Design considerations for PAMAM dendrimer therapeutics

We have previously shown that methotrexate (MTX) conjugated to a cancer-specific poly amido amine (PAMAM) dendrimer has a higher therapeutic index than MTX alone. Unfortunately, these therapeutics have been difficult to advance because of the complicated syntheses and an incomplete understanding of the dendrimer properties. We wished to address these obstacles by using copper-free click chemistry to functionalize the dendrimer scaffolds and to exploring the effects of two dendrimer properties (the targeting ligand and drug linkage) on cytotoxicity. We conjugated either ester or amide-linker modified MTX to dendrimer scaffolds with or without folic acid (FA). Because of multivalency, the FA and MTX functionalized dendrimers had similar capacities to target the folate receptor on cancer cells. Additionally, we found that the ester- and amide-linker modified MTX compounds had similar cytotoxicity but the dendrimer–ester MTX conjugates were much more cytotoxic than the dendrimer–amide MTX conjugates. These results clarify the impact of these properties on therapeutic efficacy and will allow us to design more effective polymer therapeutics.     

LPS下调小鼠肝脏、肠道羧酸酯酶表达

目的:研究细菌脂多糖(LPS)对小鼠肝脏、肠道羧酸酯酶表达及酶活性的影响。方法:小鼠经腹腔注射5.0mg/kg的LPS,对照组给予生理盐水,注射后24h处死小鼠,取肝脏和肠道组织。用qRT-PCR技术检测小鼠肝脏、肠道组织羧酸酯酶1和2(mCES1和mCES2)mRNA水平;Westernblot技术检测小鼠肝脏、肠道组织mCES1和mCES2蛋白表达水平。用分光光度计检测小鼠肝脏、肠道组织羧酸酯酶总活性。结果:LPS显著降低小鼠肝脏、肠道组织羧酸酯酶1和2的mRNA水平(P<0.05),同时LPS也显著降低小鼠肝脏、肠道组织羧酸酯酶的蛋白表达水平及酶活性(P<0.05)。结论:LPS造成的炎症状态可显著降低小鼠肝脏、肠道组织羧酸酯酶的表达及酶活性。     

Carboxylesterase in the liver and small intestine of experimental animals and human

Native polyacrylamide gel electrophoresis showed carboxylesterase (CES) to be the most abundant hydrolase in the liver and small intestine of humans, monkeys, dogs, rabbits and rats. The liver contains both CES1 and CES2 enzymes in all these species. The small intestine contains only enzymes from the CES2 family in humans and rats, while in rabbits and monkeys, enzymes from both CES1 and CES2 families are present. Interestingly, no hydrolase activity at all was found in dog small intestine. Flurbiprofen derivatives were R-preferentially hydrolyzed in the liver microsomes of all species, but hardly hydrolyzed in the small intestine microsomes of any species except rabbit. Propranolol derivatives were hydrolyzed in the small intestine and liver microsomes of all species except dog small intestine. Monkeys and rabbits showed R-preferential and non-enantio-selective hydrolysis, respectively, for propranolol derivatives in both organs. Human and rat liver showed R- and S-preferential hydrolysis, respectively, in spite of non-enantio-selective hydrolysis in their small intestines. The proximal-to-distal gradient of CES activity in human small intestine (1.1-1.5) was less steep than that of CYP 3A4 and 2C9 activity (three-fold difference). These findings indicate that human small intestine and liver show extensive hydrolase activity attributed to CES, which is different from that in species commonly used as experimental animals.     

New targeting system for antimycotic drugs: beta-glucosidase sensitive amphotericin B-star poly(ethylene glycol) conjugate

A new targeting potentially intravenous conjugate Amphotericin B (AMB)–star poly(ethylene glycol) (sPEG) (M = 25,160) has been synthesized and characterized. It contains a β-d-glucopyranoside molecular switch which is sensitive to β-glucosidases (E.C.3.2.1.21). The β-glucosidase-catalyzed release of AMB from the polymeric carrier was proved in vitro by means of spectrophotometry and HPLC.     

