Synthetic Oligonucleotides in SPECT/CT In Vivo Imaging: Chemical Modifications, In111 Complex Formation, Incorporation into Drug Delivery Systems.

Here in we describe a solid phase synthesis of oligonucleotides bearing unnatural moiety appropriate for complex formation with In111 as well as their deprotection, isolation, and purification. We also present methods for oligonucleotides/In111 complex formulation with single and double stranded oligonucleotides of RNA nature and give an example of preparation method for one supramolecular drug delivery system (DDS) consisting of radiolabeled siRNA and positively charged peptide.

Efficient cartridge purification for producing high molar activity [18F]fluoro-glycoconjugates via oxime formation.

INTRODUCTION: 18F-fluoroglycosylation via oxime formation is a chemoselective and mild radiolabeling method for sensitive molecules. Glycosylation can also improve the bioavailability, in vivo kinetics, and stability of the compound in blood, as well as accelerate clearance of biomolecules. A typical synthesis procedure for 18F-fluoroglycosylation with [18F]FDG (2-deoxy-2-[18F]fluoro-d-glucose) and [18F]FDR (5-deoxy-5-[18F]fluoro-d-ribose) involves two HPLC (high performance liquid chromatography) purifications: one after 18F-fluorination of the carbohydrate to remove its labeling precursor, and a second one after the oxime formation step to remove the aminooxy precursor. The two HPLC purifications can be time consuming and complicate the adaptation of the synthetic strategy in nuclear medicine applications and automated synthesis. We have developed a procedure in which SPE (solid phase extraction) and resin purification methods replace both of the needed HPLC purification steps. METHODS: We used [18F]FDR and [18F]FDG as prosthetic groups to radiolabel two aminooxy-modified model molecules, a tetrazine and a PSMA (prostate specific membrane antigen) inhibitor. After fluorination, the excess carbohydrate precursor was removed by derivatizing it with 4,4'-dimethoxytrityl chloride (DMT-Cl). The DMT moiety increases the hydrophobicity of the unreacted precursor making the separation from the fluorinated precursor possible with simple C18 Sep-Pak cartridge. For removal of the aminooxy precursor, we used a commercially available aldehyde resin (AminoLink, Thermo Fisher Scientific). C18 Sep-Pak SPE cartridge was used to separate [18F]FDR and [18F]FDG from the 18F-fluoroglycoconjugate end product. RESULTS: [18F]FDR and [18F]FDG were efficiently purified from their precursors, free fluorine-18, and other impurities. The aldehyde resin quantitatively removed the unreacted aminooxy precursors after the oxime formation. The fluorine-18 labeled oxime end products were obtained with high radiochemical purity (>99%) and molar activity (>600 GBq µmol-1). CONCLUSIONS: We have developed an efficient cartridge purification method for producing high molar activity 18F-glycoconjugates synthesized via oxime formation.

Pharmacokinetic aspects of retinal drug delivery.

Drug delivery to the posterior eye segment is an important challenge in ophthalmology, because many diseases affect the retina and choroid leading to impaired vision or blindness. Currently, intravitreal injections are the method of choice to administer drugs to the retina, but this approach is applicable only in selected cases (e.g. anti-VEGF antibodies and soluble receptors). There are two basic approaches that can be adopted to improve retinal drug delivery: prolonged and/or retina targeted delivery of intravitreal drugs and use of other routes of drug administration, such as periocular, suprachoroidal, sub-retinal, systemic, or topical. Properties of the administration route, drug and delivery system determine the efficacy and safety of these approaches. Pharmacokinetic and pharmacodynamic factors determine the required dosing rates and doses that are needed for drug action. In addition, tolerability factors limit the use of many materials in ocular drug delivery. This review article provides a critical discussion of retinal drug delivery, particularly from the pharmacokinetic point of view. This article does not include an extensive review of drug delivery technologies, because they have already been reviewed several times recently. Instead, we aim to provide a systematic and quantitative view on the pharmacokinetic factors in drug delivery to the posterior eye segment. This review is based on the literature and unpublished data from the authors' laboratory.

Oncolytic adenoviruses coated with MHC-I tumor epitopes increase the antitumor immunity and efficacy against melanoma.

