Virotherapeutics: conditionally replicative adenoviruses for viral oncolysis

Viral oncolysis, or virotherapy, is an endeavor to use viruses as therapeutic agents in an effort to exploit their highly evolved qualities of host cell killing and simultaneous multiplication and spread. This review describes the concept of oncolytic adenoviruses, also called conditionally replicative adenoviruses (CRAds), and recent developments--inspired by early clinical results--that aim at the optimization of CRAd efficacy. Molecular strategies applied for the development of oncolytic adenoviruses include (i) the genetic manipulation of the expression and/or function of key regulatory viral proteins in order to restrict viral replication and spread to tumor cells, (ii) the engineering of the adenoviral capsid for efficient and tumor-targeted infection, and (iii) the incorporation of heterologous genes to facilitate combination therapies or tracking of the virus. Initial clinical trials have provided proof-of-concept for adenoviral oncolysis in patients and a favorable safety profile for oncolytic adenoviruses has been demonstrated. In conclusion, adenoviral oncolysis, with its distinct therapeutic mechanism, shows remarkable therapeutic potential. Advanced generations of virotherapeutics are currently in development.     

应用在药物滥用研究中的SPECT和PET分子显像剂的研究进展

1 前言 毒品长期以来就是一个全球性的社会问题,自从药物滥用的基础研究发现其可以引起脑内生物分子的病理变化和脑功能损害后,药物滥用又成为一个全球性的医学难题.     

PET and SPET molecular imaging: focus on serotonin system

Emission tomography techniques and, in particular, positron emission tomography (PET) enable the in vivo study of several physiological and neurochemical variables in human subjects using methods originally developed for quantitative autoradiography. In particular, PET allows one to evaluate in human subjects: (a) the effect of specific neurochemical challenges on regional brain function at rest or under activation; (b) the activity of neurotransmitters and the regional expression of specific molecular targets during pathology including their modulation by drug treatment; (c) the kinetics of drug disposition and activity directly in the target organ. This is of primary interest in the field of biological psychiatry and in psychoactive drugs development, where it is particularly difficult to reproduce human diseases using animal models in view of the peculiarity of this field and the large heterogeneity of each psychiatric illness also inside the same clinical definition. The aim of this paper is to review the principal strategies and the main results of the use of PET or single photon emission tomography (SPET) molecular imaging for the in vivo study of serotonin receptors and the main results obtained from their application in the study of major depression.     

Receptor imaging in the thorax with PET

This review focuses on positron emission tomography (PET)-imaging of receptors in the sympathetic and the parasympathetic systems of heart and lung and highlights the human applications of PET. For the alpha-adrenoceptor, only [11C]GB67 (N2-[6-[(4-amino-6,7-dimethoxy-2-quinazolinyl)(methyl)amino]hexyl]-N2-[11C]methyl-2-furamide hydrochloride) has been developed. Its potential for application in patients needs to be assessed. For both the beta-adrenergic and the muscarinic systems, potent PET radioligands have been prepared and evaluated in patients. It has been possible to measure receptor densities quantitatively in human heart [[11C]MQNB: [11C]methylquinuclidinyl benzilate, [11C]CGP12177: S-(3'-t-butylamino-2'-hydroxypropoxy)-benzimidazol-2-[11C]one and [11C]CGP12388: (S)-4-(3-(2'-[11C]isopropylamino)-2-hydroxypropoxy)-2H-benzimidazol-2-one] and qualitatively in lung [[11C]VC002: N-[11C]-methyl-piperidin-4-yl-2-cyclohexyl-2-hydroxy-2-phenylacetate and [11C]CGP12177]. Besides these subtype nonselective radioligands, the development of compounds that are selective for one subtype are ongoing and have not found successful application in humans yet.     

