Extra-articular screw placement strategy in Stoppa approach based on three-dimensional reconstruction model.

The study aimed to explore an extra-articular screw placement strategy in Stoppa approach. Radiographic data of patients who underwent pelvic computed tomography from January 2016 to June 2017 were imported into Materiaise's interactive medical image control system software for three-dimensional reconstruction. Superior and lower margins of acetabulum and ipsilateral pelvic brim could be observed simultaneously through inlet-obturator view. A horizontal line from superior acetabular margin intersected pelvic brim at point "A" and another vertical line from lower margin intersected pelvic brim at point "B" were drawn, respectively. Lengths form sacroiliac joint to "A" (a), "A" to "B" (b), and "B" to pubic symphysis (c) were measured. Patients were divided into four groups depending on gender and side difference of measured hemi-pelvis: male left, male right, female left, and female right. Lengths of adjacent holes (d) and spanning different holes (e) of different plates were also measured. Mean lengths of a, b, c in four groups were 40.94 ± 1.85 mm, 40.09 ± 1.93 mm, 41.78 ± 3.62 mm, and 39.77 ± 2.23 mm (P = 0.078); 40.65 ± 1.58 mm, 41.48 ± 1.64 mm, 40.40 ± 1.96 mm, and 40.66 ± 1.70 mm (P = 0.265); 57.03 ± 3.41 mm, 57.51 ± 3.71 mm, 57.84 ± 4.40 mm, and 59.84 ± 4.35 mm (P = 0.165), respectively. Mean d length of different plates was 12.23 mm. Average lengths spanning 1, 2, 3 and 4 holes were 19.33 mm, 31.58 mm, 43.80 mm, and 55.93 mm. Our data showed that zones a and c could be safely inserted three and four screws. Penetration into hip joint could be avoided when vacant 3-hole drilling was conducted in zone b. Fracture line in zone b could serve as a landmark for screw placement.

A nonhuman primate model with Alzheimer's disease-like pathology induced by hippocampal overexpression of human tau.

BACKGROUND: Alzheimer's disease (AD) is one of the most burdening diseases of the century with no disease-modifying treatment at this time. Nonhuman primates (NHPs) share genetic, anatomical, and physiological similarities with humans, making them ideal model animals for investigating the pathogenesis of AD and potential therapies. However, the use of NHPs in AD research has been hindered by the paucity of AD monkey models due to their long generation time, ethical considerations, and technical challenges in genetically modifying monkeys. METHODS: Here, we developed an AD-like NHP model by overexpressing human tau in the bilateral hippocampi of adult rhesus macaque monkeys. We evaluated the pathological features of these monkeys with immunostaining, Nissl staining, cerebrospinal fluid (CSF) analysis, magnetic resonance imaging (MRI), positron emission tomography (PET), and behavioural tests. RESULTS: We demonstrated that after hippocampal overexpression of tau protein, these monkeys displayed multiple pathological features of AD, including 3-repeat (3R)/4-repeat (4R) tau accumulation, tau hyperphosphorylation, tau propagation, neuronal loss, hippocampal atrophy, neuroinflammation, Aß clearance deficits, blood vessel damage, and cognitive decline. More interestingly, the accumulation of both 3R and 4R tau is specific to NHPs but not found in adult rodents. CONCLUSIONS: This work establishes a tau-induced AD-like NHP model with many key pathological and behavioural features of AD. In addition, our model may potentially become one of the AD NHP models adopted by researchers worldwide since it can be generated within 2 ~ 3 months through a single injection of AAVs into the monkey brains. Hence, our model NHPs may facilitate mechanistic studies and therapeutic treatments for AD.

Age-related changes in lumbar bone mineral density measured using quantitative computed tomography in healthy female cynomolgus monkeys.

