Evolving acidic microenvironments during colitis provide selective analgesic targets for a pH-sensitive opioid.

ABSTRACT: Targeting the acidified inflammatory microenvironment with pH-sensitive opioids is a novel approach for managing visceral pain while mitigating side effects. The analgesic efficacy of pH-dependent opioids has not been studied during the evolution of inflammation, where fluctuating tissue pH and repeated therapeutic dosing could influence analgesia and side effects. Whether pH-dependent opioids can inhibit human nociceptors during extracellular acidification is unexplored. We studied the analgesic efficacy and side-effect profile of a pH-sensitive fentanyl analog, (±)- N -(3-fluoro-1-phenethylpiperidine-4-yl)- N -phenyl propionamide (NFEPP), during the evolution of colitis induced in mice with dextran sulphate sodium. Colitis was characterized by granulocyte infiltration, histological damage, and acidification of the mucosa and submucosa at sites of immune cell infiltration. Changes in nociception were determined by measuring visceromotor responses to noxious colorectal distension in conscious mice. Repeated doses of NFEPP inhibited nociception throughout the course of disease, with maximal efficacy at the peak of inflammation. Fentanyl was antinociceptive regardless of the stage of inflammation. Fentanyl inhibited gastrointestinal transit, blocked defaecation, and induced hypoxemia, whereas NFEPP had no such side effects. In proof-of-principle experiments, NFEPP inhibited mechanically provoked activation of human colonic nociceptors under acidic conditions mimicking the inflamed state. Thus, NFEPP provides analgesia throughout the evolution of colitis with maximal activity at peak inflammation. The actions of NFEPP are restricted to acidified layers of the colon, without common side effects in normal tissues. N -(3-fluoro-1-phenethylpiperidine-4-yl)- N -phenyl propionamide could provide safe and effective analgesia during acute colitis, such as flares of ulcerative colitis.

Electron Leak From the Mitochondrial Electron Transport Chain Complex I at Site IQ Is Crucial for Oxygen Sensing in Rabbit and Human Ductus Arteriosus.

Background As partial pressure of oxygen (pO2) rises with the first breath, the ductus arteriosus (DA) constricts, diverting blood flow to the pulmonary circulation. The DA's O2 sensor resides within smooth muscle cells. The DA smooth muscle cells' mitochondrial electron transport chain (ETC) produces reactive oxygen species (ROS) in proportion to oxygen tension, causing vasoconstriction by regulating redox-sensitive ion channels and enzymes. To identify which ETC complex contributes most to DA O2 sensing and determine whether ROS mediate O2 sensing independent of metabolism, we used electron leak suppressors, S1QEL (suppressor of site IQ electron leak) and S3QEL (suppressor of site IIIQo electron leak), which decrease ROS production by inhibiting electron leak from quinone sites IQ and IIIQo, respectively. Methods and Results The effects of S1QEL, S3QEL, and ETC inhibitors (rotenone and antimycin A) on DA tone, mitochondrial metabolism, O2-induced changes in intracellular calcium, and ROS were studied in rabbit DA rings, and human and rabbit DA smooth muscle cells. S1QEL's effects on DA patency were assessed in rabbit kits, using micro computed tomography. In DA rings, S1QEL, but not S3QEL, reversed O2-induced constriction (P=0.0034) without reducing phenylephrine-induced constriction. S1QEL did not inhibit mitochondrial metabolism or ETC-I activity. In human DA smooth muscle cells, S1QEL and rotenone inhibited O2-induced increases in intracellular calcium (P=0.02 and 0.001, respectively), a surrogate for DA constriction. S1QEL inhibited O2-induced ROS generation (P=0.02). In vivo, S1QEL prevented O2-induced DA closure (P<0.0001). Conclusions S1QEL, but not S3QEL, inhibited O2-induced rises in ROS and DA constriction ex vivo and in vivo. DA O2 sensing relies on pO2-dependent changes in electron leak at site IQ in ETC-I, independent of metabolism. S1QEL offers a therapeutic means to maintain DA patency.

Efficacy of Drpitor1a, a Dynamin-Related Protein 1 inhibitor, in Pulmonary Arterial Hypertension.

Rationale: Dynamin-related protein 1 (Drp1), a large GTPase, mediates mitochondrial fission. Increased Drp1-mediated fission permits accelerated mitosis, contributing to hyperproliferation of pulmonary artery smooth muscle cells (PASMC), which characterizes pulmonary arterial hypertension (PAH). We developed a Drp1 inhibitor, Drpitor1a, and tested its ability to regress PAH. Objectives: Assess Drpitor1a's efficacy and toxicity in: a)normal and PAH human PASMC (hPASMC); b)normal rats versus rats with established monocrotaline (MCT)-induced PAH. Methods: Drpitor1a's effects on recombinant and endogenous Drp1-GTPase activity, mitochondrial fission, and cell proliferation were studied in hPASMCs (normal=3; PAH=5). Drpitor1a's pharmacokinetics and tissue concentrations were measured (n=3 rats/sex). In a pilot study (n=3-4/sex/dose), Drpitor1a (1mg/kg/48-hours, intravenous) reduced adverse PA remodeling only in females. Consequently, we compared Drpitor1a to vehicle in normal (n=6 versus 8) and MCT-PAH (n=9 and 11) females, respectively. Drpitor1a treatment began 17-days post-MCT with echocardiography and cardiac catheterization performed 28-29 days post-MCT. Results: Drpitor1a inhibited recombinant and endogenous Drp1 GTPase activity, which was increased in PAH hPASMC. Drpitor1a inhibited mitochondrial fission and proliferation and induced apoptosis, in PAH hPASMC but not normal hPASMC. Drpitor1a tissue levels were higher in female versus male RVs. In MCT-PAH females, Drpitor1a regressed PA obstruction, lowered pulmonary vascular resistance, and improved RV function, without hematologic, renal, or hepatic toxicity. Conclusions: Drpitor1a inhibits Drp1 GTPase, reduces mitochondrial fission, and inhibits cell proliferation in PAH hPASMC. Drpitor1a caused no toxicity in MCT-PAH and had no significant effect on normal rats or hPASMCs. Drpitor1a is a potential PAH therapeutic which displays an interesting therapeutic sexual dimorphism.