Approaches using molecular imaging technology -- use of PET in clinical microdose studies

Positron emission tomography (PET) imaging uses minute amounts of radiolabeled drug tracers and thereby meets the criteria for clinical microdose studies. The advantage of PET, when compared to other analytical methods used in microdose studies, is that the pharmacokinetics (PK) of a drug can be determined in the tissue targeted for drug treatment. PET microdosing already offers interesting applications in clinical oncology and in the development of central nervous system pharmaceuticals and is extending its range of application to many other fields of pharmaceutical medicine. Although requirements for preclinical safety testing for microdose studies have been cut down by regulatory authorities, radiopharmaceuticals increasingly need to be produced under good manufacturing practice (GMP) conditions, which increases the costs of PET microdosing studies. Further challenges in PET microdosing include combining PET with other ultrasensitive analytical methods, such as accelerator mass spectrometry (AMS), to gain plasma PK data of drugs, beyond the short PET examination periods. Finally, conducting clinical PET studies with radiolabeled drugs both at micro- and therapeutic doses is encouraged to answer the question of dose linearity in clinical microdosing.     

Establishment and use of a cell line expressing HSV-1 thymidine kinase to characterize viral thymidine kinase-dependent drug-resistance

To understand the mechanisms of antiviral drug resistance and to have a system to examine the cytotoxicity of herpes simplex virus type 1 (HSV-1) inhibitors that are thymidine kinase (TK)-dependent, we have constructed a plasmid pFTK1 by inserting a DNA fragment containing the TK gene of HSV-1 strain F into the eukaryotic expression vector pcDNA3.1/His A. TK-deficient 143B cells were transfected with this vector and neomycin-resistant cells were selected. Cell survival in HAT medium and TK activity of the cell lysates were examined to ascertain HSV-1 TK expression. A cell line expressing the viral TK gene, FTK143B (FTK), was established and used for characterization of two laboratory-derived TK-deficient drug-resistant HSV-1 mutants of strain F. The antiviral activities of several drugs, mostly nucleoside analogues, were compared in the Vero, 143B and FTK cell culture systems. We showed that both mutant viruses lost their resistance to acyclovir and to other HSV-1 TK-dependent compounds in FTK cells but not in Vero and 143B cells. Significantly increased cytotoxicity of ganciclovir and (E)-5-(2-bromovinyl)-2'-deoxyuridine was also observed in the FTK cells. This HSV-1 TK gene-transfected cell model is a useful tool to rapidly determine HSV-1 drug resistance at the viral TK level.     

Pentagalloylglucose downregulates cofilin1 and inhibits HSV-1 infection

To investigate the anti-herpesvirus mechanism of pentagalloylglucose (PGG), we compared the proteomic changes between herpes simplex virus type 1 (HSV-1) infected MRC-5 cells with or without PGG-treatment, and between non-infected MRC-5 cells with or without PGG-treatment by 2-DE and MS-based analysis. Differentially expressed cellular proteins were mainly involved with actin cytoskeleton regulation. Significantly, PGG can down-regulate cofilin1, a key regulator of actin cytoskeleton dynamics. PGG can inhibit HSV-1-induced rearrangements of actin cytoskeleton which is important for infectivity. Furthermore, cofilin1 knockdown by siRNA also inhibited the HSV-1-induced actin-skeleton rearrangements. Both PGG-treatment and cofilin1 knockdown can reduce HSV-1 DNA, mRNA, protein synthesis and virus yields. Altogether, the results suggested that down-regulating cofilin1 plays a role in PGG inhibiting HSV-1 infection. PGG may be a promising anti-herpesvirus agent for drug development.     

Inhibition of herpes simplex virus type 2 (HSV-2) viral replication by the dominant negative mutant polypeptide of HSV-1 origin binding protein.

