Effects of transcranial magnetic stimulation on sleep structure and quality in children with autism.

Introduction: Sleep disorders are common in children with autism spectrum disorder (ASD). Transcranial magnetic stimulation (TMS) can influence the excitability of neuronal cells in stimulated areas, leading to improvements in sleep and other autistic symptoms. However, studies on clinical mechanisms of TMS in treating sleep disorders associated with ASD are limited. Therefore, we aimed to explore the effects of TMS on sleep structure and quality in children with ASD. Methods: Between January 2020 and December 2021, recruitment was advertised through child and adolescent outpatient clinics and online platforms by the Hangzhou Seventh People's Hospital and Lishui Second People's Hospital. Sixty children with ASD who met the diagnostic criteria of the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition, were selected and randomly divided into the active TMS and sham TMS treatment groups. Thirty healthy children of the same age were recruited as controls. The active TMS group received bilateral low-frequency (0.5 Hz) TMS targeting the dorsolateral prefrontal cortex on both sides in children with ASD, whereas the sham TMS group received sham stimulation with the same stimulation time and location as the experimental group. Both groups were treated for 6 weeks, and the participants were assessed using the Sleep Disturbance Scale for Children (SDSC) before treatment, at 3 weeks, and at 6 weeks of intervention. Independent sample t-tests and difference t-tests were used for statistical analysis of the data. Results: No significant differences were observed in general demographic variables, such as age and sex, between the ASD and control groups (P>0.05). Independent sample t-test analysis showed that the total SDSC score, difficulty falling asleep, sleep maintenance, awakening disorders, sleep-wake transition disorders, excessive daytime sleepiness, and nocturnal hyperhidrosis scores were significantly higher in the ASD group than in the control group (P<0.05). Before treatment, no significant differences were observed in the factor or total SDSC scores between the sham TMS group and the active TMS group (P>0.05). After 15 and 30 treatment sessions, the total SDSC score, difficulty falling asleep, sleep maintenance, sleep-wake transition disorders, and excessive daytime sleepiness scores were significantly higher in the sham TMS group than in the active TMS group (P<0.05). The difference t-test analysis showed that after 30 treatment sessions, the reduction rates of the total SDSC score, difficulty falling asleep, sleep maintenance, awakening disorders, sleep-wake transition disorders, excessive daytime sleepiness, and nocturnal hyperhidrosis dimensions were significantly higher in the active TMS group than in the sham TMS group (P<0.05). Conclusion: Low-frequency TMS targeting the dorsolateral prefrontal cortex in children with ASD can effectively improve their sleep status, and significant improvement can be achieved after 6 weeks (30 sessions) of treatment.

Cognitive enhancing effect of rTMS combined with tDCS in patients with major depressive disorder: a double-blind, randomized, sham-controlled study.

BACKGROUND: Cognitive dysfunction is one of the common symptoms in patients with major depressive disorder (MDD). Repetitive transcranial magnetic stimulation (rTMS) and transcranial direct current stimulation (tDCS) have been studied separately in the treatment of cognitive dysfunction in MDD patients. We aimed to investigate the effectiveness and safety of rTMS combined with tDCS as a new therapy to improve neurocognitive impairment in MDD patients. METHODS: In this brief 2-week, double-blind, randomized, and sham-controlled trial, a total of 550 patients were screened, and 240 MDD inpatients were randomized into four groups (active rTMS + active tDCS, active rTMS + sham tDCS, sham rTMS + active tDCS, sham rTMS + sham tDCS). Finally, 203 patients completed the study and received 10 treatment sessions over a 2-week period. The Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) was performed to assess patients' cognitive function at baseline and week 2. Also, we applied the 24-item Hamilton Depression Rating Scale (HDRS-24) to assess patients' depressive symptoms at baseline and week 2. RESULTS: After 10 sessions of treatment, the rTMS combined with the tDCS group showed more significant improvements in the RBANS total score, immediate memory, and visuospatial/constructional index score (all p < 0.05). Moreover, post hoc tests revealed a significant increase in the RBANS total score and Visuospatial/Constructional in the combined treatment group compared to the other three groups but in the immediate memory, the combined treatment group only showed a better improvement than the sham group. The results also showed the RBANS total score increased significantly higher in the active rTMS group compared with the sham group. However, rTMS or tDCS alone was not superior to the sham group in terms of other cognitive performance. In addition, the rTMS combined with the tDCS group showed a greater reduction in HDRS-24 total score and a better depression response rate than the other three groups. CONCLUSIONS: rTMS combined with tDCS treatment is more effective than any single intervention in treating cognitive dysfunction and depressive symptoms in MDD patients. TRIAL REGISTRATION: Chinese Clinical Trial Registry (ChiCTR2100052122).