SARS-CoV-2 mitochondriopathy in COVID-19 pneumonia exacerbates hypoxemia.

RATIONALE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19 pneumonia. We hypothesize that SARS-CoV-2 causes alveolar injury and hypoxemia by damaging mitochondria in airway epithelial cells (AEC) and pulmonary artery smooth muscle cells (PASMC), triggering apoptosis and bioenergetic impairment, and impairing hypoxic pulmonary vasoconstriction (HPV), respectively. OBJECTIVES: We examined the effects of: A) human betacoronaviruses, SARS-CoV-2 and HCoV-OC43, and individual SARS-CoV-2 proteins on apoptosis, mitochondrial fission, and bioenergetics in AEC; and B) SARS-CoV-2 proteins and mouse hepatitis virus (MHV-1) infection on HPV. METHODS: We used transcriptomic data to identify temporal changes in mitochondrial-relevant gene ontology (GO) pathways post-SARS-CoV-2 infection. We also transduced AECs with SARS-CoV-2 proteins (M, Nsp7 or Nsp9) and determined effects on mitochondrial permeability transition pore (mPTP) activity, relative membrane potential, apoptosis, mitochondrial fission, and oxygen consumption rates (OCR). In human PASMC, we assessed the effects of SARS-CoV-2 proteins on hypoxic increases in cytosolic calcium, an HPV proxy. In MHV-1 pneumonia, we assessed HPV via cardiac catheterization and apoptosis using the TUNEL assay. RESULTS: SARS-CoV-2 regulated mitochondrial apoptosis, mitochondrial membrane permeabilization and electron transport chain (ETC) GO pathways within 2 hours of infection. SARS-CoV-2 downregulated ETC Complex I and ATP synthase genes, and upregulated apoptosis-inducing genes. SARS-CoV-2 and HCoV-OC43 upregulated and activated dynamin-related protein 1 (Drp1) and increased mitochondrial fission. SARS-CoV-2 and transduced SARS-CoV-2 proteins increased apoptosis inducing factor (AIF) expression and activated caspase 7, resulting in apoptosis. Coronaviruses also reduced OCR, decreased ETC Complex I activity and lowered ATP levels in AEC. M protein transduction also increased mPTP opening. In human PASMC, M and Nsp9 proteins inhibited HPV. In MHV-1 pneumonia, infected AEC displayed apoptosis and HPV was suppressed. BAY K8644, a calcium channel agonist, increased HPV and improved SpO2. CONCLUSIONS: Coronaviruses, including SARS-CoV-2, cause AEC apoptosis, mitochondrial fission, and bioenergetic impairment. SARS-CoV-2 also suppresses HPV by targeting mitochondria. This mitochondriopathy is replicated by transduction with SARS-CoV-2 proteins, indicating a mechanistic role for viral-host mitochondrial protein interactions. Mitochondriopathy is a conserved feature of coronaviral pneumonia that may exacerbate hypoxemia and constitutes a therapeutic target.

Concussion/Mild Traumatic Brain Injury (TBI) Induces Brain Insulin Resistance: A Positron Emission Tomography (PET) Scanning Study.

Brain injury/concussion is a growing epidemic throughout the world. Although evidence supports association between traumatic brain injury (TBI) and disturbance in brain glucose metabolism, the underlying molecular mechanisms are not well established. Previously, we reported the release of cellular prion protein (PrPc) from the brain to circulation following TBI. The PrPc level was also found to be decreased in insulin-resistant rat brains. In the present study, we investigated the molecular link between PrPc and brain insulin resistance in a single and repeated mild TBI-induced mouse model. Mild TBI was induced in mice by dropping a weight (~95 g at 1 m high) on the right side of the head. The procedure was performed once and thrice (once daily) for single (SI) and repeated induction (RI), respectively. Micro PET/CT imaging revealed that RI mice showed significant reduction in cortical, hippocampal and cerebellum glucose uptake compared to SI and control. Mice that received RI also showed significant motor and cognitive deficits. In co-immunoprecipitation, the interaction between PrPc, flotillin and Cbl-associated protein (CAP) observed in the control mice brains was disrupted by RI. Lipid raft isolation showed decreased levels of PrPc, flotillin and CAP in the RI mice brains. Based on observation, it is clear that PrPc has an interaction with CAP and the dislodgment of PrPc from cell membranes may lead to brain insulin resistance in a mild TBI mouse model. The present study generated a new insight into the pathogenesis of brain injury, which may result in the development of novel therapy.