The stimulation of the immune system using oncolytic adenoviruses (OAds) has attracted significant interest and several studies suggested that OAds immunogenicity might be important for their efficacy. Therefore, we developed a versatile and rapid system to adsorb tumor-specific major histocompatibility complex class I (MHC-I) peptides onto the viral surface to drive the immune response toward the tumor epitopes. By studying the model epitope SIINFEKL, we demonstrated that the peptide-coated OAd (PeptiCRAd) retains its infectivity and the cross presentation of the modified-exogenous epitope on MHC-I is not hindered. We then showed that the SIINFEKL-targeting PeptiCRAd achieves a superior antitumor efficacy and increases the percentage of antitumor CD8+ T cells and mature epitope-specific dendritic cells in vivo. PeptiCRAds loaded with clinically relevant tumor epitopes derived from tyrosinase-related protein 2 (TRP-2) and human gp100 could reduce the growth of primary-treated tumors and secondary-untreated melanomas, promoting the expansion of antigen-specific T-cell populations. Finally, we tested PeptiCRAd in humanized mice bearing human melanomas. In this model, a PeptiCRAd targeting the human melanoma-associated antigen A1 (MAGE-A1) and expressing granulocyte and macrophage colony-stimulating factor (GM-CSF) was able to eradicate established tumors and increased the human MAGE-A1-specific CD8+ T cell population. Herein, we show that the immunogenicity of OAds plays a key role in their efficacy and it can be exploited to direct the immune response system toward exogenous tumor epitopes. This versatile and rapid system overcomes the immunodominance of the virus and elicits a tumor-specific immune response, making PeptiCRAd a promising approach for clinical testing.

Impact of protein binding on the analytical detectability and anticancer activity of thymoquinone.

UNLABELLED: Thymoquinone (TQ), an active component of Nigella sativa L., is known to have anti-cancer and anti-inflammatory effects; however, no studies on its analytical detection in serum and its protein binding have been published. Using high performance liquid chromatography analysis, we show that the average recovery of TQ from serum is 2.5% at 10 µg/ml of TQ and 72% at 100 µg/ml. The low recovery of TQ from serum is due to its extensive binding to plasma proteins, as more than 99% of TQ was bound within 30 min of incubation. The binding of TQ to the major plasma proteins, bovine serum albumin (BSA) and alpha -1 acid glycoprotein (AGP), was studied and found to be 94.5 ± 1.7% for BSA and 99.1 ± 0.1% for AGP. Mass spectrometric analysis revealed that TQ was bound covalently to BSA, specifically on Cyst-34. Using WST-1 proliferation assay, we showed that BSA plays a protective role against TQ-induced cell death; pre-incubation with BSA prevented TQ from exerting its anti-proliferative effects against DLD-1 and HCT-116 human colon cancer cells. On the other hand, binding of TQ to AGP did not alter its anti-proliferative activity against both cell lines. When TQ was pre-incubated with AGP prior to the addition of BSA, the activity of TQ against DLD-1 was maintained, suggesting that AGP prevented the binding of TQ to BSA. This is the first time the covalent binding and inhibitory effect of BSA on TQ is documented. These data offer new grounds for TQ future pharmacokinetic analysis in vivo. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12154-010-0052-4) contains supplementary material, which is available to authorized users.

Peptide-oligonucleotide conjugates form stable and selective complexes with antibody and DNA.

The present work demonstrates that the relatively low molecular weight synthetic peptide-oligonucleotide conjugates are capable of stable and selective three-component complex formation with complementary 72-100mer DNA oligonucleotides and a cardiac troponin I monoclonal antibody. Neither the Watson-Crick-type interaction between peptide-oligonucleotide conjugate and DNA nor the conjugate-antibody interaction dramatically hampers the other. These interactions remain selective and specific in the presence of several other conjugates not specific to cardiac troponin I monoclonal antibody as well as in the presence of control 100mer DNA oligonucleotides. The data herein demonstrate the feasibility of the synthetic peptide-oligonucleotide conjugates as convenient molecular tools, e.g., for antibody epitope mapping.

Characterization of antisense oligonucleotide-peptide conjugates with negative ionization electrospray mass spectrometry and liquid chromatography-mass spectrometry.

Covalent post-synthesis or solid-phase conjugation of peptides to oligonucleotides has been reported as a possible method of delivering antisense oligonucleotides into cells. While synthesis strategies for preparing these conjugates have been widely addressed, few detailed reports on their structural characterization have been published. This paper discusses the negative ion electrospray ionization mass spectrometric (ESI-MS) and liquid chromatography-mass spectrometric (LC-MS) analysis of various peptide-oligonucleotide conjugates ranging from small T(6)-nucleopeptides to large peptide-oligonucleotide phosphorothioate conjugates and ribozyme-peptide hybrids (3-13 kDa). Molecular weight determination with mass errors of 0.1-3.1 amu were conducted, employing on-line IP-RP-HPLC and high m/z range mode to facilitate the analysis of large compounds and difficult modifications.