Capacity limits of asialoglycoprotein receptor-mediated liver targeting

The abundant cell surface asialoglycoprotein receptor (ASGPR) is a highly selective receptor found on hepatocytes that potentially can be exploited as a selective shuttle for delivery. Various nucleic acid therapeutics that bind ASGPR are already in clinical development, but this receptor-mediated delivery mechanism can be saturated, which will likely result in reduced selectivity for the liver and therefore increase the likelihood for systemic adverse effects. Therefore, when aiming to utilize this mechanism, it is important to optimize both the administration protocol and the molecular properties. We here present a study using a novel ASGPR-targeted antibody to estimate ASGPR expression, turnover and internalization rates in vivo in mice. Using pharmacokinetic data (intravenous and subcutaneous dosing) and an in-silico target-mediated drug disposition (TMDD) model, we estimate an ASGPR expression level of 1.8 million molecules per hepatocyte. The half-life of the degradation of the receptor was found to be equal to 15 hours and the formed ligand-receptor complex is internalized with a half-life of 5 days. A biodistribution study was performed and confirmed the accuracy of the TMDD model predictions. The kinetics of the ASGPR shows that saturation of the shuttle at therapeutic concentrations is possible; however, simulation allows the dosing schedule to be optimized. The developed TMDD model can be used to support the development of therapies that use the ASGPR as a shuttle into hepatocytes.     

Palmitoylation of stathmin family proteins domain A controls Golgi versus mitochondrial subcellular targeting

Background information. Precise localization of proteins to specialized subcellular domains is fundamental for proper neuronal development and function. The neural microtubule‐regulatory phosphoproteins of the stathmin family are such proteins whose specific functions are controlled by subcellular localization. Whereas stathmin is cytosolic, SCG10, SCLIP and RB3/RB3′/RB3″ are localized to the Golgi and vesicle‐like structures along neurites and at growth cones. We examined the molecular determinants involved in the regulation of this specific subcellular localization in hippocampal neurons in culture. Results. We show that their conserved N‐terminal domain A carrying two palmitoylation sites is dominant over the others for Golgi and vesicle‐like localization. Using palmitoylation‐deficient GFP (green fluorescent protein) fusion mutants, we demonstrate that domains A of stathmin proteins have the particular ability to control protein targeting to either Golgi or mitochondria, depending on their palmitoylation. This regulation involves the co‐operation of two subdomains within domain A, and seems also to be under the control of its SLD (stathmin‐like domain) extension. Conclusions. Our results unravel that, in specific biological conditions, palmitoylation of stathmin proteins might be able to control their targeting to express their functional activities at appropriate subcellular sites. They, more generally, open new perspectives regarding the role of palmitoylation as a signalling mechanism orienting proteins to their functional subcellular compartments.     

The use of solid lipid nanoparticles to target a lipophilic molecule to the liver after intravenous administration to mice

Taspine solid lipid nanoparticles (Ta-SLN) and taspine solid lipid nanoparticles modified by galactoside (Ta-G(2)SLN) were prepared by the film evaporation-extrusion method. The nanoparticles were spherical or near-spherical particles with smooth surface, small size and high encapsulation efficiency. Ta-G(2)SLN and Ta-SLN showed significant inhibition on 7721 cell growth. Intravenous injection of either Ta-SLN or Ta-G(2)SLN resulted in a higher plasma and liver concentration and a longer retention time in mice compared with the administration of Ta. These results suggested that SLN tended to be preferentially delivered to the liver and Ta-G(2)SLN may further enhance liver targeting.     

Design and regioselective synthesis of a new generation of targeted therapeutics. Part 3: Folate conjugates of aminopterin hydrazide for the treatment of inflammation

Efficient syntheses of folate receptor (FR) targeting conjugates of the anti-inflammatory, aminopterin hydrazide, are described. 2-{4-Benzoylamino}-5-oxo-5-{N′-[2-(pyridin-2-yldisulfanyl)-ethoxycarbonyl]-hydrazino}-pentanoic acid is synthesized from commercially available 4-[(2-amino-4-imino-3,4-dihydro-pteridin-6-yl-methyl)-amino]-benzoic acid. Conjugation of this novel, activated aminopterin hydrazide to folic acid through cysteine-terminating (C-terminus), peptide/carbohydrate spacers results in highly water soluble conjugates which allow for the release of free aminopterin hydrazide within the endosomes of targeted cells.     

Immune cell-specific delivery of beta-glucan-coated iron oxide nanoparticles for diagnosing liver metastasis by MR imaging