89Zr-Labeled Domain II-Specific scFv-Fc ImmunoPET Probe for Imaging Epidermal Growth Factor Receptor In Vivo.

Epidermal growth factor receptor I (EGFR) is overexpressed in many cancers. The extracellular domain of EGFR has four binding epitopes (domains I- IV). All clinically approved anti-EGFR antibodies bind to domain III. Imaging agents that bind to domains other than domain III of EGFR are needed for accurate quantification of EGFR, patient selection for anti-EGFR therapeutics and monitoring of response to therapies. We recently developed a domain II-specific antibody fragment 8709. In this study, we have evaluated the in vitro and in vivo properties of 89Zr-8709-scFv-Fc (105 kDa). We conjugated 8709-scFv-Fc with the deferoxamine (DFO) chelator and radiolabeled the DFO-8970-scFv with 89Zr. We evaluated the binding of 89Zr-DFO-8709-scFv-Fc in EGFR positive and negative cell lines DLD-1, MDA-MB-231 and MDA-MB-435, respectively, and in mouse xenograft models. Simultaneously, we have compared the binding of 89Zr-8709-scFv-Fc with 111In-nimotuzumab, a domain III anti-EGFR antibody. DFO-8709-scFv-Fc displayed similar cell binding specificity as 8709-scFv-Fc. Saturation cell binding assay and immunoreactive fraction showed that radiolabeling did not alter the binding of 8709-scFv-Fc. Biodistribution and microPET showed good uptake of 89Zr-8709-scFv-Fc in xenografts after 120 h post injection (p.i). and was domain-specific to EGFR domain II. 89Zr-8709-scFv-Fc did not compete for binding in vitro and in vivo with a known domain III binder nimotuzumab. The results show that 89Zr-8709-scFv-Fc is specific to domain II of EGFR making it favorable for quantification of EGFR in vivo, hence, patient selection and monitoring of response to treatment with anti-EGFR antibodies.

Oxygen sensing, mitochondrial biology and experimental therapeutics for pulmonary hypertension and cancer.

The homeostatic oxygen sensing system (HOSS) optimizes systemic oxygen delivery. Specialized tissues utilize a conserved mitochondrial sensor, often involving NDUFS2 in complex I of the mitochondrial electron transport chain, as a site of pO2-responsive production of reactive oxygen species (ROS). These ROS are converted to a diffusible signaling molecule, hydrogen peroxide (H2O2), by superoxide dismutase (SOD2). H2O2 exits the mitochondria and regulates ion channels and enzymes, altering plasma membrane potential, intracellular Ca2+ and Ca2+-sensitization and controlling acute, adaptive, responses to hypoxia that involve changes in ventilation, vascular tone and neurotransmitter release. Subversion of this O2-sensing pathway creates a pseudohypoxic state that promotes disease progression in pulmonary arterial hypertension (PAH) and cancer. Pseudohypoxia is a state in which biochemical changes, normally associated with hypoxia, occur despite normal pO2. Epigenetic silencing of SOD2 by DNA methylation alters H2O2 production, activating hypoxia-inducible factor 1α, thereby disrupting mitochondrial metabolism and dynamics, accelerating cell proliferation and inhibiting apoptosis. Other epigenetic mechanisms, including dysregulation of microRNAs (miR), increase pyruvate dehydrogenase kinase and pyruvate kinase muscle isoform 2 expression in both diseases, favoring uncoupled aerobic glycolysis. This Warburg metabolic shift also accelerates cell proliferation and impairs apoptosis. Disordered mitochondrial dynamics, usually increased mitotic fission and impaired fusion, promotes disease progression in PAH and cancer. Epigenetic upregulation of dynamin-related protein 1 (Drp1) and its binding partners, MiD49 and MiD51, contributes to the pathogenesis of PAH and cancer. Finally, dysregulation of intramitochondrial Ca2+, resulting from impaired mitochondrial calcium uniporter complex (MCUC) function, links abnormal mitochondrial metabolism and dynamics. MiR-mediated decreases in MCUC function reduce intramitochondrial Ca2+, promoting Warburg metabolism, whilst increasing cytosolic Ca2+, promoting fission. Epigenetically disordered mitochondrial O2-sensing, metabolism, dynamics, and Ca2+ homeostasis offer new therapeutic targets for PAH and cancer. Promoting glucose oxidation, restoring the fission/fusion balance, and restoring mitochondrial calcium regulation are promising experimental therapeutic strategies.

Nimotuzumab Site-Specifically Labeled with 89Zr and 225Ac Using SpyTag/SpyCatcher for PET Imaging and Alpha Particle Radioimmunotherapy of Epidermal Growth Factor Receptor Positive Cancers.