TMS-evoked potential in the dorsolateral prefrontal cortex to assess the severity of depression disease: a TMS-EEG study.

Objective: The combined use of transcranial magnetic stimulation and electroencephalography (TMS-EEG), as a powerful technique that can non-invasively probe the state of the brain, can be used as a method to study neurophysiological markers in the field of psychiatric disorders and discover potential diagnostic predictors. This study used TMS-evoked potentials (TEPs) to study the cortical activity of patients with major depressive disorder depression (MDD) and the correlation with clinical symptoms to provide an electrophysiological basis for the clinical diagnosis. Methods: A total of 41 patients and 42 healthy controls were recruited to study. Using TMS-EEG techniques to measure the left dorsolateral prefrontal cortex (DLPFC) 's TEP index and evaluate the clinical symptoms of MDD patients using the Hamilton Depression Scale-24 (HAMD-24). Results: MDD subjects performing TMS-EEG on the DLPFC showed lower cortical excitability P60 index levels than healthy controls. Further analysis revealed that the degree of P60 excitability within the DLPFC of MDD patients was significantly negatively correlated with the severity of depression. Conclusion: The low levels of P60 exhibited in DLPFC reflect low excitability in MDD; the P60 component can be used as a biomarker for MDD in clinical assessment tools.

Motor cortical plasticity as a predictor of treatment response to high frequency repetitive transcranial magnetic stimulation (rTMS) for cognitive function in drug-naive patients with major depressive disorder.

BACKGROUND: There is growing evidence that repetitive transcranial magnetic stimulation (rTMS) can improve cognitive function in patients with major depressive disorder (MDD). Few biomarkers are currently available to predict cognitive response in MDD patients. This study aimed to examine whether cortical plasticity played an important role in improving cognitive deficits in MDD patients treated with rTMS. METHODS: A total of 66 MDD patients and 53 healthy controls were recruited. MDD patients were randomly assigned to receive 10 Hz active or sham rTMS 5 days per week for 4 weeks. Cognitive function was assessed using the Repeatable Battery for assessing Neuropsychological Status (RBANS), while depressive symptoms were assessed with the Hamilton Rating Scale for Depression (HRSD-24) before and after treatment. We combined transcranial magnetic stimulation and muscle surface electrophysiological recording to measure plasticity in motor cortex areas in healthy controls at baseline and MDD patients before and after treatment. RESULTS: Compared with healthy controls, cortical plasticity was impaired in MDD patients. Moreover, cortical plasticity was correlated with RBANS total score at baseline in MDD patients. After 4-week 10 Hz rTMS treatment, the impaired cortical plasticity was restored to some extent. Interestingly, 10 Hz rTMS treatment produced effective therapeutic effects on immediate memory, attention, and RBANS total score. Pearson correlation analysis shows that improvements in plasticity were positively correlated with improvement of immediate memory and RBANS total score. CONCLUSIONS: Our results show for the first time that 10 Hz rTMS can effectively treat impaired cortical plasticity and cognitive impairment in MDD patients and that changes in plasticity and cognitive function are closely related, which may indicate that motor cortical plasticity may play a vital role in cognitive impairment and that cortical plasticity may serve as a potential predictive biomarker for cognitive improvement in MDD patients.

Glioma-associated mesenchymal stem cells-mediated PD-L1 expression is attenuated by Ad5-Ki67/IL-15 in GBM treatment.

