Low-volume jet injection for efficient nonviral in vivo gene transfer

The transfer of naked deoxyribonucleic acid (DNA) represents an alternative to viral and liposomal gene transfer technologies for gene therapy applications. Various procedures are employed to deliver naked DNA into the desired cells or tissues in vitro and in vivo, such as by simple needle injection, particle bombardment, in vivo electroporation or jet injection. Among the various nonviral gene delivery technologies jet injection is gaining increasing acceptance because it allows gene transfer into different tissues with deeper penetration of the applied naked DNA. The versatile hand-held Swiss jet injector uses pressurized air to force small volumes of 3 to 10 μL of naked DNA into targeted tissues. The β-galactosidase (LacZ) reporter gene construct and tumor necrosis factor α gene-expressing vectors were successfully jet injected at a pressure of 3.0 bar into xenotransplanted human tumor models of colon carcinoma. Qualitative and quantitative expression analysis of jet injected tumor tissues revealed the efficient expression of these genes in the tumors. Using this Swiss jet-injector prototype repeated jet injections of low volumes (3–10 μL) into one target tissue can easily be performed. The key parameters of in vivo jet injection such as jet injection volume, pressure, jet penetration into the tumor tissue, DNA stability have been defined for optimized nonviral gene therapy. These studies demonstrate the applicability of the jet injection technology for the efficient and simultaneous in vivo gene transfer of two different plasmid DNAs into tumors. It can be employed for nonviral gene therapy of cancer using minimal amounts of naked DNA.     

Use of the nuclease inhibitor aurintricarboxylic acid (ATA) for improved non-viral intratumoral in vivo gene transfer by jet-injection

BACKGROUND: Stability, integrity and retention of the DNA within the targeted tissue is decisive for efficient gene transfer using naked DNA. Pre-clinical and clinical studies require reproducible transfection rates by preventing rapid degradation of naked DNA in the transduced tissue. Tumor tissues contain nuclease activity, which can affect DNA stability if naked DNA is used. Therefore, inhibition of nuclease-mediated DNA degradation by the nuclease inhibitor aurintricarboxylic acid (ATA) might lead to improved gene transfer efficiency in tumor tissues. METHODS: For both, DNA-degradation analysis and in vivo gene transfer experiments, the beta-galactosidase (LacZ)-expressing pCMVbeta and the cytosine deaminase (CD)-expressing pCMV-CD plasmid were used. Influence of the nuclease inhibitor ATA was determined in tumors, in which naked pCMVbeta or pCMV-CD DNA and ATA was co-administered by jet-injection. The nuclease activity and inhibition by ATA was analyzed using the DNase Alert detection system. The influence of ATA on LacZ expression was determined by specific ELISA and its effect on the therapeutic efficacy of CD gene transfer on tumor growth was determined in vivo. RESULTS: The screening of different human mammary and colon carcinoma models revealed strong nuclease activity rapidly degrading naked plasmid DNA. Co-administration of ATA with pCMVbeta or pCMV-CD for in vivo jet-injection of tumors prevented DNA from nuclease degradation associated with either increased LacZ gene expression or improved reduction in tumor growth. CONCLUSIONS: Tumor-associated nuclease activity is a notable hurdle in gene transfer of naked DNA and therefore inhibition of nucleolytic degradation of plasmid DNA facilitates intratumoral gene expression.     

Metabolomic Analysis of the Effect of Postnatal Hypoxia on the Retina in a Newly Born Piglet Model

The availability of reliable biomarkers of brain injury secondary to birth asphyxia could substantially improve clinical grading, therapeutic intervention strategies, and prognosis. In this study, changes in the metabolome of retinal tissue caused by profound hypoxia in an established neonatal piglet model were investigated using an ultra performance liquid chromatography – quadrupole time of flight mass spectrometry (UPLC-QTOFMS) untargeted metabolomic approach, which included Partial Least Squares – Discriminant Analysis (PLSDA) multivariate data analysis. The initial identification of a set of discriminant metabolites from UPLC-QTOFMS data was confirmed by target UPLC-MS/MS and allowed the selection of endogenous CDP-choline as a promising candidate biomarker for hypoxia-derived brain damage assessing intensity of retinal hypoxia. Results from this study will foster further research on CDP-choline changes occurring during resuscitation.     

Enzymatic determination of BPA by means of tyrosinase immobilized on different carbon carriers

Different tyrosinase carbon paste modified electrodes to determine bisphenol A (BPA) concentration in aqueous solutions have been constructed. Variables examined were in the carbon paste composition and in particular: (i) the immobilized enzyme amount; (ii) the carbon type (powder, single or multi-walled nanotubes); (iii) the nature of the pasting oil (mineral oil, hexadecane and dodecane). For each biosensor type the amperometric response was evaluated with reference to the linear range and sensitivity. Constant reference has been made to the amperometric signals obtained, under the same experimental conditions, towards the catechol, a specific phenolic substrate for tyrosinase. The most efficient biosensors were those constructed by using the following composition for the carbon paste: 10% of tyrosinase, 45% of single wall carbon nanotubes (SWCN) and 45% of mineral oil. This biosensor formulation displayed the following electrochemical characteristics: a sensitivity equal to 138 microA/mM, LOD of 0.02 microM (based on three times the S/N ratio), linear range of 0.1-12 microM and response time of 6 min. This experimental work represents a first attempt at construction of a new carbon nanotube-tyrosinase based biosensor able to determine the concentration of BPA, one of the most ubiquitous and hazardous endocrine disruptors which can pollute the drinking and surface water, as well as many products of the food chain.     