To develop imaging and therapeutic agents, antibodies are often conjugated randomly to a chelator/radioisotope or drug using a primary amine (NH2) of lysine or sulfhydryl (SH) of cysteine. Random conjugation to NH2 or SH groups can require extreme conditions and may affect target recognition/binding and must therefore be tested. In the present study, nimotuzumab was site-specifically labeled using ∆N-SpyCatcher/SpyTag with different chelators and radiometals. Nimotuzumab is a well-tolerated anti-EGFR antibody with low skin toxicities. First, ΔN-SpyCatcher was reduced using tris(2-carboxyethyl)phosphine (TCEP), which was followed by desferoxamine-maleimide (DFO-mal) conjugation to yield a reactive ΔN-SpyCatcher-DFO. The ΔN-SpyCatcher-DFO was reacted with nimotuzumab-SpyTag to obtain stable nimotuzumab-SpyTag-∆N-SpyCatcher-DFO. Radiolabeling was performed with 89Zr, and the conjugate was used for the in vivo microPET imaging of EGFR-positive MDA-MB-468 xenografts. Similarly, ∆N-SpyCatcher was conjugated to an eighteen-membered macrocyclic chelator macropa-maleimide and used to radiolabel nimotuzumab-SpyTag with actinium-225 (225Ac) for in vivo radiotherapy studies. All constructs were characterized using biolayer interferometry, flow cytometry, radioligand binding assays, HPLC, and bioanalyzer. MicroPET/CT imaging showed a good tumor uptake of 89Zr-nimotuzumab-SpyTag-∆N-SpyCatcher with 6.0 ± 0.6%IA/cc (n = 3) at 48 h post injection. The EC50 of 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher and 225Ac-control-IgG-SpyTag-∆N-SpyCatcher against an EGFR-positive cell-line (MDA-MB-468) was 3.7 ± 3.3 Bq/mL (0.04 ± 0.03 nM) and 18.5 ± 4.4 Bq/mL (0.2 ± 0.04 nM), respectively. In mice bearing MDA-MB-468 EGFR-positive xenografts, 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher significantly (p = 0.0017) prolonged the survival of mice (64 days) compared to 225Ac-control IgG (28.5 days), nimotuzumab (28.5 days), or PBS-treated mice (30 days). The results showed that the conjugation and labeling using SpyTag/∆N-SpyCatcher to nimotuzumab did not significantly (p > 0.05) alter the receptor binding of nimotuzumab compared with a non-specific conjugation approach. 225Ac-nimotuzumab-SpyTag-∆N-SpyCatcher was effective in vitro and in an EGFR-positive triple negative breast cancer xenograft model.

Development and preclinical evaluation of cixutumumab drug conjugates in a model of insulin growth factor receptor I (IGF-1R) positive cancer.

Overexpression of insulin growth factor receptor type 1 (IGF-1R) is observed in many cancers. Antibody drug conjugates (ADCs) with PEGylated maytansine (PEG6-DM1) show promise in vitro. We developed PEG6-DM1 ADCs with low and high drug to antibody ratios (DAR) using an anti-IGF-1R antibody cixutumumab (IMC-A12). Conjugates with low (cixutumumab-PEG6-DM1-Low) and high (cixutumumab-PEG6-DM1-High) DAR as 3.4 and 7.2, respectively, were generated. QC was performed by UV spectrophotometry, HPLC, bioanalyzer, and biolayer-interferometry. We compared the in vitro binding and internalization rates of the ADCs in IGF-1R-positive MCF-7/Her18 cells. We radiolabeled the ADCs with 111In and used microSPECT/CT imaging and ex vivo biodistribution to understand their in vivo behavior in MCF-7/Her18 xenograft mice. The therapeutic potential of the ADC was studied in vitro and in mouse xenograft. Internalization rates of all ADCs was high and increased over 48 h and EC50 was in the low nanomolar range. MicroSPECT/CT imaging and ex vivo biodistribution showed significantly lower tumor uptake of 111In-cixutumumab-PEG6-DM1-High compared to 111In-cixutumumab-PEG6-DM1-Low and 111In-cixutumumab. Cixutumumab-PEG6-DM1-Low significantly prolonged the survival of mice bearing MCF-7/Her18 xenograft compared with cixutumumab, cixutumumab-PEG6-DM1-High, or the PBS control group. Cixutumumab-PEG6-DM1-Low ADC was more effective. The study highlights the potential utility of cixutumumab-ADCs as theranostics against IGF-1R positive cancers.

Large-Scale Image Analysis for Investigating Spatio-Temporal Changes in Nuclear DNA Damage Caused by Nitrogen Atmospheric Pressure Plasma Jets.

The effective clinical application of atmospheric pressure plasma jet (APPJ) treatments requires a well-founded methodology that can describe the interactions between the plasma jet and a treated sample and the temporal and spatial changes that result from the treatment. In this study, we developed a large-scale image analysis method to identify the cell-cycle stage and quantify damage to nuclear DNA in single cells. The method was then tested and used to examine spatio-temporal distributions of nuclear DNA damage in two cell lines from the same anatomic location, namely the oral cavity, after treatment with a nitrogen APPJ. One cell line was malignant, and the other, nonmalignant. The results showed that DNA damage in cancer cells was maximized at the plasma jet treatment region, where the APPJ directly contacted the sample, and declined radially outward. As incubation continued, DNA damage in cancer cells decreased slightly over the first 4 h before rapidly decreasing by approximately 60% at 8 h post-treatment. In nonmalignant cells, no damage was observed within 1 h after treatment, but damage was detected 2 h after treatment. Notably, the damage was 5-fold less than that detected in irradiated cancer cells. Moreover, examining damage with respect to the cell cycle showed that S phase cells were more susceptible to DNA damage than either G1 or G2 phase cells. The proposed methodology for large-scale image analysis is not limited to APPJ post-treatment applications and can be utilized to evaluate biological samples affected by any type of radiation, and, more so, the cell-cycle classification can be used on any cell type with any nuclear DNA staining.