BACKGROUND: Glioblastoma (GBM) is a highly immunosuppressive and vascular malignant brain tumor. Current therapeutic strategies targeting tumor cells have limited efficacy because of the immunosuppressive microenvironment and vascularization. Glioma-associated mesenchymal stem cells (GA-MSCs) have been identified as important stromal components of the tumor microenvironment, owing to their contribution to tumor angiogenesis and their potential to drive glioma stem cells. However, there are no reports on the effect of oncolytic Ad5-Ki67/IL-15 on programmed death ligand 1 (PD-L1) expression and angiogenesis induced by GA-MSCs. METHODS: Flow cytometry was respectively performed to detect the PD-L1 of glioma cells and programmed death protein 1 (PD-1), CD3, CD4 and CD8 in lymphocytes, as well as distribution of the cell cycle. CCK-8 assay investigated the proliferation of glioma cells and GA-MSCs in vitro. Tumor-bearing nude mice were established with U87-Luc cells and treated with the viruses, and further the IVIS spectrum was utilized to obtain luciferase images. Finally, the expression of PD-L1 in tumor tissues was also investigated using western blotting. RESULTS: We found that GA-MSCs had potential to induce PD-L1 upregulation and involved in vascular mimicry in vitro. Importantly, Ad5-Ki67/IL-15 reduced PD-L1 expression of glioma cells and neovascularization by targeting GA-MSCs. Furthermore, despite the presence of GA-MSCs, the virus has the ability to generate potent antitumor efficacy in vitro and vivo. CONCLUSIONS: These findings suggest the use of oncolytic Ad5-Ki67/IL-15 targeting GA-MSCs to treat GBM, indicating potential clinical applications.

Enhancement of CD70-specific CAR T treatment by IFN-γ released from oHSV-1-infected glioblastoma.

Even with progressive combination treatments, the prognosis of patients with glioblastoma (GBM) remains extremely poor. OV is one of the new promising therapeutic strategies to treat human GBM. OVs stimulate immune cells to release cytokines such as IFN-γ during oncolysis, further improve tumor microenvironment (TME) and enhance therapeutic efficacy. IFN-γ plays vital role in the apoptosis of tumor cells and recruitment of tumor-infiltrating T cells. We hypothesized that oncolytic herpes simplex virus-1 (oHSV-1) enhanced the antitumor efficacy of novel CD70-specific chimeric antigen receptor (CAR) T cells by T cell infiltration and IFN-γ release. In this study, oHSV-1 has the potential to stimulate IFN-γ secretion of tumor cells rather than T cell secretion and lead to an increase of T cell activity, as well as CD70-specific CAR T cells can specifically recognize and kill tumor cells in vitro. Specifically, combinational therapy with CD70-specific CAR T and oHSV-1 promotes tumor degradation by enhancing pro-inflammatory circumstances and reducing anti-inflammatory factors in vitro. More importantly, combined therapy generated potent antitumor efficacy, increased the proportion of T cells and natural killer cells in TME, and reduced regulatory T cells and transformed growth factor-ß1 expression in orthotopic xenotransplanted animal model of GBM. In summary, we reveal that oHSV-1 enhance the therapeutic efficacy of CD70-spefific CAR T cells by intratumoral T cell infiltration and IFN-γ release, supporting the use of CAR T therapy in GBM therapeutic strategies.

Targeting Tumor-Associated Antigen: A Promising CAR-T Therapeutic Strategy for Glioblastoma Treatment.

Chimeric antigen receptor T cells (CAR-T) therapy is a prospective therapeutic strategy for blood cancers tumor, especially leukemia, but it is not effective for solid tumors. Glioblastoma (GBM) is a highly immunosuppressive and deadly malignant tumor with poor responses to immunotherapies. Although CAR-T therapeutic strategies were used for glioma in preclinical trials, the current proliferation activity of CAR-T is not sufficient, and malignant glioma usually recruit immunosuppressive cells to form a tumor microenvironment that hinders CAR-T infiltration, depletes CAR-T, and impairs their efficacy. Moreover, specific environments such as hypoxia and nutritional deficiency can hinder the killing effect of CAR-T, limiting their therapeutic effect. The normal brain lack lymphocytes, but CAR-T usually can recognize specific antigens and regulate the tumor immune microenvironment to increase and decrease pro- and anti-inflammatory factors, respectively. This increases the number of T cells and ultimately enhances anti-tumor effects. CAR-T therapy has become an indispensable modality for glioma due to the specific tumor-associated antigens (TAAs). This review describes the characteristics of CAR-T specific antigen recognition and changing tumor immune microenvironment, as well as ongoing research into CAR-T therapy targeting TAAs in GBM and their potential clinical application.

Balancing Properties with Carboxylates: A Lead Optimization Campaign for Selective and Orally Active CDK9 Inhibitors.