UL9-C535C, the trans-dominant negative mutant polypeptide of herpes simplex virus type 1 (HSV-1) UL9 origin binding protein, is a potent inhibitor of HSV-1 viral DNA replication. This study focused on testing whether HSV-1 UL9-C535C and a genetically engineered UL9-C535C-encoding HSV-1 recombinant virus CJ83193 could inhibit herpes simplex virus type 2 (HSV-2) infection. First, a stable cell line, R-C535C, expressing a high level of UL9-C535C in the presence of tetracycline and little or no UL9-C535C in the absence of tetracycline was established. The single step growth experiment showed that like HSV-1, the de novo synthesis of HSV-2 could be suppressed approximately 1000-fold by UL9-C535C expressed in R-C535C cells in the presence of tetracycline. Secondly, compared with cells singly infected with HSV-2, co-infection of Vero cells with HSV-2 and CJ83193 reduced the replication efficiency of HSV-2 in co-infected cells by 30-40 fold in a single-step growth assay, which coincided with marked reduction in viral late gene expression, but not the expression of viral immediate-early genes. Taken together, in view of our recent demonstration that CJ83193 can serve as an effective vaccine in preventing HSV-1 infection in mice, one can generate a CJ83193-like HSV-2 recombinant virus that could potentially function as a new therapeutic class of recombinant viral vaccine against HSV-2 infection.     

PET imaging of brain cancer with positron emitter-labeled liposomes

Since nanocarriers such as liposomes are known to accumulate in tumors of tumor-bearing animals, and those that have entrapped a positron emitter can be used to image a tumor by PET, we applied (18)F-labeled 100-nm-sized liposomes for the imaging of brain tumors. Polyethylene glycol (PEG)-modified liposomes, which are known to accumulate in tumors by passive targeting and those modified with Ala-Pro-Arg-Pro-Gly, which are known to home into angiogenic sites were used. Those liposomes labeled with DiI fluorescence accumulated in a glioma implanted in a rat brain 1h after the injection, although they did not accumulate in the normal brain tissues due to the protection afforded by the blood-brain barrier. Preformed liposomes were easily labeled with 1-[(18)F]fluoro-3,6-dioxatetracosane, and enabled the imaging of gliomas by PET with higher contrast than that obtained with [(18)F]deoxyfluoroglucose. In addition, the smallest tumor among those tested, having a diameter of 1mm was successfully imaged by the liposomal (18)F. Therefore, nanocarrier-based imaging of brain tumors is promising for the diagnosis of brain cancer and possible drug delivery-based therapy.     

PET imaging in multiple sclerosis

Positron emission tomography (PET) is a non-invasive technique for quantitative imaging of biochemical and physiological processes in animals and humans. PET uses probes labeled with a radioactive isotope, called PET tracers, which can bind to or be converted by a specific biological target and thus can be applied to detect and monitor different aspects of diseases. The number of applications of PET imaging in multiple sclerosis is still limited. Clinical studies using PET are basically focused on monitoring changes in glucose metabolism and the presence of activated microglia/macrophages in sclerotic lesions. In preclinical studies, PET imaging of targets for other processes, like demyelination and remyelination, has been investigated and may soon be translated to clinical applications. Moreover, more PET tracers that could be relevant for MS are available now, but have not been studied in this context yet. In this review, we summarize the PET imaging studies performed in multiple sclerosis up to now. In addition, we will identify potential applications of PET imaging of processes or targets that are of interest to MS research, but have yet remained largely unexplored.     

PET imaging of norepinephrine transporters

The involvement of the norepinephrine transporter (NET) in the pathophysiology and treatment of attention deficit hyperactivity disorder (ADHD), substance abuse, neurodegenerative disorders (e.g., Alzheimer's disease (AD) and Parkinson's disease (PD)) and depression has long been recognized. However, many of these important findings have resulted from studies in vitro using postmortem tissues; as of now, these results have never been verified via in vivo methods because brain imaging of NET in living systems has been hampered due to the lack of suitable radioligands. The fact that all three monoamine (dopamine, norepinephrine, and serotonin) transporters (DAT, NET and SERT) are involved in various neurological and psychiatric diseases further emphasizes the need to develop suitable NET ligands so that researchers will be able to probe the contributions of each monoamine transporter system to specific CNS disorders. In this review article, the design and biological evaluation of several radioligands for imaging the brain NET system with PET are discussed. Based on these characterization studies, including C-11 labeled desipramine (DMI), 2-hydroxydesipramine (HDMI), talopram, talsupram, nisoxetine (Nis), oxaprotiline (Oxap), lortalamine (Lort) and C-11 and F-18 derivatives of reboxetine (RB), methylreboxetine (MRB) and their individual (R, R) and (S, S) enantiomers, in conjunction with studies with radiolabeled 4-iodo-tomoxetine and 2-iodo-nisoxetine, we have identified the superiority of (S, S)-[(11)C]MRB and the suitability of the MRB analogs as potential NET ligands for PET. In contrast, Nis, Oxap and Lort displayed high uptake in striatum (higher than thalamus). The use of these ligands is further limited by high non-specific binding and relatively low specific signal, as is characteristic of many earlier NET ligands. Thus, to our knowledge, (S, S)-[(11)C]MRB remains by far the most promising NET ligand for PET studies.     