Supra-coronary aortic banding improves right ventricular function in experimental pulmonary arterial hypertension in rats by increasing systolic right coronary artery perfusion.

AIM: Pulmonary arterial hypertension (PAH) results in right ventricular (RV) dysfunction owing, in part, to RV ischemia. The relative contribution of RV microvascular rarefaction vs reduced right coronary artery perfusion pressure (RCA-PP) to RV ischemia remains unknown. We hypothesize that increasing RCA-PP improves RV function in PAH by increasing RV systolic perfusion. METHODS: Supra-coronary aortic banding (SAB) or sham surgery was performed on male Sprague-Dawley rats. Seven to ten days later, rats received either monocrotaline (MCT; 60 mg/kg) or saline. After 1 month, echocardiography, cardiac catheterization, 99m Tc-sestamibi single-photon emission computed tomography (SPECT) and microsphere infusion studies were performed. The RV was harvested for measurement of hypertrophy (RVH), fibrosis and immunoblotting, and the lung was harvested for pulmonary artery (PA) histology. RESULTS: Supra-coronary aortic banding increased systolic pressures in proximal aorta and systolic RCA-PP in SAB + MCT vs MCT rats (114 ± 12 vs 5 ± 9 mm Hg), without altering diastolic RCA-PP. SAB + MCT rats had improved RV function vs MCT rats, evident from their significantly increased cardiac output (CO), RV free wall (RVFW) thickening, tricuspid annular plane systolic excursion (TAPSE) and RV-PA coupling indices. RV-PA coupling indices and CO correlated directly with systolic RCA-PP. RV perfusion was increased in SAB + MCT vs MCT rats and correlated well with CO; whereas microvascular rarefaction was unaltered. SAB + MCT rats had less RVH and fibrosis and lower PA pressures vs MCT rats. SAB + MCT rats had significantly lower RV pyruvate kinase muscle isoform 2/1 ratios than MCT rats, consistent with restoration of oxidative metabolism. CONCLUSION: A SAB-induced increase in systolic RCA-PP improves RV perfusion and function in MCT rats. Maintaining systolic RCA perfusion can preserve RV function in PAH.

Mitochondria in the Pulmonary Vasculature in Health and Disease: Oxygen-Sensing, Metabolism, and Dynamics.

In lung vascular cells, mitochondria serve a canonical metabolic role, governing energy homeostasis. In addition, mitochondria exist in dynamic networks, which serve noncanonical functions, including regulation of redox signaling, cell cycle, apoptosis, and mitochondrial quality control. Mitochondria in pulmonary artery smooth muscle cells (PASMC) are oxygen sensors and initiate hypoxic pulmonary vasoconstriction. Acquired dysfunction of mitochondrial metabolism and dynamics contribute to a cancer-like phenotype in pulmonary arterial hypertension (PAH). Acquired mitochondrial abnormalities, such as increased pyruvate dehydrogenase kinase (PDK) and pyruvate kinase muscle isoform 2 (PKM2) expression, which increase uncoupled glycolysis (the Warburg phenomenon), are implicated in PAH. Warburg metabolism sustains energy homeostasis by the inhibition of oxidative metabolism that reduces mitochondrial apoptosis, allowing unchecked cell accumulation. Warburg metabolism is initiated by the induction of a pseudohypoxic state, in which DNA methyltransferase (DNMT)-mediated changes in redox signaling cause normoxic activation of HIF-1α and increase PDK expression. Furthermore, mitochondrial division is coordinated with nuclear division through a process called mitotic fission. Increased mitotic fission in PAH, driven by increased fission and reduced fusion favors rapid cell cycle progression and apoptosis resistance. Downregulation of the mitochondrial calcium uniporter complex (MCUC) occurs in PAH and is one potential unifying mechanism linking Warburg metabolism and mitochondrial fission. Mitochondrial metabolic and dynamic disorders combine to promote the hyperproliferative, apoptosis-resistant, phenotype in PAH PASMC, endothelial cells, and fibroblasts. Understanding the molecular mechanism regulating mitochondrial metabolism and dynamics has permitted identification of new biomarkers, nuclear and CT imaging modalities, and new therapeutic targets for PAH. © 2020 American Physiological Society. Compr Physiol 10:713-765, 2020.

Identification of novel dynamin-related protein 1 (Drp1) GTPase inhibitors: Therapeutic potential of Drpitor1 and Drpitor1a in cancer and cardiac ischemia-reperfusion injury.