Cyclin-dependent kinase 9 (CDK9) is a serine/threonine kinase involved in the regulation of transcription elongation. An inhibition of CDK9 downregulates a number of short-lived proteins responsible for tumor maintenance and survival, including the antiapoptotic BCL-2 family member MCL-1. As pan-CDK inhibitors under development have faced dosing and toxicity challenges in the clinical setting, we generated selective CDK9 inhibitors that could be amenable to an oral administration. Here, we report the lead optimization of a series of azaindole-based inhibitors. To overcome early challenges with promiscuity and cardiovascular toxicity, carboxylates were introduced into the pharmacophore en route to compounds such as 14 and 16. These CDK9 inhibitors demonstrated a reduced toxicity, adequate pharmacokinetic properties, and a robust in vivo efficacy in mice upon oral dosing.

Application value of UPLC-MS/MS in detecting serum concentration of anti-schizophrenic drugs in patients with mental illnes.

OBJECTIVE: This study was designed to demonstrate the accuracy of ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) in detecting serum concentration of anti-schizophrenic drugs in patients with mental illness. METHODS: The study participants were 186 schizophrenia patients treated in our hospital. Serum concentrations of anti-schizophrenic drugs in Chinese patients with mental illness were evaluated according to the reference intervals of drug therapy recommended in the guidelines. RESULTS: Five drugs, namely Aripiprazole (ARI), Amisulpride (AMI), Olanzapine (OLA), Paliperidone (PAL) and Ziprasidone (ZIP) all showed good linearity within the linear range. The within-day precision of the above five drugs was all between 1.3%-8.9% and the inter-day precision was between 1.8%-7.6%, with within-day and inter-day relative standard deviations (RSDs) less than 15.00% and accuracy ranging from 87.00% to 106.73%. However, AMI had a mean blood concentration of 436.31±241.05 ng/mL (median concentration: 379.34 ng/mL), which was significantly higher than the reference range (100-320 ng/mL) recommended in the guidelines. Good recovery rates (86.21%-99.77%) were obtained after the samples were stored at room temperature for 24 h, at 4°C for 48h and at -20°C for half a year. CONCLUSIONS: Given that UPLC-MS/MS renders more accurate results in detecting the concentration of psychotropic drugs, it can be applied clinically to detect the concentration of therapeutic drugs in patients with mental illness.

Macrophage polarization contributes to the efficacy of an oncolytic HSV-1 targeting human uveal melanoma in a murine xenograft model.

Uveal melanoma (UM), the most common primary malignant tumor of the eye in adults, is difficult-to-treat. UM has a relatively high mortality secondary to distant metastasis and poor overall survival with existing therapies. The oncolytic virus herpes simplex virus type-1 (HSV-1) has been approved for clinical use in melanoma. This double-stranded DNA virus was suspected to directly activate lysis specifically in neoplastic cells. We tested the antitumor efficacy of recombinant oncolytic HSV-1-EGFP (oHSV-EGFP) in UM and characterized the local and systemic antitumor innate immune response in a murine xenograft model. We first determined the efficacy of the oncolytic virus in 92.1, MUM2B and MP41 cell lines. In murine xenograft models, oHSV-EGFP reduced intraocular tumors as well as systemic subcutaneous tumors. A significant change in cytokines was observed in viral infected cells, especially a rise in IFNγ. In vivo analyses showed increased anti-tumorigenic M1 macrophages and decreased pro-tumorigenic M2 macrophages in peripheral blood, and intraocular and distant tumors after intravitreal viral treatment. Increased infiltration of natural killer cells and mature dendritic cells was also detected after viral treatment. In addition, no virus was detected in major organs after the treatment. Our data support that oHSV-EGFP is effective, neoplasm specific, immune active and safe, providing possible clinical translatable options to treat ocular and metastatic UM.

Efficacy of a novel double-controlled oncolytic adenovirus driven by the Ki67 core promoter and armed with IL-15 against glioblastoma cells.

