Identification of Novel Inhibitors for ERα Target of Breast Cancer By In Silico Approach.

BACKGROUND: Estrogen alpha has been recognized as a perilous factor in breast cancer cell proliferation and has been proficiently treated in breast cancer chemotherapy with the development of selective estrogen receptor modulators (SERMs). OBJECTIVES: The major aim of this study was to identify the potential inhibitors against the most influential target ERα receptor by in silico studies of 115 phytochemicals from 17 medicinal plants using in silico molecular docking studies. METHODS: The molecular docking investigation was carried out by a genetic algorithm using the Auto Dock Vina program, and the validation of docking was also performed using molecular dynamic (MD) simulation by the Desmond tool of Schrödinger molecular modeling. The ADME( T) studies were performed by SWISS ADME and ProTox-II. RESULTS: The top ten highest binding energy phytochemicals identified were amyrin acetate (- 10.7 kcal/mol), uscharine (-10.5 kcal/mol), voruscharin (-10.0 kcal/mol), cyclitols (-10.0 kcal/mol), taraxeryl acetate (-9.9 kcal/mol), amyrin (-9.9 kcal/mol), barringtogenol C (-9.9 kcal/mol), calactin (-9.9 kcal/mol), 3-beta taraxerol (-9.8 kcal/mol), and calotoxin (-9.8 kcal/mol). A molecular docking study revealed that these phytochemical constituents showed higher binding affinity compared to the reference standard tamoxifen (-6.6 kcal/mol) towards the target protein ERα. The results of MD studies showed that all four tested compounds possess comparatively stable ligand-protein complexes with ERα target as compared to the tamoxifen- ERα complex. CONCLUSION: Among the ten compounds, phytochemical amyrin acetate (triterpenoids) formed a more stable complex as well as exhibited greater binding affinity than standard tamoxifen. ADMET studies for the top ten phytochemicals showed a good safety profile. Additionally, these compounds are being reported for the first time in this study as possible inhibitors of ERα for the treatment of breast cancer by adopting the concept of drug repurposing. Hence, these phytochemicals can be further studied and can be used as a parent core molecule to develop novel lead molecules for breast cancer therapy.

An Overview of Acridine Analogs: Pharmacological Significance and Recent Developments.

The clinical effectiveness of the available anticancer drugs has been reduced due to the development of drug resistance and serious adverse effects, which have restricted chemotherapy for cancer. Therefore, there is a persistent need for new anticancer medications with reduced side effects. Medical researchers are pursuing various methods to find new, potent, specifically targeted molecules for cancer treatment. Through various techniques, numerous molecules are discovered. However, among them, acridine stands out as a promising heterocycle that has captured the interest of medicinal chemists and acquired significant pharmacological value. The synthetic adaptability of acridine has enabled the creation of numerous derivatives with a wide range of architectural properties, further accelerating this broad spectrum of pharmacological activities. Recent studies have looked at the mechanisms by which acridine and its analogs inhibit tyrosine kinases, topoisomerases, telomerase, and DNA repair interaction. We have compiled our knowledge of acridine compounds for their anticancer activities, mechanisms of action, structure-activity relationship (SAR), and selective, specific activity against different cancer drug targets, as well as in vitro and in vivo anticancer activities of acridine and its analogs from the perspective of cancer drug discovery, in this review.

Computational Screening of Some Phytochemicals to Identify Best Modulators for Ligand Binding Domain of Estrogen Receptor Alpha.

