transgenic mice from memory impairment. Our data demonstrate that G4HisMal has significantly improved biocompatibility and ability to cross the blood-brain barrier in vivo. Therefore, we suggest that a maltose-histidine shell can be used to improve biocompatibility and ability to cross the blood-brain barrier of dendrimers. Moreover, G4HisMal demonstrated properties for synapse and memory protection when administered to Alzheimer disease transgenic mice. Therefore, G4HisMal can be considered as a promising drug candidate to prevent Alzheimer disease via synapse protection.

Metabolite profiling of l-isocorypalmine in rat urine, plasma, and feces after oral administration using high performance liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry.

l-Isocorypalmine, an active alkaloid compound isolated from Rhizoma Corydalis yanhusuo, has been reported to possess biological activity for treating cocaine use disorder. A high-performance liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry method was established for identification of the metabolites of l-isocorypalmine in urine, plasma and feces samples of rats after a single intragastric gavage of l-isocorypalmine at a dose of 15 mg/kg. As a result, a total of 21 metabolites (six phase І metabolites and fifteen phase II metabolites) were detected and tentatively identified by mass spectrometry and fragment ions from tandem mass spectrometry spectra. All metabolites were present in the urine samples, nine metabolites were found in the plasma samples and three metabolites were found in the feces samples. Results indicated that metabolic pathways of l-isocorypalmine included oxidation, dehydrogenation, demethylation, sulfate conjugation, and glucuronide conjugation. In addition, glucuronidation was the major metabolic reaction. Results of this investigation could provide significant experimental basis for efficacy, safety and action mechanism of l-isocorypalmine, which will be advantageous to new drug development for treating cocaine addiction.

Semi-Mechanistic Population Pharmacokinetic Modeling of L-Histidine Disposition and Brain Uptake in Wildtype and Pht1 Null Mice.

PURPOSE: To develop a semi-mechanistic population pharmacokinetic (PK) model to quantitate the disposition kinetics of L-histidine, a peptide-histidine transporter 1 (PHT1) substrate, in the plasma, cerebrospinal fluid and brain parenchyma of wildtype (WT) and Pht1 knockout (KO) mice. METHODS: L-[14C]Hisidine (L-His) was administrated to WT and KO mice via tail vein injection, after which plasma, cerebrospinal fluid (CSF) and brain parenchyma samples were collected. A PK model was developed using non-linear mixed effects modeling (NONMEM). The disposition of L-His between the plasma, brain, and CSF was described by a combination of PHT1-mediated uptake, CSF bulk flow and first-order micro-rate constants. RESULTS: The PK profile of L-His was best described by a four-compartment model. A more rapid uptake of L-His in brain parenchyma was observed in WT mice due to PHT1-mediated uptake, a process characterized by a Michaelis-Menten component (Vmax = 0.051 nmoL/min and Km = 34.94 µM). CONCLUSIONS: A semi-mechanistic population PK model was successfully developed, for the first time, to quantitatively characterize the disposition kinetics of L-His in brain under in vivo conditions. This model may prove a useful tool in predicting the uptake of L-His, and possibly other PHT1 peptide/mimetic substrates, for drug delivery to the brain.

Efeitos da ingestão de semente de linhaça e seu possivel efeito hipocolesterolêmico / Effects of linseed intake and its possible hypocholesterolemic effects

Introdução: O colesterol é essencial para a vida. Em excesso no sangue, deposita-se nas paredes das artérias promovendo seu estreitamento ou obstrução. Objetivos: O objetivo foi verificar o possível efeito hipocolesterolêmico da semente de linhaça. Materiais e Métodos: Utilizaram-se 30 ratos Wistar fêmeas divididos em três grupos: Controle, Hipercolesterolêmico, e Linhaça. Os parâmetros avaliados foram: ingestão hídrica; ingestão de ração; peso corporal; gordura peritoneal e níveis de colesterol; Resultados e Discussão: O grupo controle apresentou maior ingestão hídrica (57,46 ± 0,13mL) e ração (30,68 ± 1,96g) quando comparados ao hipercolesterolêmico e linhaça; Para peso corporal total, somente o controle não sofreu alteração; Em relação ao acúmulo de gordura peritoneal, o grupo hipercolesterolêmico se sobressaiu (17,06 ± 3,99g) em comparação ao linhaça e ao controle; para os níveis de colesterol sérico, o grupo controle obteve (1,14 ± 0,14g), sendo observado portanto no grupo linhaça (1,66 ± 0,16g), uma maior aproximação para o resultado do grupo controle do que do hipercolesterolêmico (2,20 ± 6,32g), resultado que nos leva a reconhecer que a linhaça surtiu efeito benéfico sobre o colesterol. Conclusão: Os resultados obtidos demonstram benefícios na utilização da semente para redução dos níveis de gordura peritoneal, peso total e colesterol