BACKGROUND: Glioblastoma (GBM) is an immunosuppressive, highly vascular and devastating malignant brain tumor. Even with progressive combination treatment that includes surgery, radiotherapy, and chemotherapy, the prognosis for GBM patients is still extremely poor. Oncolytic adenovirus (OAd) can specifically replicate in GBM cells, permitting the rapid copy of the therapeutic genes it carries. Moreover, E1A is an essential gene in adenoviral replication and is the first gene expressed upon viral infection. E1A expression can be regulated by the Ki67 promoter, while the CMV promoter drives therapeutic gene expression. However, the efficacy of a double-controlled OAd driven by the Ki67 core promoter and armed with IL-15 against GBM cells has not been investigated. METHODS: Fluorescence microscopy was performed to evaluate infection ability in the viruses. Cell viability was detected by CCK-8 assay. Levels of cytokines in different supernatants were determined by ELISA, and IL-15 gene expression was measured by RT-PCR. Angiogenic capacity was analyzed by tube formation assay. RESULTS: We successfully constructed a double-controlled oncolytic adenovirus driven by the Ki67 core promoter and armed with IL-15 that selectively infected and killed GBM cells while sparing normal cells. The adenoviruses prime IL-15 gene expression to significantly enhance anti-GBM efficacy through effective activation of microglial cells. Moreover, OAd not only directly inhibits angiogenesis but exhibits potent antiangiogenic capacity mediated by the reduction of VEGF secretion. CONCLUSIONS: These results provide new insight into the effects of a novel double-controlled OAd driven by the Ki67 core promoter and armed with IL-15 in glioblastoma treatment, which may help in the development of novel therapies in solid tumors.

Antitumor efficacy of oncolytic HSV-1 expressing cytosine deaminase is synergistically enhanced by DPD down-regulation and EMT inhibition in uveal melanoma xenograft.

Uveal melanoma (UM) is the most common intraocular tumor in adults and has a high incidence of metastases. Possible treatments remain limited in UM with enucleation and radiation, leading to poor prognosis in this chemo-resistant carcinoma. Thus, urging demand for novel treatment is needed. We examined the antitumor efficacy of a new recombinant oncolytic herpes simplex virus type 1 (oHSV-1) armed with E.coli cytosine deaminase (CD). We determined the efficacy of the oncolytic virus in UM cell lines. In vivo experiments showed that oHSV-CD/5-fluorocytosine (5-FC) treatment reduce tumor volume and prolonged survival. We further demonstrated the molecular mechanisms of oHSV-CD/5-FC treatment. The oncolytic virus down-regulated IL-6 expression and thereby reversed the epithelial-mesenchymal transition (EMT) phenotype. Dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme in 5-fluorouracil (5-FU) metabolism, was also down-regulated. Therefore, the efficacy of oHSV-CD/5-FC was synergistically enhanced by DPD down-regulation and EMT inhibition. This study provides solid evidence for the antitumor efficacy of oHSV-CD/5-FC treatment in vitro and in vivo. The molecular mechanisms of this treatment may bring a new therapeutic approach for future treatment of UM.

Decreased Intrinsic Functional Connectivity of the Salience Network in Drug-Naïve Patients With Obsessive-Compulsive Disorder.

Obsessive-compulsive disorder (OCD) patients have difficulty in switching between obsessive thought and compulsive behavior, which may be related to the dysfunction of the salience network (SN). However, little is known about the changes in intra- and inter- intrinsic functional connectivity (iFC) of the SN in patients with OCD. In this study, we parceled the SN into 19 subregions and investigated iFC changes for each of these subregions in 40 drug-naïve patients with OCD and 40 healthy controls (HCs) using seed-based functional connectivity resting-state functional magnetic resonance imaging (rs-fMRI). We found that patients with OCD exhibited decreased iFC strength between subregions of the SN, as well as decreased inter-network connectivity between SN and DMN, and ECN. These findings highlight a specific alteration in iFC patterns associated with SN in patients with OCD and provide new insights into the dysfunctional brain organization of the SN in patients with OCD.

Enhanced antitumor efficacy of an oncolytic herpes simplex virus expressing an endostatin-angiostatin fusion gene in human glioblastoma stem cell xenografts.

Viruses have demonstrated strong potential for the therapeutic targeting of glioblastoma stem cells (GSCs). In this study, the use of a herpes simplex virus carrying endostatin-angiostatin (VAE) as a novel therapeutic targeting strategy for glioblastoma-derived cancer stem cells was investigated. We isolated six stable GSC-enriched cultures from 36 human glioblastoma specimens and selected one of the stable GSCs lines for establishing GSC-carrying orthotopic nude mouse models. The following results were obtained: (a) VAE rapidly proliferated in GSCs and expressed endo-angio in vitro and in vivo 48 h and 10 d after infection, respectively; (b) compared with the control gliomas treated with rHSV or Endostar, the subcutaneous gliomas derived from the GSCs showed a significant reduction in microvessel density after VAE treatment; (c) compared with the control, a significant improvement was observed in the length of the survival of mice with intracranial and subcutaneous gliomas treated with VAE; (d) MRI analysis showed that the tumor volumes of the intracranial gliomas generated by GSCs remarkably decreased after 10 d of VAE treatment compared with the controls. In conclusion, VAE demonstrated oncolytic therapeutic efficacy in animal models of human GSCs and expressed an endostatin-angiostatin fusion gene, which enhanced antitumor efficacy most likely by restricting tumor microvasculature development.