Glucans are reported to elicit immune responses through activation of macrophages by a specific interaction of β-glucan with an immune cell-specific (1,3)-β-d-glucan receptor or Dectin-1 receptor. In this study, superparamagnetic iron oxide nanoparticles (SPIONs) were coated with β-glucan in order to target the immune cells residing in the metastatic liver as an aid for discriminating metastasized tumor regions from normal hepatic parenchymal tissue. The morphology of prepared β-glucan-coated SPIONs (Glu-SPIONs) was characterized with dynamic light scattering (DLS) and transmission electron microscopy (TEM). The cytotoxicity of Glu-SPIONs was analyzed and compared to that of dextran- and PVA-coated SPIONs. The uptake of Glu-SPIONs by peritoneal macrophages was also confirmed with Prussian blue staining and MRI phantom tube imaging. The in vivo uptake of Glu-SPIONs in liver and lymph nodes in a metastatic mouse liver model was tracked by MR imaging after the systemic injection. The Glu-SPIONs predominantly accumulated in the macrophages surrounding the metastatic regions of the liver thereby indicating the distribution of tumor cells in the liver. MR imaging of the Glu-SPIONs clearly revealed macro- or micro-metastasized tumor regions throughout the liver, due to the preferential uptake of Glu-SPIONs into macrophages, not tumor cells. The Glu-SPION-accumulating regions were further confirmed with H&E and Prussian blue stainings after tissue sectioning. Based on our study, we propose that Glu-SPIONs can be successfully applied for diagnosing hepatic metastasis.     

Interfacial versus homogeneous enzymatic cleavage of mandelonitrile by hydroxynitrile lyase in a biphasic system

The question of an interfacial versus a homogeneous reaction is carefully addressed for the enzymatic biphasic cleavage of mandelonitrile to benzaldehyde by Prunus amygdalus hydroxynitrile lyase (pa-Hnl) (Hickel et al. [1999] Biotechnol Bioeng 36:425-436). Experimental evidence, including 1) the reaction ceases when the interface is populated by previously adsorbed denatured pa-Hnl, 2) the reaction continues even after washout of the bulk enzyme from the aqueous phase, 3) highly nonpolar organic solvents initially promote fast reaction kinetics that relatively quickly decay to zero product production, and 4) the reaction rate is nonlinear in the bulk enzyme concentration, provide robust grounds for an interfacial reaction. We also model enzymatic mandelonitrile cleavage assuming a homogeneous aqueous-phase reaction. The homogeneous reaction scheme does not simultaneously account for the experimental observations of a linear dependence of the reaction rate on organic/water interfacial area, no dependence on the aqueous-phase volume, and a nonlinear dependence on pa-Hnl aqueous concentration. Further, simple calculations demonstrate that the homogeneous reaction rate is at least three orders of magnitude slower than those observed by Hickel et al. (1999). We again conclude that enzyme adsorbed at the organic solvent/water interface primarily catalyzes the biphasic mandelonitrile cleavage reaction.     

Increased efficiency of homologous recombination in ES cells by cleavage at both ends of homology in the targeting vector

Transgenic Research -     

Different approaches of katira gum formulations for colon targeting

The objective of the study is to compare the different formulations prepared by using gum, grafted gum and hydrogel of katira as a carrier for colon-specific drug delivery using in vitro methods with and without enzymes. Katira gum is naturally occurring polysaccharides containing mainly l-rhamnose and d-galactose sugar unit and small percent of d-galactouronic acid. Compared to grafted gum and hydrogel, all proportions of katira gum protect the drug from being released completely in the physiological environment of the stomach and small intestine. In vitro release studies in enzymes (Pectinex Ultra SP-L having galactouronidase activity) have demonstrated the susceptibility of katira gum to the colonic bacterial enzyme (galactouronidase activity from Pectinex Ultra SP-L) with a consequent drug release. It illustrates that katira gum, a natural polysaccharide may be suitable as a carrier for colon targeting.     

肝移植患者真菌感染的流行病学特点及耐药性分析

目的了解肝移植术后真菌感染的种类及耐药特性,为临床治疗提供依据。方法分析2003年6月至2006年6月,我院67例肝移植患者术后感染的标本,鉴定真菌种类,分析其耐药性。结果67例肝移植患者有21例发生真菌感染,占肝移植患者的31.3%;共检出73株真菌,以酵母菌感染为主,占98.6%,其中近平滑念珠菌、白色念珠菌、热带念珠菌、季也蒙念珠菌、克柔念珠菌的检出率分别是53.4%、21.9%、9.6%、8.2%、2.7%。曲霉菌感染1例。药敏试验显示73株真菌对两性霉素B(AMB)、5-氟胞嘧啶(5-FC)、制霉菌素(MYS)、酮康唑(KTC)、益康唑(ECO)和咪康唑(MIC)的平均敏感率分别为98.6%、95.7%、87.1%、70.0%、65.7%和64.3%。结论加强肝移植术后真菌的鉴定和耐药性监测,对指导临床治疗具有重要意义。     

Plastid targeting and transient expression of rat liver ATP: citrate lyase in pea protoplasts