OBJECTIVE: The peculiar aim of this study is to discover and identify the most effective and potential inhibitors against the most influential target ERα receptor by in silico studies of 45 phytochemicals from six diverse ayurvedic medicinal plants. METHODS: The molecular docking investigation was carried out by the genetic algorithm program of AutoDock Vina. The molecular dynamic (MD) simulation investigations were conducted using the Desmond tool of Schrödinger molecular modelling. This study identified the top ten highest binding energy phytochemicals that were taken for drug-likeness test and ADMET profile prediction with the help of the web-based server QikpropADME. RESULTS: Molecular docking study revealed that ellagic acid (-9.3 kcal/mol), emodin (-9.1 kcal/mol), rhein (-9.1 kcal/mol), andquercetin (-9.0 kcal/mol) phytochemicals showed similar binding affinity as standard tamoxifen towards the target protein ERα. MD studies showed that all four compounds possess comparatively stable ligand-protein complexes with ERα target compared to the tamoxifen-ERα complex. Among the four compounds, phytochemical rhein formed a more stable complex than standard tamoxifen. ADMET studies for the top ten highest binding energy phytochemicals showed a better safety profile. CONCLUSION: Additionally, these compounds are being reported for the first time in this study as possible inhibitors of ERα for treating breast cancer, according to the notion of drug repurposing. Hence, these phytochemicals can be further studied and used as a parent core molecule to develop innovative lead molecules for breast cancer therapy.

Fabrication of Quercetin-Functionalized Morpholine and Pyridine Motifs-Laden Silk Fibroin Nanofibers for Effective Wound Healing in Preclinical Study.

Choosing suitable wound dressings is crucial for effective wound healing. Spun scaffolds with bioactive molecule functionalization are gaining attention as a promising approach to expedite tissue repair and regeneration. Here, we present the synthesis of novel multifunctional quercetin with morpholine and pyridine functional motifs (QFM) embedded in silk fibroin (SF)-spun fibers (SF-QFM) for preclinical skin repair therapies. The verification of the novel QFM structural arrangement was characterized using ATR-FTIR, NMR, and ESI-MS spectroscopy analysis. Extensive characterization of the spun SF-QFM fibrous mats revealed their excellent antibacterial and antioxidant properties, biocompatibility, biodegradability, and remarkable mechanical and controlled drug release capabilities. SF-QFM mats were studied for drug release in pH 7.4 PBS over 72 h. The QFM-controlled release is mainly driven by diffusion and follows Fickian's law. Significant QFM release (40%) occurred within the first 6 h, with a total release of 79% at the end of 72 h, which is considered beneficial in effectively reducing bacterial load and helping expedite the healing process. Interestingly, the SF-QFM-spun mat demonstrated significantly improved NIH 3T3 cell proliferation and migration compared to the pure SF mat, as evidenced by the complete migration of NIH 3T3 cells within 24 h in the scratch assay. Furthermore, the in vivo outcome of SF-QFM was demonstrated by the regeneration of fresh fibroblasts and the realignment of collagen fibers deposition at 9 days post-operation in a preclinical rat full-thickness skin defect model. Our findings collectively indicate that the SF-QFM electrospun nanofiber scaffolds hold significant capability as a cost-effective and efficient bioactive spun architecture for use in wound healing applications.

Liquid chromatography-tandem mass spectrometry determination of bumetanide in human plasma and application to a clinical pharmacokinetic study.

Determining a drug's bioavailability and bioequivalence is important for developing and approving a drug product. The procedure supports applications for generic drug products and novel therapeutic substances, makes important decisions regarding safety and efficacy, and measures a drug's concentration in biological matrices. This study aimed to develop and validate a specific, simple, sensitive, and accurate method using liquid chromatography-tandem mass spectrometry (LC-MS) for measuring bumetanide (BUM) in human plasma. Chromatographic separation was achieved using a Hypurity C18 column (4.6 × 50 mm, 5 µm) under isocratic conditions, and LC-MS detected positive ionization acquisition modes. Protonated precursor to product ion transitions were observed at m/z 365.08 → 240.10 and 370.04 → 244.52 for BUM and internal standard, respectively. The linear range of BUM in plasma samples was 3.490-401.192 ng/mL. The inter-precision value ranged from 1.76% to 4.75%. The inter-accuracy value ranged from 96.46% to 99.95%. The method was adequately validated per the U.S. Food and Drug Administration guidelines, and the results were within permissible bounds. The Cmax and Tmax values were ~53.097 ± 13.537 ng/mL and 1.25 (0.67-5.00) h, respectively. The new approach showed satisfactory results for studying BUM in human plasma with potential use in pharmacokinetic and bioequivalence investigations.