Background: Cynomolgus monkeys are widely used in studies related to osteoporosis, and there is no evidence of age-related changes in volumetric bone mineral density (vBMD) measured using quantitative computed tomography (QCT) in nonhuman primates. This study aimed to describe changes in the characteristics of lumbar vBMD with age, to analyze the relationship between lumbar vBMD and body composition, and to investigate the precision of QCT measurements in healthy female cynomolgus monkeys. Methods: Age-related changes in lumbar vBMD were described using cubic regression models, and the accumulated bone loss rates (ABLR) of the lumbar spine were calculated. Spearman rank correlation and ridge regression analysis were used to investigate the relationship of the average lumbar vBMD and body components. Thirty animals were selected to analyze the short-term in vivo precision of the QCT measurements. The precision was expressed as the root-mean-square coefficient of variation (RMS-CV%) or root-mean-square standard deviation (RMS-SD). Results: A total of 72 healthy female cynomolgus monkeys, aged 1-25 years, were included in this study. The average lumbar vBMD of female cynomolgus monkeys increased with age until the age of 10 years, around which it reached peak bone mass, with a relatively marked decline after the age of 13 years. The ABLRs of female cynomolgus monkeys in the premenopausal (13-19 years) and postmenopausal age groups (20-25 years) were -4.9% and -21.2%, respectively. Ridge regression analysis showed that age and subcutaneous adipose tissue (SAT) contributed positively to the average lumbar vBMD in animals aged ≤10 years, whereas in animals aged >10 years, age contributed negatively to lumbar vBMD. The RMS-CV% (RMS-SD) of the lumbar vBMD measured using QCT ranged from 0.47% to 1.60% (1.91-6.31 mg/cm3). Conclusions: Age-related changes in lumbar vBMD measured using QCT in healthy female monkeys showed similar trends to those in humans. Age and SAT may affect the lumbar vBMD in female cynomolgus monkeys. QCT revealed good precision in measuring the lumbar vBMD in female cynomolgus monkeys.

Establishing age-adjusted trabecular bone score curves using dual-energy X-ray absorptiometry in Chinese women and men: a cross-sectional study.

Background: Trabecular bone score (TBS) is a relatively new gray-level textural parameter that provides information on bone microarchitecture. TBS has been shown to be a good predictor of fragility fractures independent of bone density and clinical risk factors. Estimating the normal reference values of TBS in both sexes among the Chinese population is necessary to improve the clinical fracture risk assessment. Methods: This retrospective study enrolled healthy Chinese participants living in Guangzhou, China, including 1,018 men and 3,061 women (aged 20-74 years). Bone mineral density images were obtained with dual-energy X-ray absorptiometry (DXA) scanning of the lumbar region (L1-4). Lumbar spine TBS values were calculated. The correlations between the scores and bone mineral density, age, height, and weight were calculated for men and women. A TBS reference plot was established in relation to age (20-74 years). Values 2 standard deviations below the mean score for each sex were used as the cutoff values for low-quality bone. Results: The TBS (L1-4) was significantly higher in Chinese men than in Chinese women. The scores peaked at 25-29 years (1.47±0.08 years) in men and at 20-24 years (1.43±0.08 years) in women. According to the statistical confidence interval, in Chinese males, a TBS ≥1.39 is considered normal, a TBS between 1.31 and 1.39 indicates partially degraded microarchitecture, and a TBS ≤1.31 indicates degraded microarchitecture. In Chinese females, a TBS ≥1.35 is considered normal, a TBS between 1.27 and 1.35 indicates partially degraded microarchitecture, and a TBS ≤1.27 indicates degraded microarchitecture. Conclusions: This study provides normative reference ranges for the TBS in Chinese men and women. Chinese men with a TBS score ≤1.31 and Chinese women with a TBS score ≤1.27 are can be considered to have reduced bone microarchitecture and may be at higher risk of having osteoporosis fractures.

Quantitative assessment of translocator protein (TSPO) in the non-human primate brain and clinical translation of [18F]LW223 as a TSPO-targeted PET radioligand.