Protoplasts isolated from pea leaves (Pisum sativum L. cv. Hurst Greenshaft) were electroporated in the presence of plasmid pDR#1, which contains the rat liver ATP:citrate lyase gene fused to a duplex 35S cauliflower mosaic virus promoter with a transit peptide sequence of the Rubisco small subunit. The level of enzyme expression and viability of protoplasts were both influenced by polyethylene glycol treatment before electroporation. Under the optimised electroporation conditions, an average increase of ATP:citrate lyase activity of 14% was observed in the transfected cells after 24 h, with a similar magnitude of change in the abundance of the corresponding mRNA. Immunoblot analysis confirmed the correct expression and targeting of ATP:citrate lyase protein in the chloroplasts of pea protoplasts. These results provide a basis for the establishment of a procedure for targeting heterologous protein into pea plastids in the presence of a transit peptide. Received: 14 June 1996 / Revision received: 24 November 1996 / Accepted: 4 January 1997     

Novel therapeutic strategies for targeting liver cancer stem cells

The cancer stem cell (CSC) hypothesis was first proposed over 40 years ago. Advances in CSC isolation were first achieved in hematological malignancies, with the first CSC demonstrated in acute myeloid leukemia. However, using similar strategies and technologies, and taking advantage of available surface markers, CSCs have been more recently demonstrated in a growing range of epithelial and other solid organ malignancies, suggesting that the majority of malignancies are dependent on such a compartment.Primary liver cancer consists predominantly of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). It is believed that hepatic progenitor cells (HPCs) could be the origin of some HCCs and ICCs. Furthermore, stem cell activators such as Wnt/β-catenin, TGF-β, Notch and Hedgehog signaling pathways also expedite tumorigenesis, and these pathways could serve as molecular targets to assist in designing cancer prevention strategies. Recent studies indicate that additional factors such as EpCAM, Lin28 or miR-181 may also contribute to HCC progression by targeting HCC CSCs. Various therapeutic drugs that directly modulate CSCs have been examined in vivo and in vitro. However, CSCs clearly have a complex pathogenesis, with a considerable crosstalk and redundancy in signaling pathways, and hence targeting single molecules or pathways may have a limited benefit for treatment. Many of the key signaling molecules are shared by both CSCs and normal stem cells, which add further challenges for designing molecularly targeted strategies specific to CSCs but sparing normal stem cells to avoid side effects. In addition to the direct control of CSCs, many other factors that are needed for the maintenance of CSCs, such as angiogenesis, vasculogenesis, invasion and migration, hypoxia, immune evasion, multiple drug resistance, and radioresistance, should be taken into consideration when designing therapeutic strategies for HCC. Here we provide a brief review of molecular signaling in liver CSCs and present insights into new therapeutic strategies for targeting liver CSCs.     

Eudragit-coated pectin microspheres of 5-fluorouracil for colon targeting

An objective of the present investigation was to prepare and evaluate Eudragit-coated pectin microspheres for colon targeting of 5-fluorouracil (FU). Pectin microspheres were prepared by emulsion dehydration method using different ratios of FU and pectin (1:3 to 1:6), stirring speeds (500–2000 rpm) and emulsifier concentrations (0.75%–1.5% wt/vol). The yield of preparation and the encapsulation efficiencies were high for all pectin microspheres. Microspheres prepared by using drug:polymer ratio 1:4, stirring speed 1000 rpm, and 1.25% wt/vol concentration of emulsifying agent were selected as an optimized formulation. Eudragit-coating of pectin microspheres was performed by oil-in-oil solvent evaporation method using coat: core ratio (5:1). Pectin microspheres and Eudragit-coated pectin microspheres were evaluated for surface morphology, particle size and size distribution, swellability, percentage drug entrapment, and in vitro drug release in simulated gastrointestinal fluids (SGF). The in vitro drug release study of optimized formulation was also performed in simulated colonic fluid in the presence of 2% rat cecal content. Organ distribution study in albino rats was performed to establish the targeting potential of optimized formulation in the colon. The release profile of FU from Eudragit-coated pectin microspheres was pH dependent. In acidic medium, the release rate was much slower; however, the drug was released quickly at pH 7.4. It is concluded from the present investigation that Eudragit-coated pectin microspheres are promising controlled release carriers for colon-targeted delivery of FU. Published: February 16, 2007     

Scaffold-free 3D bio-printed human liver tissue stably maintains metabolic functions useful for drug discovery