Mitochondrial fission is important in physiological processes, including coordination of mitochondrial and nuclear division during mitosis, and pathologic processes, such as the production of reactive oxygen species (ROS) during cardiac ischemia-reperfusion injury (IR). Mitochondrial fission is mainly mediated by dynamin-related protein 1 (Drp1), a large GTPase. The GTPase activity of Drp1 is essential for its fissogenic activity. Therefore, we aimed to identify Drp1 inhibitors and evaluate their anti-neoplastic and cardioprotective properties in five cancer cell lines (A549, SK-MES-1, SK-LU-1, SW 900, and MCF7) and an experimental cardiac IR injury model. Virtual screening of a chemical library revealed 17 compounds with high predicted affinity to the GTPase domain of Drp1. In silico screening identified an ellipticine compound, Drpitor1, as a putative, potent Drp1 inhibitor. We also synthesized a congener of Drpitor1 to remove the methoxymethyl group and reduce hydrolytic lability (Drpitor1a). Drpitor1 and Drpitor1a inhibited the GTPase activity of Drp1 without inhibiting the GTPase of dynamin 1. Drpitor1 and Drpitor1a have greater potency than the current standard Drp1 GTPase inhibitor, mdivi-1, (IC50 for mitochondrial fragmentation are 0.09, 0.06, and 10 µM, respectively). Both Drpitors reduced proliferation and induced apoptosis in cancer cells. Drpitor1a suppressed lung cancer tumor growth in a mouse xenograft model. Drpitor1a also inhibited mitochondrial ROS production, prevented mitochondrial fission, and improved right ventricular diastolic dysfunction during IR injury. In conclusion, Drpitors are useful tools for understanding mitochondrial dynamics and have therapeutic potential in treating cancer and cardiac IR injury.

111In- and 225Ac-Labeled Cixutumumab for Imaging and α-Particle Radiotherapy of IGF-1R Positive Triple-Negative Breast Cancer.

Insulin growth factor receptor (IGF-1R) is overexpressed in many cancers of epithelial origin, where it confers enhanced proliferation and resistance to therapies targeted at other receptors. Anti-IGF-1R monoclonal antibodies have not demonstrated significant improvements in patient outcomes in clinical trials. Humanized monoclonal antibody cixutumumab (IMC-A12) binds to IGF-1R with low nM affinity. In this study, cixutumumab was conjugated with p-SCN-Bn-DOTA and radiolabeled with 111In or 225Ac for imaging or radiotherapy using a triple-negative breast cancer (TNBC) model SUM149PT. The antibody conjugate showed low nM affinity to IGF-1R, which was not affected by conjugation and radiolabeling procedures. Cixutumumab immunoconjugates were effectively internalized in SUM149PT and were cytotoxic to the cells with an EC50 of 225Ac-cixutumumab (0.02 nM) that was almost 5000-fold less than that of unlabeled cixutumumab (95.2 nM). MicroSPECT imaging of the SUM149PT xenograft showed the highest tumor uptake occurred at 48 h post injection and was 9.9 ± 0.5% injected activity per gram (%IA/cc). In radiotherapy studies, we evaluated the effect of the specific activity of 225Ac-cixutumumab on efficacy following a tail vein injection of two doses (days 0 and 10) of the investigation agent or controls. Cixutumumab (2.5 mg/kg) prolonged the survival of the SUM149PT tumor-bearing mice with a median survival of 87 days compared to the PBS control group (median survival of 62 days). Median survival of high specific activity 225Ac-cixutumumab (8 kBq/µg, 225 nCi, 0.05 mg/kg) was 103.5 days compared to 122 days for low specific activity 225Ac-cixutumumab (0.15 kBq/µg, 225 nCi, 2.5 mg/kg). Additionally, low specific activity radioimmunoconjugate led to complete tumor remission in 2/6 mice. The data suggest that the efficacy of cixutumumab can be enhanced by radiolabeling with 225Ac at a low specific activity.

111In-Labeled Glycoprotein Nonmetastatic b (GPNMB) Targeted Gemini Surfactant-Based Nanoparticles against Melanoma: In Vitro Characterization and in Vivo Evaluation in Melanoma Mouse Xenograft Model.

Melanoma is a devastating form of skin cancer with high tendency to metastasis. This work addresses the development of new targeted nanoparticles that can be used for single-photon emission computed tomography (SPECT) imaging of melanoma. Melanoma-specific glycoprotein nonmetastatic b (GPNMB) antigen targeted and nontargeted gemini nanoparticles were prepared, characterized, and radiolabeled with 111In. 111In-labeled nanoparticles were composed of gemini surfactant grafted with monoclonal antibody Fab fragment that targeted GPNMB. Specific uptake of GPNMB-Fab was studied in six melanoma cell lines using flow cytometry. In vitro cellular uptake and internalization were studied using flow cytometry, confocal laser scanning microscopy, and radiometric techniques. Specific uptake of anti-GPNMB targeted nanoparticles was observed in GPNMB expressing cells, which was higher than low expressing or control cells. In vitro studies showed that conjugation of GPNMB targeted nanoparticles led to enhanced intracellular uptake of the nanodelivery system, which is critical for drug delivery. In vivo distribution of the nanoparticles was studied by microSPECT/CT imaging and ex vivo biodistribution. Tumor uptake was significantly higher ( p < 0.05) in nontargeted nanoparticles (5.47 ± 0.46%IA/cc) compared to GPNMB targeted nanoparticles (1.87 ± 0.27% ID/cc), which might be attributed to the high spleen uptake of the targeted formulation. These findings demonstrated that the radiolabeled gemini nanoparticles are promising for image-guided radiotherapy of melanoma. Formulation optimization is needed to improved tumor uptake and in vivo intracellular delivery for radiotherapeutic applications.