OBJECTIVES: Translocator protein 18 kDa (TSPO) positron emission tomography (PET) can be harnessed for the non-invasive detection of macrophage-driven inflammation. [18F]LW223, a newly reported TSPO PET tracer which was insensitive to rs6971 polymorphism, showed favorable performance characteristics in a recent imaging study involving a rat myocardial infarction model. To enable quantitative neuroimaging with [18F]LW223, we conducted kinetic analysis in the non-human primate (NHP) brain. Further, we sought to assess the utility of [18F]LW223-based TSPO imaging in a first-in-human study. METHODS: Radiosynthesis of [18F]LW223 was accomplished on an automated module, whereas molar activities, stability in formulation, lipophilicity and unbound free fraction (fu) of the probe were measured. Brain penetration and target specificity of [18F]LW223 in NHPs were corroborated by PET-MR imaging under baseline and pre-blocking conditions using the validated TSPO inhibitor, (R)-PK11195, at doses ranging from 5 to 10 mg/kg. Kinetic modeling was performed using one-tissue compartment model (1TCM), two-tissue compartment model (2TCM) and Logan graphical analyses, using dynamic PET data acquisition, arterial blood collection and metabolic stability testing. Clinical PET scans were performed in two healthy volunteers (HVs). Regional brain standard uptake value ratio (SUVr) was assessed for different time intervals. RESULTS: [18F]LW223 was synthesized in non-decay corrected radiochemical yields (n.d.c. RCYs) of 33.3 ± 6.5% with molar activities ranging from 1.8 ± 0.7 Ci/µmol (n = 11). [18F]LW223 was stable in formulation for up to 4 h and LogD7.4 of 2.31 ± 0.13 (n = 6) and fu of 5.80 ± 1.42% (n = 6) were determined. [18F]LW223 exhibited good brain penetration in NHPs, with a peak SUV value of ca. 1.79 in the whole brain. Pre-treatment with (R)-PK11195 substantially accelerated the washout and attenuated the area under the time-activity curve, indicating in vivo specificity of [18F]LW223 towards TSPO. Kinetic modeling demonstrated that 2TCM was the most suitable model for [18F]LW223-based neuroimaging. Global transfer rate constants (K1) and total volumes of distribution (VT) were found to be 0.10 ± 0.01 mL/cm3/min and 2.30 ± 0.17 mL/cm3, respectively. Dynamic PET data analyses across distinct time windows revealed that the VT values were relatively stable after 60 min post-injection. In a preliminary clinical study with two healthy volunteers, [18F]LW223 exhibited good brain uptake and considerable tracer retention across all analyzed brain regions. Of note, an excellent correlation between SUVr with VT was obtained when assessing the time interval from 20 to 40 min post tracer injection (SUVr(20-40 min), R2 = 0.94, p < 0.0001), suggesting this time window may be suitable to estimate specific binding to TSPO in human brain. CONCLUSION: Our findings indicate that [18F]LW223 is suitable for quantitative TSPO-targeted PET imaging in higher species. Employing state-of-the-art kinetic modeling, we found that [18F]LW223 was effective in mapping TSPO throughout the NHP brain, with best model fits obtained from 2TCM and Logan graphical analyses. Overall, our results indicate that [18F]LW223 exhibits favorable tracer performance characteristics in higher species, and this novel imaging tool may hold promise to provide effective neuroinflammation imaging in patients with neurological disease.

Recent developments on the application of molecular probes in multiple myeloma: Beyond [18F]FDG.

Multiple myeloma (MM) is a neoplastic plasma cell proliferative disorder characterized by various osteolytic bone destruction as a radiological morphological marker. Functional imaging, particularly nuclear medicine imaging, is a promising method to visualize disease processes before the appearance of structural changes by targeting specific biomarkers related to metabolism ability, tumor microenvironment as well as neoplastic receptors. In addition, by targeting particular antigens with therapeutic antibodies, immuno-PET imaging can support the development of personalized theranostics. At present, various imaging agents have been prepared and evaluated in MM at preclinical and clinical levels. A summary overview of molecular functional imaging in MM is provided, and commonly used radiotracers are characterized.

Easily automated radiosynthesis of [18F]P10A-1910 and its clinical translation to quantify phosphodiesterase 10A in human brain.

Our previous work showed that [18F]P10A-1910 was a potential radioligand for use in imaging phosphodiesterase 10A (PDE10A). Specifically, it had high brain penetration and specific binding that was demonstrated in both rodents and non-human primates. Here, we present the first automatic cGMP-level production of [18F]P10A-1910 and translational PET/MRI study in living human brains. Successful one-step radiolabeling of [18F]P10A-1910 on a GE TRACERlab FX2N synthesis module was realized via two different methods. First, formulated [18F]P10A-1910 was derived from heating spirocyclic iodonium ylide in a tetra-n-butyl ammonium methanesulfonate solution. At the end of synthesis, it was obtained in non-decay corrected radiochemical yields (n.d.c. RCYs) of 12.4 ± 1.3%, with molar activities (MAs) of 90.3 ± 12.6 µmol (n = 7) (Method I). The boronic pinacol ester combined with copper and oxygen also delivered the radioligand with 16.8 ± 1.0% n. d.c. RCYs and 77.3 ± 20.7 GBq/µmol (n = 7) MAs after formulation (Method II). The radiochemical purity, radionuclidic purity, solvent residue, sterility, endotoxin content and other parameters were all validated for human use. Consistent with the distribution of PDE10A in the brain, escalating uptake of [18F]P10A-1910 was observed in the order of cerebellum (reference region), substantial nigra, caudate and putamen. The non-displaceable binding potential (BP ND) was estimated by simplified reference-tissue model (SRTM); linear regressions demonstrated that BP ND was well correlated with the most widely used semiquantitative parameter SUV. The strongest correlation was observed with SUV(50-60 min) (R 2 = 0.966, p < 0.01). Collectively, these results indicated that a static scan protocol could be easily performed for PET imaging of PDE10A. Most importantly, that [18F]P10A-1910 is a promising radioligand to clinically quantify PDE10A.