[Design of next generation antibody drug conjugates].

Chemotherapy remains one of the major tools, along with surgery, radiotherapy, and more recently targeted therapy, in the war against cancer. There have appeared a plethora of highly potent cytotoxic drugs but the poor discriminability between cancerous and healthy cells of these agents limits their broader application in clinical settings. Therapeutic antibodies have emerged as an important class of biological anticancer agents, thanks to their ability in specific binding to tumor-associated antigens. While this important class of biologics can be used as single agents for the treatment of cancer through antibody-dependent cell cytotoxicity (ADCC), their therapeutical efficacy is often limited. Antitumor antibody drug conjugates (ADCs) combine the target-specificity of monoclonal antibody (mAb) and the highly active cell-killing drugs, taking advantages of the best characteristics out of both components. Thus, insufficiency of most naked mAbs in cancer therapy has been circumvented by arming the immunoglobulin with cytotoxic drugs. Here mAbs are used as vehicles to transport potent payloads to tumor cells. ADCs contain three main components: antibody, linker and cytotoxics (also frequently referred as payload). Antibodies can recognize and specifically bind to the tumor-specific antigens, leading to an antibody-assisted internalization, and payload release. While ADC has demonstrated tremendous success, a number of practical challenges limit the broader applications of this new class of anticancer therapy, including inefficient cellular uptake, low cytotoxicity, and off-target effects. This review article aims to cover recent advances in optimizing linkers with increased stability in circulation while allowing efficient payload release within tumor cells. We also attempt to provide some practical strategies in resolving the current challenges in this attractive research area, particularly to those new to the field.

VP22 and cytosine deaminase fusion gene modified tissue-engineered neural stem cells for glioma therapy.

PURPOSE: The herpes simplex virus type 1 tegument protein VP22 has the remarkable property of intercellular trafficking, thus making it a promising tool for improving gene transfer efficiency. METHODS: To investigate whether the fusion of VP22 to the cytosine deaminase (CD) suicide gene could enhance the therapeutic efficiency of neural stem cells (NSCs) in the treatment for C6 glioma, the lentiviral vectors pHIV-VP(22)-EGFP, pHIV-CD, and pHIV-VP(22)-CD were constructed based on the pHIV-EGFP vector. After packaging, vectors were transduced into rat NSCs. RESULTS: Fluorescence-activated cell sorting analysis revealed that the fusion of VP22-EGFP increased the expression rate of EGFP in NSCs compared with lenti-EGFP transduced cells. Under incubation with the prodrug 5-fluorocytosine (5-FC), the survival rates of C6 cells co-cultured with NSCs/VP(22)-CD (NSCs transduced with lenti-VP(22)-CD) decreased tremendously compared with those of C6 and NSCs/CD. Similar results were also observed in vivo; a significant reduction in tumor volumes in C6 glioma-bearing rats was observed in the NSCs/VP(22)-CD therapy group when compared with other control groups. CONCLUSIONS: Our results reveal that VP22 increases the transduction efficiency of lentivirus into NSCs and enhances the therapeutic efficacy of CD-engineered rat NSCs in the treatment for C6 glioma, demonstrating that VP22 might be a useful tool for the gene therapy of engineered NSCs and providing a potential novel strategy for enhancing the effectiveness of gene therapy in other diseases.

Discovery and SAR of orally efficacious tetrahydropyridopyridazinone PARP inhibitors for the treatment of cancer.

PARP-1, the most abundant member of the PARP superfamily of nuclear enzymes, has emerged as a promising molecular target in the past decade particularly for the treatment of cancer. A number of PARP-1 inhibitors, including veliparab discovered at Abbott, have advanced into different stages of clinical trials. Herein we describe the development of a new tetrahydropyridopyridazinone series of PARP-1 inhibitors. Many compounds in this class, such as 20w, displayed excellent potency against the PARP-1 enzyme with a K(i) value of <1nM and an EC(50) value of 1nM in a C41 whole cell assay. The presence of the NH in the tetrahydropyridyl ring of the tetrahydropyridopyridazinone scaffold improved the pharmacokinetic properties over similar carbon based analogs. Compounds 8c and 20u are orally available, and have demonstrated significant efficacy in a B16 murine xenograft model, potentiating the efficacy of temozolomide (TMZ).