Preclinical Evaluation of 111In-Labeled PEGylated Maytansine Nimotuzumab Drug Conjugates in EGFR-Positive Cancer Models.

Epidermal growth factor receptor I (EGFR) is overexpressed in most cancers of epithelial origin. Antibody drug conjugates (ADCs) with PEGylated-maytansine (PEG-DM1) show promise in vitro and in vivo. However, in vivo biodistribution data for ADCs with PEG-DM1 have not been reported. Development of methods to understand the real-time in vivo behavior of these ADCs is needed to move these compounds to the clinic. Methods: Here we have used noninvasive small-animal SPECT/CT imaging and ex vivo biodistribution to understand the in vivo behavior of PEG6-DM1 ADCs. We developed nimotuzumab ADCs conjugated to PEG6-DM1. We generated immunoconjugates with low (nimotuzumab-PEG6-DM1-Low) and high (nimotuzumab-PEG6-DM1-High) drug-to-antibody ratios. The drug-to-antibody of nimotuzumab-PEG6-DM1-Low and nimotuzumab-PEG6-DM1-High was 3.5 and 7.3, respectively. Quality control was performed using ultraviolet spectrophotometry, size-exclusion high-performance liquid chromatography, bioanalyzer, biolayer interferometry, and flow cytometry in EGFR-positive DLD-1 cells. These immunoconjugates were conjugated with DOTA and radiolabeled with 111In. The in vitro binding and internalization rates of 111In-nimotuzumab, 111In-nimotuzumab-PEG6-DM1-Low, and 111In-nimotuzumab-PEG6-DM1-High were characterized. Furthermore, the pharmacokinetics, biodistribution, and imaging characteristics were evaluated in normal and DLD-1 tumor-bearing mice. Results: Flow cytometry and biolayer interferometry showed a trend toward decreasing EGFR affinity with increasing number of PEG6-DM1 on the antibody. Despite the lower overall cellular binding of the PEG6-DM1 radioimmunoconjugates, internalization was higher for PEG6-DM1 ADCs than for the non-PEGylated ADC in the following order: 111In-nimotuzumab-PEG6-DM1-High > 111In-nimotuzumab-PEG6-DM1-Low > 111In-nimotuzumab. Nuclear uptake of 111In-nimotuzumab-PEG6-DM1-High was 4.4-fold higher than 111In-nimotuzumab. Pharmacokinetics and biodistribution showed that 111In-nimotuzumab-PEG6-DM1-High had the slowest blood and whole-body clearance rate. Uptake in DLD-1 tumors of 111In-nimotuzumab was similar to 111In-nimotuzumab-PEG6-DM1-Low but was significantly higher than for 111In-nimotuzumab-PEG6-DM1-High. Tumor-to-background ratios for 111In-nimotuzumab and 111In-nimotuzumab-PEG6-DM1-Low were higher than for 111In-nimotuzumab-PEG6-DM1-High. Conclusion: The results show that conjugation of multiple PEG6-DM1 reduces the affinity for EGFR in vitro. However, the reduced affinity is counteracted by the high internalization rate of constructs with PEG6-DM1 ADCs in vitro. The decreased affinity resulted in low tumor uptake of 111In-nimotuzumab-PEG6-DM1-High, with a slow overall whole-body clearance rate. These data provide insights for evaluating the pharmacokinetics and normal -tissue toxicity and in determining dosing rate of PEGylated ADCs.

99mTc(CO)3+ labeled domain I/II-specific anti-EGFR (scFv)2 antibody fragment for imaging EGFR expression.

Bifunctional chelators (BFCs) are covalently linked to biologically active targeting molecules and radiolabeled with radiometals. Technetium-99 m (99mTc) is the most widely used isotope in nuclear medicine because of its excellent physical properties. The objective of this study was to synthesize and characterize a novel BFC that allows for the labeling of antibodies and antibody fragments using the 99mTc(CO)3+ core which forms a very stable complex with 99mTc in the +1 oxidation sate. This study reports the synthesis of a BFC 1-pyrrolidinyl-2,5-dione-11-(bis((1-(carboxymethyl)-1H-imidazol-2-yl)methyl)amino)undecanoic acid (SAAC-CIM NHS ester), and the in vitro and in vivo evaluation of 99mTc(CO)3-SAAC-CIM-DLO6-(scFv)2 (99mTc(CO)3-DLO6-(scFv)2), a domain I/II-specific anti-epidermal growth factor receptor I (anti-EGFR) antibody fragment. The chelator allowed radiolabeling the (scFv)2 antibody fragment in very mild conditions with no significant decrease in binding to EGFR. Radiochemical yields of >50% (radiochemical purity > 95%) of the resulting anti-EGFR (scFv)2 immunoconjugate 99mTc(CO)3-DLO6-(scFv)2 was obtained. The radioimmunoconjugate was stable in histidine challenge experiments with less than 20% transchelation at 24 h after challenge in the presence of a 1500-fold excess of histidine. In vivo biodistribution of 99mTc(CO)3-DLO6-(scFv)2 indicates that the tracer was mainly cleared via renal excretion and to a lesser extent via the hepatobiliary pathway. The microSPECT imaging studies performed in mice confirmed the in vitro affinity results. The 99mTc(CO)3-DLO6-(scFv)2 shows some promising properties and warrants further investigation for imaging EGFR.