Discovery of a highly specific 18F-labeled PET ligand for phosphodiesterase 10A enabled by novel spirocyclic iodonium ylide radiofluorination.

As a member of cyclic nucleotide phosphodiesterase (PDE) enzyme family, PDE10A is in charge of the degradation of cyclic adenosine (cAMP) and guanosine monophosphates (cGMP). While PDE10A is primarily expressed in the medium spiny neurons of the striatum, it has been implicated in a variety of neurological disorders. Indeed, inhibition of PDE10A has proven to be of potential use for the treatment of central nervous system (CNS) pathologies caused by dysfunction of the basal ganglia-of which the striatum constitutes the largest component. A PDE10A-targeted positron emission tomography (PET) radioligand would enable a better assessment of the pathophysiologic role of PDE10A, as well as confirm the relationship between target occupancy and administrated dose of a given drug candidate, thus accelerating the development of effective PDE10A inhibitors. In this study, we designed and synthesized a novel 18F-aryl PDE10A PET radioligand, codenamed [18F]P10A-1910 ([18F]9), in high radiochemical yield and molar activity via spirocyclic iodonium ylide-mediated radiofluorination. [18F]9 possessed good in vitro binding affinity (IC50 = 2.1 nmol/L) and selectivity towards PDE10A. Further, [18F]9 exhibited reasonable lipophilicity (logD = 3.50) and brain permeability (P app > 10 × 10-6 cm/s in MDCK-MDR1 cells). PET imaging studies of [18F]9 revealed high striatal uptake and excellent in vivo specificity with reversible tracer kinetics. Preclinical studies in rodents revealed an improved plasma and brain stability of [18F]9 when compared to the current reference standard for PDE10A-targeted PET, [18F]MNI659. Further, dose-response experiments with a series of escalating doses of PDE10A inhibitor 1 in rhesus monkey brains confirmed the utility of [18F]9 for evaluating target occupancy in vivo in higher species. In conclusion, our results indicated that [18F]9 is a promising PDE10A PET radioligand for clinical translation.

Positron Emission Tomography in the Neuroimaging of Autism Spectrum Disorder: A Review.

Autism spectrum disorder (ASD) is a basket term for neurodevelopmental disorders characterized by marked impairments in social interactions, repetitive and stereotypical behaviors, and restricted interests and activities. Subtypes include (A) disorders with known genetic abnormalities including fragile X syndrome, Rett syndrome, and tuberous sclerosis and (B) idiopathic ASD, conditions with unknown etiologies. Positron emission tomography (PET) is a molecular imaging technology that can be utilized in vivo for dynamic and quantitative research, and is a valuable tool for exploring pathophysiological mechanisms, evaluating therapeutic efficacy, and accelerating drug development in ASD. Recently, several imaging studies on ASD have been published and physiological changes during ASD progression was disclosed by PET. This paper reviews the specific radioligands for PET imaging of critical biomarkers in ASD, and summarizes and discusses the similar and different discoveries in outcomes of previous studies. It is of great importance to identify general physiological changes in cerebral glucose metabolism, cerebral blood flow perfusion, abnormalities in neurotransmitter systems, and inflammation in the central nervous system in ASD, which may provide excellent points for further ASD research.

Recent developments on PET radiotracers for TSPO and their applications in neuroimaging.