The liver plays a central role in metabolism. Although many studies have described in vitro liver models for drug discovery, to date, no model has been described that can stably maintain liver function. Here, we used a unique, scaffold-free 3D bio-printing technology to construct a small portion of liver tissue that could stably maintain drug, glucose, and lipid metabolism, in addition to bile acid secretion. This bio-printed normal human liver tissue maintained expression of several kinds of hepatic drug transporters and metabolic enzymes that functioned for several weeks. The bio-printed liver tissue displayed glucose production via cAMP/protein kinase A signaling, which could be suppressed with insulin. Bile acid secretion was also observed from the printed liver tissue, and it accumulated in the culture medium over time. We observed both bile duct and sinusoid-like structures in the bio-printed liver tissue, which suggested that bile acid secretion occurred via a sinusoid-hepatocyte-bile duct route. These results demonstrated that our bio-printed liver tissue was unique, because it exerted diverse liver metabolic functions for several weeks. In future, we expect our bio-printed liver tissue to be applied to developing new models that can be used to improve preclinical predictions of long-term toxicity in humans, generate novel targets for metabolic liver disease, and evaluate biliary excretion in drug development.     

Peroxisomal and mitochondrial targeting of serine: Pyruvate/alanine: Glyoxylate aminotransferase in rat liver

Serine: pyruvate/alanine:glyoxylate aminotransferase (SPT or SPT/AGT) of rat liver is a unique enzyme of dual subcellular localization, and exists in both mitochondria and peroxisomes. To characterize a peroxisomal targeting signal of rat liver SPT, a number of C-terminal mutants were constructed and their subcellular localization in transfected COS-1 cells was examined. Deletion of C-terminal NKL, and point mutation of K2 (the second Lys from the C-terminus), K4 and E15 caused accumulation of translated products in the cytoplasm. This suggests that the PTS of SPT is not identical to PTS1 (the C-terminal SKL motif) in that it is not restricted to the C-terminal tripeptide. In vitro synthesized precursor for mitochondrial SPT was highly sensitive to the proteinase K digestion, whereas peroxisomal SPT (SPTp) was fairly resistant to the protease. In in vitro import experiment with purified peroxisomes, however, STPp recovered in the peroxisomal fraction was very sensitive to the protease. These results suggest that the mitochondrial precursor is synthesized as an unfolded form and is translocated into the mitochondrial matrix, whereas SPTp is synthesized as a folded form and its conformation changes to an unfolded form just before translocation into peroxisomes.     

Processing of somatostatin precursors: evidence for enzymatic cleavage by hypothalamic extract

The action of enzymes extracted from rat hypothalamus on the previously characterized high molecular weight forms of hypothalamic somatostatin-like immunoreactivity (4 K SLI and 25 K SLI) has been investigated in vitro in order to further define the role of these molecules as possible precursors for tetradecapeptide somatostatin (SRIF). Studies of the degradation of endogenous SLI and of synthetic SRIF by hypothalamic enzymes showed that the time course of breakdown of endogenous SLI is markedly slower than that of synthetic SRIF due to the relative stability of 25 K SLI as well as the generation of at least two new immunoreactive molecules. Incubation of purified 25 K SLI with SLI-free hypothalamic extract showed after 10 to 30 min newly formed immunoreactive material of an intermediate size between 25 K SLI and 4 K SLI and after 60 min the emergence of material coeluting with SRIF. These data show that the hypothalamus contains the enzymes necessary for degrading endogenous SLI and for processing the 25 K SLI molecule to SRIF providing further evidence that 25 K SLI might be a biosynthetic precursor for SRIF.     

Design of a Tumor Homing Cell-Penetrating Peptide for Drug Delivery

The major drawbacks with conventional cancer chemotherapy are the lack of satisfactory specificity towards tumor cells and poor antitumor activity. In order to improve these characteristics, chemotherapeutic drugs can be conjugated to targeting moieties e.g. to peptides with the ability to recognize cancer cells. We have previously reported that combining a tumor homing peptide with a cell-penetrating peptide yields a chimeric peptide with tumor cell specificity that can carry cargo molecules inside the cells. In the present study, we have used a linear breast tumor homing peptide, CREKA, in conjunction with a cell-penetrating peptide, pVEC. This new chimeric peptide, CREKA–pVEC, is more convenient to synthesize and moreover it is better in translocating cargo molecules inside cancer cells as compared to previously published PEGA–pVEC peptide. This study demonstrates that CREKA–pVEC is a suitable vehicle for targeted intracellular delivery of a DNA alkylating agent, chlorambucil, as the chlorambucil–peptide conjugate was substantially better at killing cancer cells in vitro than the anticancer drug alone.