Introduction: Cholesterol is essential for life, when in excess in blood, is deposited on the artery walls promoting its narrowing or obstruction. Objectives: The objective was to verify the possible hypocholesterolemic effect of linseed. Methods: We used 30 female Wistar rats divided into three groups: control, hypercholesterolemic, and linseed. The evaluated parameters were: water intake, feed intake, body weight, peritoneal fat, and cholesterol levels; Results and Discussion: The results were: the control group had a higher fluid intake (57.46 ± 0.13ml) and feed (30.68 ± 1.96g) when compared to hypercholesterolemic and linseed; for total body weight, only the control group did not change; in relation to the accumulation of peritoneal fat, the hypercholesterolemic group stood out (17.06 ± 3.99g) compared to linseed and control; for serum cholesterol levels, the control group obtained (1.14 ± 0.14g), thus being observed in linseed group (1.66 ± 0.16g), closer to the result of the control group than the hypercholesterolemic (2.20 ± 6.32g), a result that leads us to recognize that linseed has had a beneficial effect on cholesterol.Conclusion: The results obtained demonstrate benefits in the use of the seed to reduce levels of peritoneal fat, total weight and cholesterol

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A novel role for PHT1 in the disposition of l-histidine in brain: In vitro slice and in vivo pharmacokinetic studies in wildtype and Pht1 null mice.

PHT1 (SLC15A4) is responsible for translocating l-histidine (l-His), di/tripeptides and peptide-like drugs across biological membranes. Previous studies have indicated that PHT1 is located in brain parenchyma, however, its role and significance in brain along with effect on the biodistribution of substrates is unknown. In this study, adult gender-matched Pht1-competent (wildtype) and Pht1-deficient (null) mice were used to investigate the effect of PHT1 on l-His brain disposition via in vitro slice and in vivo pharmacokinetic approaches. We also evaluated the serum clinical chemistry and expression levels of select transporters and enzymes in the two genotypes. No significant differences were observed between genotypes in serum chemistry, body weight, viability and fertility. PCR analyses indicated that Pept2 had a compensatory up-regulation in Pht1 null mice (about 2-fold) as compared to wildtype animals, which was consistent in different brain regions and confirmed by immunoblots. The uptake of l-His was reduced in brain slices by 50% during PHT1 ablation. The l-amino acid transporters accounted for 30% of the uptake, and passive (other) pathways for 20% of the uptake. During the in vivo pharmacokinetic studies, plasma concentration-time profiles of l-His were comparable between the two genotypes after intravenous administration. Still, biodistribution studies revealed that, when sampled 5min after dosing, l-His values were 28-48% lower in Pht1 null mice, as compared to wildtype animals, in brain parenchyma but not cerebrospinal fluid. These findings suggest that PHT1 may play an important role in histidine transport in brain, and resultant effects on histidine/histamine homeostasis and neuropeptide regulation.

Novel self-assembled pH-responsive biomimetic nanocarriers for drug delivery.

Novel pH-responsive biodegradable biomimetic nanocarriers were prepared by the self-assembly of N-acetyl-l-histidine-phosphorylcholine-chitosan conjugate (NAcHis-PCCs), which was synthesized via Atherton-Todd reaction to couple biomembrane-like phosphorylcholine (PC) groups, and N,N'-carbonyldiimidazole (CDI) coupling reaction to link pH-responsive N-acetyl-l-histidine (NAcHis) moieties to chitosan. In vitro biological assay revealed that NAcHis-PCCs nanoparticles had excellent biocompatibility to avoid adverse biological response mainly owing to their biomimetic PC shell, and DLS results confirmed their pH-responsive behavior in acidic aqueous solution (pH≤6.0). Quercetin (QUE), an anti-inflammatory, antioxidant and potential anti-tumor hydrophobic drug, was effectively loaded in NAcHis-PCCs nanocarriers and showed a pH-triggered release behavior with the enhanced QUE release at acidic pH5.5 compared to neutral pH7.4. The results indicated that pH-responsive biomimetic NAcHis-PCCs nanocarriers might have great potential for site-specific delivery to pathological acidic microenvironment avoiding unfavorable biological response.