The 18 kDa translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor, is predominately localized to the outer mitochondrial membrane in steroidogenic cells. Brain TSPO expression is relatively low under physiological conditions, but is upregulated in response to glial cell activation. As the primary index of neuroinflammation, TSPO is implicated in the pathogenesis and progression of numerous neuropsychiatric disorders and neurodegenerative diseases, including Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), multiple sclerosis (MS), major depressive disorder (MDD) and obsessive compulsive disorder (OCD). In this context, numerous TSPO-targeted positron emission tomography (PET) tracers have been developed. Among them, several radioligands have advanced to clinical research studies. In this review, we will overview the recent development of TSPO PET tracers, focusing on the radioligand design, radioisotope labeling, pharmacokinetics, and PET imaging evaluation. Additionally, we will consider current limitations, as well as translational potential for future application of TSPO radiopharmaceuticals. This review aims to not only present the challenges in current TSPO PET imaging, but to also provide a new perspective on TSPO targeted PET tracer discovery efforts. Addressing these challenges will facilitate the translation of TSPO in clinical studies of neuroinflammation associated with central nervous system diseases.

Localizing Epileptic Foci Before Surgery in Patients With MRI-Negative Refractory Epilepsy Using Statistical Parameter Mapping and Three-Dimensional Stereotactic Surface Projection Based on 18F-FDG PET.

Patients with refractory epilepsy are not only free of seizures after resecting epileptic foci, but also experience significantly improved quality of life. Fluorine-18-fluorodeoxyglucose positron-emission tomography (18F-FDG PET) is a promising avenue for detecting epileptic foci in patients with magnetic resonance imaging (MRI)-negative refractory epilepsy. However, the detection of epileptic foci by visual assessment based on 18F-FDG PET is often complicated by a variety of factors in clinical practice. Easy imaging methods based on 18F-FDG PET images, such as statistical parameter mapping (SPM) and three-dimensional stereotactic surface projection (3D-SSP), can objectively detect epileptic foci. In this study, the regions of surgical resection of patients with over 1 year follow-up and no seizures were defined as standard epileptic foci. We retrospectively analyzed the sensitivity of visual assessment, SPM and 3D-SSP based on 18F-FDG PET to detect epileptic foci in MRI-negative refractory epilepsy patients and obtained the sensitivities of visual assessment, SPM and 3D-SSP are 57, 70 and 60% respectively. Visual assessment combined with SPM or 3D-SSP can improve the sensitivity of detecting epileptic foci. The sensitivity was highest when the three methods were combined, but decreased consistency, in localizing epileptic foci. We conclude that SPM and 3D-SSP can be used as objective methods to detect epileptic foci before surgery in patients with MRI-negative refractory epilepsy. Visual assessment is the preferred method for PET image analysis in MRI-negative refractory epilepsy. When the visual assessment is inconsistent with the patient's electroclinical information, SPM or 3D-SSP was further selected to assess the epileptic foci. If the combination of the two methods still fails to accurately locate the epileptic foci, comprehensive evaluation can be performed by combining the three methods.

F-Box Gene D5RF Is Regulated by Agrobacterium Virulence Protein VirD5 and Essential for Agrobacterium-Mediated Plant Transformation.

We previously reported that the Agrobacterium virulence protein VirD5 possesses transcriptional activation activity, binds to a specific DNA element D5RE, and is required for Agrobacterium-mediated stable transformation, but not for transient transformation. However, direct evidence for a role of VirD5 in plant transcriptional regulation has been lacking. In this study, we found that the Arabidopsis gene D5RF (coding for VirD5 response F-box protein, At3G49480) is regulated by VirD5. D5RF has two alternative transcripts of 930 bp and 1594 bp that encode F-box proteins of 309 and 449 amino acids, designated as D5RF.1 and D5RF.2, respectively. D5RF.2 has a N-terminal extension of 140 amino acids compared to D5RF.1, and both of them are located in the plant cell nucleus. The promoter of the D5RF.1 contains two D5RE elements and can be activated by VirD5. The expression of D5RF is downregulated when the host plant is infected with virD5 deleted Agrobacterium. Similar to VirD5, D5RF also affects the stable but not transient transformation efficiency of Agrobacterium. Some pathogen-responsive genes are downregulated in the d5rf mutant. In conclusion, this study further confirmed Agrobacterium VirD5 as the plant transcription activator and identified Arabidopsis thalianaD5RF.1 as the first target gene of VirD5 in regulation.

Light-activatable cannabinoid prodrug for combined and target-specific photodynamic and cannabinoid therapy.