89Zr-nimotuzumab for immunoPET imaging of epidermal growth factor receptor I.

RATIONALE: Epidermal growth factor receptor (EGFR) upregulation is associated with enhanced proliferation and drug resistance in a number of cancers. Nimotuzumab is a humanized monoclonal antibody with high affinity for EGFR. The objective of this study was to determine if 89Zr-DFO-nimotuzumab could be suitable for human use as a PET probe for quantifying EGFR in vivo. METHODS: To evaluate the pharmacokinetics, biodistribution, microPET imaging, radiation dosimetry, and normal tissue toxicity in tumor and non-tumor bearing mice of 89Zr-desferoxamine-nimotuzumab (89Zr-DFO-nimotuzumab) of a product prepared under GMP conditions. Nimotuzumab was conjugated to DFO and radiolabeled with 89Zr. 89Zr-DFO-nimotuzumab was characterized by in vitro gel-electrophoresis, biolayer interferometry (BLI) and flow cytometry. 89Zr-DFO-nimotuzumab was evaluated in vivo by microPET and ex vivo by biodistribution in healthy and EGFR-positive tumor bearing mice. RESULTS: Flow cytometry with A431 cells showed no significant difference in the dissociation constant of nimotuzumab (13 ± 2 nM) compared with DFO-nimotuzumab (17 ± 4 nM). PET imaging in mice xenografts showed persistently high tumor uptake with the highest uptake obtained in DLD-1 xenograft (18.3 %IA/cc) at 168 hp.i. The projected human effective dose was low and was 0.184 mSv/MBq (0.679 rem/mCi) in females and 0.205 mSv/MBq (0.757 rem/mCi) in males. There was no apparent normal tissue toxicity as shown by cell blood counts and blood biochemistry analyses at 168-fold and 25-fold excess of the projected human radioactive and mass dose of the agent. CONCLUSION: 89Zr-DFO-nimotuzumab had low organ absorbed dose and effective dose that makes it suitable for potential human use.

Investigating the Interaction of Silicon Dioxide Nanoparticles with Human Hemoglobin and Lymphocyte Cells by Biophysical, Computational, and Cellular Studies.

Nanoparticles (NPs) have received great attention in biological and medical applications because of their unique features. However, their induced adverse effects on the biological system are not well-explored. Herein, the interaction of silicon dioxide nanoparticles (SiO2 NPs) with human hemoglobin (Hb) and lymphocyte cell line was evaluated under physiological conditions by multispectroscopic [intrinsic and synchronous fluorescence spectroscopy and circular dichrosim (CD)], molecular docking, and cellular [3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) and acridine orange/ethidium bromide (AO/EB) staining] methods. Transmission electron microscopy and dynamic light scattering revealed the nanosized and spherical shaped SiO2 particle. The fluorescence and lifetime decay results indicated that SiO2 NPs quenched the intrinsic intensity of Hb through a static quenching mechanism. The binding affinity of SiO2 NPs toward Hb was directly correlated with temperature. The sign of thermodynamic parameters demonstrated that hydrophobic forces played a pivotal role in the interaction of SiO2 NPs with Hb. The results of synchronous fluorescence experiments displayed that Tyr residues are moved to a more hydrophilic microenvironment. Molecular docking studies exhibited that SiO2 and Si NPs were bound to Hb primarily by hydrophobic residues. The findings from CD data verified no alteration in the secondary structure of Hb upon binding to SiO2 NPs. The human lymphocyte cell line was treated with SiO2 NPs at varying concentrations and time intervals and the cytotoxicity assays by MTT and AO/EB staining showed that cell viability was reduced by the SiO2 NP-induced apoptosis mechanism in a dose and time-dependent manner. Therefore, it may be suggested that comprehensive details regarding the interaction of NPs and biological systems such as cells and proteins can provide useful information in the development of NP-based systems.

Low-energy electron-induced dissociation in gas-phase nicotine, pyridine, and methyl-pyrrolidine.

Dissociative electron attachment to nicotine, pyridine, and N-methyl-pyrrolidine was studied in the gas phase in order to assess their stability with respect to low-energy electron interactions. Anion yield curves for different products at electron energies ranging from zero to 15 eV were measured, and the molecular fragmentation pathways were proposed. Nicotine does not form a stable parent anion or a dehydrogenated anion, contrary to other biological systems. However, we have observed complex dissociation pathways involving fragmentation at the pyrrolidine side accompanied by isomerization mechanisms. Combining structure optimization and enthalpy calculations, performed with the Gaussian09 package, with the comparison with a deuterium-labeled N-methyl-d3-pyrrolidine allowed for the determination of the fragmentation pathways. In contrast to nicotine and N-methylpyrrolidine, the dominant pathway in dissociative electron attachment to pyridine is the loss of hydrogen, leading to the formation of an [M-H]- anion. The presented results provide important new information about the stability of nicotine and its constituent parts and contribute to a better understanding of the fragmentation mechanisms and their effects on the biological environment.