Membrane adsorber for endotoxin removal

ABSTRACT The surface of flat-sheet nylon membranes was modified using bisoxirane as the spacer and polyvinyl alcohol as the coating polymer. The amino acid histidine was explored as a ligand for endotoxins, aiming at its application for endotoxin removal from aqueous solutions. Characterization of the membrane adsorber, analysis of the depyrogenation procedures and the evaluation of endotoxin removal efficiency in static mode are discussed. Ligand density of the membranes was around 7 mg/g dry membrane, allowing removal of up to 65% of the endotoxins. The performance of the membrane adsorber prepared using nylon coated with polyvinyl alcohol and containing histidine as the ligand proved superior to other membrane adsorbers reported in the literature. The lack of endotoxin adsorption on nylon membranes without histidine confirmed that endotoxin removal was due to the presence of the ligand at the membrane surface. Modified membranes were highly stable, exhibiting a lifespan of approximately thirty months.

RESUMO A superfície de membranas planas de nylon foi modificada utilizando-se bisoxirano como espaçador e poli(álcool vinílico) para recobrimento das membranas. O aminoácido histidina foi utilizado como ligante para endotoxinas, visando à sua aplicação na remoção de endotoxinas a partir de soluções aquosas. São discutidas as etapas de caracterização do adsorvedor com membranas, análise do procedimento de despirogenização e avaliação da eficiência de remoção em modo estático. A densidade de ligantes nas membranas foi em torno de 7 mg/g membrana (massa seca), permitindo uma remoção de endotoxinas de até 65%. O desempenho das membranas preparadas com nylon e recobertas com poli(álcool vinílico) contendo histidina como ligante foi superior ao de outros adsorvedores com membranas descritos na literatura. A ausência de adsorção de endotoxinas em membranas sem histidina confirma que a remoção das endotoxinas deve-se exclusivamente à presença do ligante na superfície da membrana. As membranas modificadas mostraram-se bastante estáveis, exibindo um tempo de vida superior a 30 dias.

Cellular uptake and in vivo distribution of polyhistidine peptides.

Cell-penetrating peptides (CPPs) are arginine/lysine-rich sequences, and they are effectively internalized into cells. In this process, positive charge is crucial. In the present study, we found polyhistidine peptides (PHPs), as the novel CPP, which are efficiently internalized into cells in a positive charge-independent manner. Interestingly, cellular uptake of the PHPs increased as the chain length increased, reaching a maximum uptake at H16 (HHHHHHHHHHHHHHHH-NH2). This H16 peptide showed up to 14.6-fold higher cell-penetrating capacity against HT1080 human fibrosarcoma cells relative to a major CPP, the octa-arginine (RRRRRRRR-NH2) peptide. Cellular uptake of the H16 peptide is mainly due to macropinocytosis and most of the H16 peptide localizes in the lysosome and Golgi apparatus. However, a cytoplasmic pro-apoptotic domain (KLAKLAKKLAKLAK-NH2) conjugated to the H16 peptide showed cytotoxic effects. This indicates that a proportion of the H16 peptide escapes from the macropinosome to the cytoplasm. In a protein transduction study, green fluorescence protein fused to the H16 peptide (GFP-H16) was purified by Ni-NTA chromatography, detected using an anti-His-tag antibody and internalized into cells. This serial process reveals that H16 functions as a His-tag and protein transduction domain. Furthermore, in vivo distribution analysis showed that the H16 peptide accumulates immediately in tumor tissue and is retained up to 132h following injection into the tumor (HT1080 human fibrosarcoma)-bearing mice. This is the first observation of a His-polymer being internalize into cells efficiently. The findings suggest that PHPs are novel CPPs. In particular, the H16 peptide represents a promising drug delivery carrier candidate in medical and biotechnological fields.

Histidine-rich stabilized polyplexes for cMet-directed tumor-targeted gene transfer.

Overexpression of the hepatocyte growth factor receptor/c-Met proto oncogene on the surface of a variety of tumor cells gives an opportunity to specifically target cancerous tissues. Herein, we report the first use of c-Met as receptor for non-viral tumor-targeted gene delivery. Sequence-defined oligomers comprising the c-Met binding peptide ligand cMBP2 for targeting, a monodisperse polyethylene glycol (PEG) for polyplex surface shielding, and various cationic (oligoethanamino) amide cores containing terminal cysteines for redox-sensitive polyplex stabilization, were assembled by solid-phase supported syntheses. The resulting oligomers exhibited a greatly enhanced cellular uptake and gene transfer over non-targeted control sequences, confirming the efficacy and target-specificity of the formed polyplexes. Implementation of endosomal escape-promoting histidines in the cationic core was required for gene expression without additional endosomolytic agent. The histidine-enriched polyplexes demonstrated stability in serum as well as receptor-specific gene transfer in vivo upon intratumoral injection. The co-formulation with an analogous PEG-free cationic oligomer led to a further compaction of pDNA polyplexes with an obvious change of shape as demonstrated by transmission electron microscopy. Such compaction was critically required for efficient intravenous gene delivery which resulted in greatly enhanced, cMBP2 ligand-dependent gene expression in the distant tumor.