Development of albumin-binding camptothecin prodrugs using a Peptide positional scanning library

Designing truly tumor-specific prodrugs remains a challenge in the field of cancer chemotherapy. As a new strategy, we incubated homogenates of a spectrum of human colon tumor xenografts with a fluorogenic positional scanning tetrapeptide library in order to identify peptide sequences that are preferentially cleaved by colon tumors. Our screening experiments at pH 7.4 revealed that Met, Leu, and Lys were preferred amino acids in the position P(1) and Tyr, Phe, and Met in P(2), whereas in P(3) and P(4), the cleavage profiles were less characteristic. However, similar results were obtained when testing breast tumor material and homogenates from healthy murine organs. On the basis of these results, we developed albumin-binding camptothecin (CPT) prodrugs of the general formula EMC-Arg-P(4)-P(3)-P(2)-P(1)-Ala-CPT (EMC = 6-maleimidocaproic acid) that incorporated two peptide linkers: H-Arg-Ala-Phe-Met-OH and H-Arg-Phe-Tyr-Met-OH (P(4)-P(3)-P(2)-P(1)). The incorporation of two arginine residues rendered the prodrugs water-soluble (>7 mg/mL), while the use of alanine as an amino acid spacer proved to be beneficial for the release of the active agent. Incubation studies with homogenates of HT-29 colon tumor tissue and murine spleen, liver, and kidneys demonstrated cleavage of the peptide linker with CPT-peptide derivatives and CPT being the major cleavage products. Although the peptide sequence is not selectively cleaved in colon tumors, an in vivo study in a HT-29 xenograft model showed that the prodrug EMC-Arg-Arg-Ala-Phe-Met-Ala-CPT demonstrated enhanced antitumor efficacy when compared to CPT [( T/ C max: 17% for the prodrug (2 x 12.5 mg/kg CPT equivalents) and 40% for CPT (3 x 12.5 mg/kg)].     

Albumin-binding prodrugs of camptothecin and doxorubicin with an Ala-Leu-Ala-Leu-linker that are cleaved by cathepsin B: synthesis and antitumor efficacy

We have recently validated a macromolecular prodrug strategy for improved cancer chemotherapy based on two features: (a) rapid and selective binding of thiol-reactive prodrugs to the cysteine-34 position of endogenous albumin and (b) acid-sensitive promoted or enzymatic release of the drug at the tumor site [Kratz, F., Warnecke, A., Scheuemann, K., Stockmar, C., Schwab, J., Lazar, P., Druckes, P., Esser, N., Drevs, J., Rognan, D., Bissantz, C., Hinderling, C., Folkers, G., Fichtner, I., and Unger, C. (2002) J. Med. Chem. 45, 5523-33]. In the present work, we developed water-soluble camptothecin (CPT) and doxorubicin (DOXO) prodrugs that incorporate the peptide linker Ala-Leu-Ala-Leu that serves as a substrate for the tumor-associated protease, cathepsin B, which is overexpressed in several solid tumors. Consequently, two albumin-binding prodrugs were synthesized [EMC-Arg-Arg-Ala-Leu-Ala-Leu-Ala-CPT (1) and EMC-Arg-Arg-Ala-Leu-Ala-Leu-DOXO (2) (EMC = 6-maleimidocaproic acid)]. Both prodrugs exhibited excellent water-solubility and bound rapidly and selectively to the cysteine-34 position of endogenous albumin. Further in vitro studies showed that the albumin-bound form of the prodrugs was cleaved specifically by cathepsin B as well as in human tumor homogenates. Major cleavage products were CPT-peptide derivatives and CPT for the CPT prodrug and H-Leu-Ala-Leu-DOXO, H-Leu-DOXO, and DOXO for the doxorubicin prodrug. In vivo, 1 was superior to free camptothecin in an HT-29 human colon xenograft model; the antitumor efficacy of prodrug 2 was comparable to that of free doxorubicin in the M-3366 mamma carcinoma xenograft model at equimolar doses.     