VP22 enhances the expression of glucocerebrosidase in human Gaucher II fibroblast cells mediated by lentiviral vectors.

Gaucher disease is an autosomal recessive lysosomal storage disorder resulting in a deficiency of glucocerebrosidase (GC). Imiglucerase, a recombinant form of GC, has been successfully used in the treatment of Gaucher disease and has been shown to be a good potential candidate for gene therapy. However, its low transduction efficiency and short duration of expression have limited it as a gene therapy strategy. VP22, the herpes simplex virus type I tegument protein, is known to facilitate intercellular protein transport, thus making it a promising tool for improving gene transfer efficiency. To investigate whether the fusion of VP22 to GC could improve its therapeutic efficiency for Gaucher disease, the lentiviral vectors pHIV-GC and pHIV-VP(22)-GC were constructed and confirmed by PCR or RT-PCR. After packaging, the vectors were transduced into human Gaucher II fibroblast cells (GII cells). Flow cytometric analysis revealed that the GC expression rates in lenti-VP(22)-GC-transduced GII cells were higher by comparison than those in lenti-GC-transduced GII cells. A Western blot demonstrated higher levels of GC protein expression in lenti-VP(22)-GC-transduced GII cells. In addition, the long-term expression levels and increased GC activities in lenti-VP(22)-GC-transduced GII cells were also observed. These data implicate that VP22-mediated effects may be useful for enhancing the efficacy of this Gaucher disease treatment.

Glioma stem cells targeted by oncolytic virus carrying endostatin-angiostatin fusion gene and the expression of its exogenous gene in vitro.

The development of the cancer stem cell (CSCs) niche theory has provided a new target for the treatment of gliomas. Gene therapy using oncolytic viral vectors has shown great potential for the therapeutic targeting of CSCs. To explore whether a viral vector carrying an exogenous Endo-Angio fusion gene (VAE) can infect and kill glioma stem cells (GSCs), as well as inhibit their vascular niche in vitro, we have collected surgical specimens of human high-grade glioma (world health organization, WHO Classes III-VI) from which we isolated and cultured GSCs under conditions originally designed for the selective expansion of neural stem cells. Our results demonstrate the following: (1) Four lines of GSCs (isolated from 20 surgical specimens) could grow in suspension, were multipotent, had the ability to self-renew and expressed the neural stem cell markers, CD133 and nestin. (2) VAE could infect GSCs and significantly inhibit their viability. (3) The Endo-Angio fusion gene was expressed in GSCs 48 h after VAE infection and could inhibit the proliferation of human brain microvascular endothelial cells (HBMEC). (4) Residual viable cells lose the ability of self-renewal and adherent differentiation. In conclusion, VAE can significantly inhibit the activity of GSCs in vitro and the expression of exogenous Endo-Angio fusion gene can inhibit HBMEC proliferation. VAE can be used as a novel virus-gene therapy strategy for glioma.

Optimization of phenyl-substituted benzimidazole carboxamide poly(ADP-ribose) polymerase inhibitors: identification of (S)-2-(2-fluoro-4-(pyrrolidin-2-yl)phenyl)-1H-benzimidazole-4-carboxamide (A-966492), a highly potent and efficacious inhibitor.

We have developed a series of phenylpyrrolidine- and phenylpiperidine-substituted benzimidazole carboxamide poly(ADP-ribose) polymerase (PARP) inhibitors with excellent PARP enzyme potency as well as single-digit nanomolar cellular potency. These efforts led to the identification of (S)-2-(2-fluoro-4-(pyrrolidin-2-yl)phenyl)-1H-benzimidazole-4-carboxamide (22b, A-966492). Compound 22b displayed excellent potency against the PARP-1 enzyme with a K(i) of 1 nM and an EC(50) of 1 nM in a whole cell assay. In addition, 22b is orally bioavailable across multiple species, crosses the blood-brain barrier, and appears to distribute into tumor tissue. It also demonstrated good in vivo efficacy in a B16F10 subcutaneous murine melanoma model in combination with temozolomide and in an MX-1 breast cancer xenograft model both as a single agent and in combination with carboplatin.