Cannabinoids are emerging as promising antitumor drugs. However, complete tumor eradication solely by cannabinoid therapy remains challenging. In this study, we developed a far-red light activatable cannabinoid prodrug, which allows for tumor-specific and combinatory cannabinoid and photodynamic therapy. This prodrug consists of a phthalocyanine photosensitizer (PS), reactive oxygen species (ROS)-sensitive linker, and cannabinoid. It targets the type-2 cannabinoid receptor (CB2R) overexpressed in various types of cancers. Upon the 690-nm light irradiation, the PS produces cytotoxic ROS, which simultaneously cleaves the ROS-sensitive linker and subsequently releases the cannabinoid drug. We found that this unique multifunctional prodrug design offered dramatically improved therapeutic efficacy, and therefore provided a new strategy for targeted, controlled, and effective antitumor cannabinoid therapy.

Combined CB2 receptor agonist and photodynamic therapy synergistically inhibit tumor growth in triple negative breast cancer.

Triple negative breast cancer (TNBC) is the deadliest form of breast cancer because it is more aggressive, diagnosed at later stage and more likely to develop local and systemic recurrence. Many patients do not experience adequate tumor control after current clinical treatments involving surgical removal, chemotherapy and/or radiotherapy, leading to disease progression and significantly decreased quality of life. Here we report a new combinatory therapy strategy involving cannabinoid-based medicine and photodynamic therapy (PDT) for the treatment of TNBC. This combinatory therapy targets two proteins upregulated in TNBC: the cannabinoid CB2 receptor (CB2R, a G-protein coupled receptor) and translocator protein (TSPO, a mitochondria membrane receptor). We found that the combined CB2R agonist and TSPO-PDT treatment resulted in synergistic inhibition in TNBC cell and tumor growth. This combinatory therapy approach provides new opportunities to treat TNBC with high efficacy. In addition, this study provides new evidence on the therapeutic potential of CB2R agonists for cancer.

VirD5 is required for efficient Agrobacterium infection and interacts with Arabidopsis VIP2.

During Agrobacterium (Agrobacterium tumefaciens) infection, the translocated virulence proteins (VirD2, VirE2, VirE3, VirF and VirD5) play crucial roles. It is thought that, through protein-protein interactions, Agrobacterium uses and abuses host plant factors and systems to facilitate its infection. Although some molecular functions have been revealed, the roles of VirD5 still need to be further elucidated. Here, plant transformation and tumorigenesis mediated by genetically modified Agrobacterium strains were performed to examine VirD5 roles. In addition, protein-protein interaction-associated molecular and biochemistry technologies were used to reveal and elucidate VirD5 interaction with Arabidopsis VirE2 interacting protein 2 (VIP2). Our results showed that deleting virD5 from Agrobacterium reduced its tumor formation ability and stable transformation efficiency but did not affect the transient transformation efficiency. We also found that VirD5 can interact with Arabidopsis VIP2. Further experiments demonstrated that VirD5 can affect VIP2 binding to cap-binding proteins (CBP20 and CBP80). The tumorigenesis efficiency for cbp80 mutant was not significantly changed, but that for cbp20, cbp20cbp80 mutants were significantly increased. This work demonstrates experimentally that VirD5 is required for efficient Agrobacterium infection and may promote this process by competitive interaction with Arabidopsis VIP2. CBP20 is involved in the Agrobacterium infection process and its effect can be synergistically enhanced by CBP80.

Multilayer photodynamic therapy for highly effective and safe cancer treatment.