Antitumor efficacy,pharmacokinetic and biodistribution studies of the anticancer peptide CIGB‐552 in mouse models

Accumulation of the COMMD1 protein as a druggable pharmacology event to target cancer cells has not been evaluated so far in cancer animal models. We have previously demonstrated that a second‐generation peptide, with cell‐penetrating capacity, termed CIGB‐552, was able to induce apoptosis mediated by stabilization of COMMD1. Here, we explore the antitumor effect by subcutaneous administration of CIGB‐552 in a therapeutic schedule. Outstandingly, a significant delay of tumor growth was observed at 0.2 and 0.7 mg/kg (p < 0.01) or 1.4 mg/kg (p < 0.001) after CIGB‐552 administration in both syngeneic murine tumors and patient‐derived xenograft models. Furthermore, we evidenced that ¹³¹I‐CIGB‐552 peptide was actually accumulated in the tumors after administration by subcutaneous route. A typical serine‐proteases degradation pattern for CIGB‐552 in BALB/c mice serum was identified. Further, biological characterization of the main metabolites of the peptide CIGB‐552 suggests that the cell‐penetrating capacity plays an important role in the cytotoxic activity. This report is the first in describing the antitumor effect induced by systemic administration of a peptide that targets COMMD1 for stabilization. Moreover, our data reinforce the perspectives of CIGB‐552 for cancer targeted therapy. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

In vitro and in vivo evaluation of doxorubicin conjugates with the divalent peptide E-[c(RGDfK)2] that targets integrin alphavbeta3

Integrins, especially integrin alpha vbeta3, are attractive receptors for vascular targeting strategies. Recently, a divalent RGD peptidomimetic, E-[c(RGDfK)2], has been described that demonstrates increased uptake in human ovarian carcinoma OVCAR-3 xenograft tumors. Inspired by these results, we set out to develop doxorubicin conjugates with E-[c(RGDfK)2] by binding two different maleimide derivatives of doxorubicin to E-[c(RGDfK)2] that was thiolated with iminothiolane. In this way, two water-soluble derivatives were obtained, E-[c(RGDfK)2]-DOXO-1 and E-[c(RGDfK)2]-DOXO-2. In E-[c(RGDfK)2]-DOXO-1, doxorubicin was bound to the peptide through a stable amide bond, and in E-[c(RGDfK)2]-DOXO-2, a MMP-2/MMP-9 cleavable octapeptide was introduced between doxorubicin and the peptide. The rationale for a MMP-2/MMP-9-cleavable linker was that MMP-2 and MMP-9 bind to integrin alpha vbeta3 and both are overexpressed in tumor vasculature. In addition, analogous control doxorubicin-containing peptides bearing c(RADfK) that does not bind to integrin alpha vbeta3 were synthesized, i.e., c(RADfK)-DOXO-1 and c(RADfK)-DOXO-2. Whereas E-[c(RGDfK) 2]-DOXO-2 was cleaved effectively by MMP-2 and in OVCAR-3 tumor homogenates releasing a doxorubicin-tetrapeptide or doxorubicin as the final cleavage product, no release of doxorubicin was observed for E-[c(RGDfK)2]-DOXO-1. Proliferation of HUVEC in the presence of MMP-2-cleavable doxorubicin-containing peptides exhibited 6- to 10-fold increased inhibition compared to the amide-linked doxorubicin-containing peptides. In addition, inhibition of HUVEC sprouting during a 24 h exposure was approximately 3-fold stronger for E-[c(RGDfK) 2]-DOXO-2 and 20-fold stronger for the reference peptide conjugate c(RADfK)-DOXO-2 than for doxorubicin alone. In vivo studies in an OVCAR-3 xenograft model demonstrated no or only moderate antitumor efficacy for either E-[c(RGDfK)2], E-[c(RGDfK)2]-DOXO-1, E-[c(RGDfK)2]-DOXO-2, or c(RADfK)-DOXO-2, even at doses of 3 x 24 mg/kg doxorubicin equivalents, compared to an improved antitumor effect for doxorubicin at 2 x 8 mg/kg.     

Germinal‐center kinase‐like kinase co‐crystal structure reveals a swapped activation loop and C‐terminal extension

Germinal‐center kinase‐like kinase (GLK, Map4k3), a GCK‐I family kinase, plays multiple roles in regulating apoptosis, amino acid sensing, and immune signaling. We describe here the crystal structure of an activation loop mutant of GLK kinase domain bound to an inhibitor. The structure reveals a weakly associated, activation‐loop swapped dimer with more than 20 amino acids of ordered density at the carboxy‐terminus. This C‐terminal PEST region binds intermolecularly to the hydrophobic groove of the N‐terminal domain of a neighboring molecule. Although the GLK activation loop mutant crystallized demonstrates reduced kinase activity, its structure demonstrates all the hallmarks of an “active” kinase, including the salt bridge between the C‐helix glutamate and the catalytic lysine. Our compound displacement data suggests that the effect of the Ser170Ala mutation in reducing kinase activity is likely due to its effect in reducing substrate peptide binding affinity rather than reducing ATP binding or ATP turnover. This report details the first structure of GLK; comparison of its activation loop sequence and P‐loop structure to that of Map4k4 suggests ideas for designing inhibitors that can distinguish between these family members to achieve selective pharmacological inhibitors.