pH-sensitive nanoparticles of poly(L-histidine)-poly(lactide-co-glycolide)-tocopheryl polyethylene glycol succinate for anti-tumor drug delivery.

A novel pH-sensitive polymer, poly(L-histidine)-poly(lactide-co-glycolide)-tocopheryl polyethylene glycol succinate (PLH-PLGA-TPGS), was synthesized to design a biocompatible drug delivery system for cancer chemotherapy. The structure of the PLH-PLGA-TPGS copolymer was confirmed by (1)H-NMR, FTIR and GPC. The apparent pKa of the PLH-PLGA-TPGS copolymer was calculated to be 6.33 according to the acid-base titration curve. The doxorubicin (DOX)-loaded nanoparticles (PLH-PLGA-TPGS nanoparticles and PLGA-TPGS nanoparticles) and corresponding blank nanoparticles were prepared by a co-solvent evaporation method. The blank PLH-PLGA-TPGS nanoparticles showed an acidic pH-induced increase in particle size. The DOX-loaded nanoparticles based on PLH-PLGA-TPGS showed a pH-triggered drug-release behavior under acidic conditions. The results of in vitro cytotoxicity experiment on MCF-7 and MCF-7/ADR cells showed that the DOX-loaded PLH-PLGA-TPGS nanoparticles resulted in lower cell viability versus the PLGA-TPGS nanoparticles and free DOX solution. Confocal laser scanning microscopy images showed that DOX-loaded PLH-PLGA-TPGS nanoparticles were internalized by MCF-7/ADR cells after 1 and 4h incubation and most of them accumulated in lysosomes to accelerate DOX release under acidic conditions. In summary, the PLH-PLGA-TPGS nanoparticles have great potential to be used as carriers for anti-tumor drug delivery.

Effects of pH-sensitive chain length on release of doxorubicin from mPEG-b-PH-b-PLLA nanoparticles.

BACKGROUND: Two methoxyl poly(ethylene glycol)-poly(L-histidine)-poly(L-lactide) (mPEG-PH-PLLA) triblock copolymers with different poly(L-histidine) chain lengths were synthesized. The morphology and biocompatibility of these self-assembled nanoparticles was investigated. METHODS: Doxorubicin, an antitumor drug, was trapped in the nanoparticles to explore their drug-release behavior. The drug-loaded nanoparticles were incubated with HepG2 cells to evaluate their antitumor efficacy in vitro. The effects of poly(L-histidine) chain length on the properties, drug-release behavior, and antitumor efficiency of the nanoparticles were investigated. RESULTS: The nanoparticles were pH-sensitive. The mean diameters of the two types of mPEG-PH- PLLA nanoparticle were less than 200 nm when the pH values were 5.0 and 7.4. The nanoparticles were nontoxic to NIH 3T3 fibroblasts and HepG2 cells. The release of doxorubicin at pH 5.0 was much faster than that at pH 7.4. The release rate of mPEG(45)-PH(15)-PLLA(82) nanoparticles was much faster than that of mPEG(45)-PH(30)-PLLA(82) nanoparticles at pH 5.0. CONCLUSION: The inhibition effect of mPEG(45)-PH(15)-PLLA(82) nanoparticles on the growth of HepG2 cells was greater than that of mPEG(45)-PH(30)-PLLA(82) nanoparticles when the concentration of encapsulated doxorubicin was less than 15 µg/mL.

Poly(L-histidine)-tagged 5-aminolevulinic acid prodrugs: new photosensitizing precursors of protoporphyrin IX for photodynamic colon cancer therapy.