Enterocin CRL35 inhibits late stages of HSV-1 and HSV-2 replication in vitro

The replication of herpes simplex virus (HSV) type 1 and 2 in Vero cells is inhibited in the presence of enterocin CRL35 (ECRL), a bacteriocin produced by Enterococcus faecium CRL35. Attempts to resolve the mode of action of ECRL indicate that virus adsorption and penetration are not affected. Instead, a late step of virus multiplication is hindered since the addition of 100 microg/ml of ECRL at 8h post infection still causes a 90% inhibition of virus release. The effect of ECRL on HSV antigen expression was studied by immunofluorescence using a polyclonal serum and a monoclonal antibody against glycoprotein D (gamma protein). These studies indicated that ECRL impeded the second round of infection, apparently as a consequence of the inhibition of glycoprotein D expression. The replication of syncytial mutants of HSV-1 was significantly inhibited at a ECRL concentration of 25 microg/ml. Both the percentage of fused cells and the polykaryocyte size were affected. Studies on the effect of ECRL on viral protein synthesis showed that in the presence of ECRL, HSV late gamma proteins were not synthesized. From these findings, it is concluded that inhibition of HSV spreading by ECRL is due to the prevention of mainly late glycoprotein synthesis.     

The role of in vivo molecular imaging with PET and SPECT in the elucidation of psychiatric drug action and new drug development

This paper reviews the contribution of human PET and SPECT neuroreceptor occupancy studies to the understanding of drug action in psychiatric illness, and how they can aid the development of new drugs. All effective antipsychotics show significant D₂ receptor occupancy. However, at least for atypical antipsychotics, there is no clear relationship between occupancy and clinical response. The mechanisms underlying antipsychotic efficacy, and the minimal effective D₂ occupancy, remain to be elucidated, particularly for drugs with modest or transient occupancy. The low liability of some atypical antipsychotics for extrapyramidal side effects does not appear to be explained by their 5-HT_2A antagonism, and the muscarinic receptor occupancy of some drugs may be partly explanatory. Previous reports of apparent ‘limbic selectivity’ of atypical antipsychotics may be in error, and may be due to technical differences in radiotracers. For SSRIs, high occupancies at the serotonin transporter (SERT) are achieved at therapeutic doses, although the minimum SERT occupancy required for therapeutic response remains undefined. Previous attempts to augment the antidepressant effect of SSRIs by pindolol have generally used daily doses which result in inadequate 5-HT_1A receptor occupancy. For benzodiazepines, clinical doses would appear to leave a wide margin of unoccupied receptors. For methylphenidate and cocaine, typical doses occupy more than 50% of dopamine transporters, and their profiles are extremely similar. In therapeutic drug development, these techniques may be used to assess receptor occupancy profiles, likely drug dosages and dosing intervals which cannot be reliably assessed in humans by other methods.     

Dendrimer-based contrast agents for PET imaging

Abstract

Positron emission tomography (PET) imaging offers physiological and biological information through the in vivo distribution of PET agents for disease diagnosis, therapy monitoring and prognosis evaluation. Due to the unique structural characteristics allowing for facile modification of targeting ligands and radionuclides, dendrimers can be served as a versatile scaffold to build up various PET imaging agents, and significant breakthroughs have been made in this field over the past decades. This review focuses on the recent advances in dendrimer-based contrast agents for PET imaging of cancer, cardiovascular and other diseases. In particular, radiolabeling strategies for different PET isotopes are described in detail. Several challenges involved in clinical translation of radiolabeled dendrimers are also discussed.