Recent efforts to develop tumor-targeted photodynamic therapy (PDT) photosensitizers (PSs) have greatly advanced the potential of PDT in cancer therapy, although complete eradication of tumor cells by PDT alone remains challenging. As a way to improve PDT efficacy, we report a new combinatory PDT therapy technique that specifically targets multilayers of cells. Simply mixing different PDT PSs, even those that target distinct receptors (this may still lead to similar cell-killing pathways), may not achieve ideal therapeutic outcomes. Instead, significantly improved outcomes likely require synergistic therapies that target various cellular pathways. In this study, we target two proteins upregulated in cancers: the cannabinoid CB2 receptor (CB2R, a G-protein coupled receptor) and translocator protein (TSPO, a mitochondria membrane receptor). We found that the CB2R-targeted PS, IR700DX-mbc94, triggered necrotic cell death upon light irradiation, whereas PDT with the TSPO-targeted IR700DX-6T agent led to apoptotic cell death. Both PSs significantly inhibited tumor growth in vivo in a target-specific manner. As expected, the combined CB2R- and TSPO-PDT resulted in enhanced cell killing efficacy and tumor inhibition with lower drug dose. The median survival time of animals with multilayer PDT treatment was extended by as much as 2.8-fold over single PDT treatment. Overall, multilayer PDT provides new opportunities to treat cancers with high efficacy and low side effects. STATEMENT OF SIGNIFICANCE: Photodynamic therapy (PDT) is increasingly used as a minimally invasive, controllable and effective therapeutic procedure for cancer treatment. However, complete eradication of tumor cells by PDT alone remains challenging. In this study, we investigate the potential of multilayer PDT in cancer treatment with high efficacy and low side effects. Through PDT targeting two cancer biomarkers located at distinct subcellular localizations, remarkable synergistic effects in cancer cell killing and tumor inhibition were observed in both in vitro and in vivo experiments. This strategy may be widely applied to treat various cancer types by using strategically designed PDT photosensitizers that target corresponding upregulated receptors at tactical subcellular localization.

Synthesis and Evaluation of a Tetra[6,7]quinoxalinoporphyrazine-based Near Infrared Photosensitizer.

Here we report a near infrared, water-soluble, functional and dendrimeric photosensitizer (PS) based on quinoxalinoporphyrazine structure. The photophysical properties and in vitro photodynamic therapy results suggest that this quinoxalinoporphyrazine-based dendrimer may serve as an efficient near infrared (NIR) PS platform.

Discovery of N-((1-(4-(3-(3-((6,7-Dimethoxyquinolin-3-yl)oxy)phenyl)ureido)-2-(trifluoromethyl)phenyl)piperidin-4-yl)methyl)propionamide (CHMFL-KIT-8140) as a Highly Potent Type II Inhibitor Capable of Inhibiting the T670I "Gatekeeper" Mutant of cKIT Kinase.

cKIT kinase inhibitors, e.g., imatinib, could induce drug-acquired mutations such as cKIT T670I that rendered drug resistance after chronic treatment. Through a type II kinase inhibitor design approach we discovered a highly potent type II cKIT kinase inhibitor compound 35 (CHMFL-KIT-8140), which potently inhibited both cKIT wt (IC50 = 33 nM) and cKIT gatekeeper T670I mutant (IC50 = 99 nM). Compound 35 displayed strong antiproliferative effect against GISTs cancer cell lines GIST-T1 (cKIT wt, GI50 = 4 nM) and GIST-5R (cKIT T670I, GI50 = 26 nM). In the cellular context it strongly inhibited c-KIT mediated signaling pathways and induced apoptosis. In the BaF3-TEL-cKIT-T670I isogenic cell inoculated xenograft mouse model, 35 exhibited dose dependent tumor growth suppression efficacy and 100 mg/kg dosage provided 47.7% tumor growth inhibition (TGI) without obvious toxicity. We believe compound 35 would be a good pharmacological tool for exploration of the cKIT-T670I mutant mediated pathology in GISTs.

Before In Vivo Imaging: Evaluation of Fluorescent Probes Using Fluorescence Microscopy, Multiplate Reader, and Cytotoxicity Assays.

Fluorescent probes are widely utilized for noninvasive fluorescence imaging. Continuing efforts have been made in developing novel fluorescent probes with improved fluorescence quantum yield, enhanced target-specificity, and lower cytotoxicity. Before such probes are administrated into a living system, it is essential to evaluate the subcellular uptake, targeting specificity, and cytotoxicity in vitro. In this chapter, we briefly outline common methods used to evaluate fluorescent probes using fluorescence microscopy, multiplate reader, and cytotoxicity assay.

Ex Vivo Imaging, Biodistribution, and Histological Study in Addition to In Vivo Imaging.

In addition to in vivo fluorescence imaging, ex vivo evaluations including ex vivo imaging, biodistribution, and histological study are often conducted to further investigate the biological behavior of fluorescent probes. These studies can further confirm the localization of fluorescent probes at the target sites and demonstrate the probe distribution in various organs and tissues. Such studies can also be extended to cellular level for biochemical analysis. Therefore, ex vivo evaluations are valuable to fully characterizing fluorescent probes in a living system. This chapter provides an overview of techniques for evaluating pharmacological profiles of fluorescent probes ex vivo.