BACKGROUND: 5-Aminolevulinic acid (ALA) and its derivatives have been widely used in photodynamic therapy. The main drawback associated with ALA-based photodynamic therapy (ALA-PDT) and ALA fluorescence diagnosis results from the hydrophilic nature of ALA and lack of selectivity for tumor versus nontumor cells. The application of certain triggers, such as pH, into conventional sensitizers for controllable (1)O(2) release is a promising strategy for tumor-targeted treatment. METHODS: A series of pH-sensitive ALA-poly(L-histidine) [p(L-His)(n)] prodrugs were synthesized via ring opening polymerization of 1-benzyl-N-carboxy-L-histidine anhydride initiated by the amine hydrochloride group of ALA itself. As an alternative to ALA for PDT, the synthesized prodrugs were used to treat a cultured human colon cancer HCT116 cell line under different pH conditions. The effect of ALA-p(L-His)(n) derivatives was evaluated by monitoring the fluorescence intensity of protoporphyrin IX, and measuring the cell survival rate after suitable light irradiation. RESULTS: The cytotoxicity and dark toxicity of ALA and synthesized ALA-p(L-His) derivatives in HEK293T and HCT116 cells in the absence of light at pH 7.4 and 6.8 shows that the cell viability was relatively higher than 100%. ALA-p(L-His)(n) showed high phototoxicity and selectivity in different pH conditions compared with ALA alone. Because the length of the histidine chain increases in the ALA-p(L-His)(n) prodrugs, the PDT effect was found to be more powerful. In particular, high phototoxicity was observed when the cells were treated with ALA-p(L-His)(15), compared with treatment using ALA alone. CONCLUSION: The newly synthesized ALA-p(L-His)(n) derivatives are an effective alternative to ALA for enhancing protoporphyrin IX production and the selectivity of the phototoxic effect in tumor cells.

PET with 64Cu-histidine for noninvasive diagnosis of biliary copper excretion in Long-Evans cinnamon rat model of Wilson disease.

UNLABELLED: Excretion of copper into bile requires the copper transporter Atp7b, which is deficient in Wilson disease. We hypothesized that a radiocopper-histidine complex would be effective for diagnosing Wilson disease by molecular imaging and tested this hypothesis in the Long-Evans cinnamon (LEC) rat model with Atp7b deficiency. METHODS: We complexed (64)Cu to l-histidine and analyzed clearance from blood, uptake in tissues, and excretion in bile of healthy Long-Evans agouti (LEA) rats versus LEC rats modeling Wilson disease. Sixty-minute dynamic PET recordings were obtained in LEA and LEC rats. Possible effects of acute and chronic liver injury induced by carbon tetrachloride were studied in LEA rats. Atp7b deficiency in LEC rats was reconstituted by transplantation of healthy cells to establish the specificity of findings. RESULTS: Examination of blood, tissue, and bile showed that in healthy rats, radiocopper was incorporated in the liver, followed by rapid excretion in bile. Corresponding blood, tissue, and bile studies in LEC rats showed incorporation of radiocopper in the liver but without copper excretion in bile, leading to hepatic retention of the radiotracer. PET showed onset of copper clearance in the liver of LEA rats, whereas liver copper content progressively increased in LEC rats during the 1-h period. Hepatic radiocopper excretion was not altered by either acute or chronic liver injury. In LEC rats with liver repopulation by transplanted healthy hepatocytes, excretion of radiocopper confirmed that Atp7b was responsible for this effect. CONCLUSION: Imaging with the radiocopper-histidine complex successfully identified Atp7b-dependent biliary copper excretion. This principle will advance molecular imaging for Wilson disease.

Design, synthesis, hydrolysis kinetics and phamacodynamic profiles of histidine and alanine conjugates of aceclofenac.

The gastrointestinal toxicity associated with aceclofenac can be reduced by condensing its carboxylic acid group with methyl esters of amino acids like histidine and alanine to give amide linkage by the Schotten-Baumann method. Physicochemical characterization of the conjugates was carried out by various analytical and spectral methods. The synthesized conjugates were also subjected to in vitro hydrolysis in simulated gastric fluid (SGF) at pH 1.2, simulated intestinal fluid (SIF) at pH 7.4 and SIF+ 80% human plasma at pH 7.4. The release of free aceclofenac from histidine and alanine conjugated aceclofenac showed negligible hydrolysis in SGF compared to SIF. This indicated that the conjugates do not break in stomach, but release aceclofenac in SIF. Both synthesized conjugates showed excellent pharmacological response and encouraging hydrolysis rate in SIF and SIF + 80% human plasma. Marked reduction of the ulcer index and comparable increase in analgesic and anti-inflammatory activities were obtained in both cases compared to aceclofenac alone. These findings suggest that the conjugates are better in action compared to the parent drug and have fewer gastrointestinal side-effects.