Electrospun Scaffold-based Antibiotic Therapeutics for Chronic Wound Recovery.

Treatment of a wound infection caused by a multidrug-resistant (MDR) bacterium is challenging since traditional medicine is incapable of curing such infections. As a result, there is a critical need to develop wound dressings resistant to MDR bacteria. Over half of diabetic and burn wounds showed clinical symptoms of infection. Diabetes is a metabolic disorder that may have various consequences, including chronic sores, vascular damage, and neuropathy. Microbial infection and oxidative stress to the fibroblast are common causes of slow and ineffective wound healing. Since wound healing and tissue repair are complex cascades of cellular activities, prompt and ordered healing is critical throughout this process. Despite advances in medication development and sophisticated formulations, treating persistent wound infections remains difficult. The drawbacks of administering antibiotics through the digestive system have motivated the development of enhanced therapeutic dressings with antibacterial activity and the application of antibiotics by localized administration. Antimicrobial wound dressings have great promise for reducing infection risk and improving the healing rate of chronic lesions. Most current research in skin tissue engineering focuses on developing threedimensional scaffolds that mimic natural skin's extracellular matrix (ECM). Electrospinning is a wellestablished method for producing nanoscale fibers. It is a simple, cost-effective, reproducible, and efficient process for encapsulating hydrophobic and hydrophilic antimicrobial compounds in synthetic and natural polymeric carriers. This review discusses various nanofibers as novel delivery systems for antimicrobial compounds in chronic wound healing. We will discuss the significant polymers used to make nanofibers, their manufacturing processes, and, most importantly, their antibacterial effectiveness against microorganisms that typically cause chronic wound infections.

Ultrasonic-Assisted Solid-Phase Peptide Synthesis of DOTA-TATE and DOTA-linker-TATE Derivatives as a Simple and Low-Cost Method for the Facile Synthesis of Chelator-Peptide Conjugates.

Peptides have been widely adopted as biological targeting vectors for applications in molecular imaging and peptide-receptor radionuclide therapy (PRRT). Somatostatin (SST) analogues such as octreotate (TATE) are exogenous ligands for somatostatin receptors (SSTRs), which are highly expressed on neuroendocrine tumors (NETs). Recently, both [68Ga]Ga-DOTA-TATE (NETSPOT) and [177Lu]Lu-DOTA-TATE (LUTATHERA) received U.S. Food and Drug Administration approval for positron emission tomography (PET) imaging and PRRT of NETs, respectively. However, to the best of our knowledge a well-described synthesis of DOTA-TATE has not been reported in the literature. Herein, we report a fully reoptimized DOTA-TATE synthesis, including the application of a simple ultrasonic bath to greatly improve yields, reduce coupling times, and decrease the amount of reagents required for each coupling step by a half. The most prevalently used cyclizing agents such as iodine, thallium(III) trifluoroacetate, hydrogen peroxide, and dimethyl sulfoxide were compared. On-resin cyclizations using mechanical agitation showed higher yields (23% and 25% using I2 and Tl(III), respectively) than off-resin (1.3% and 11% using DMSO and H2O2, respectively), and the total synthesis time of DOTA-TATE was ∼540 min excluding the cyclization step, with a total synthesis yield of ∼23%. The same manual SPPS methods/reagents were reoptimized with ultrasonic (US) agitation, resulting in an immense reduction in the total synthesis time by ∼8-fold to ∼70 min for DOTA-TATE with a higher yield (∼29% yield), and ∼13-fold to 105 min for DOTA-PEG4-TATE (∼29% yield). Also, the use of US agitation reduces the need for excess molar equivalents of the reagents to a half, which is particularly important when coupling expensive or custom-synthesized groups such as bifunctional chelators and linkers. Finally, the synthesized DOTA-TATE was successfully radiolabeled with [68Ga]Ga3+ (t1/2 = 68 min) with high radiochemical yields (30 min, 95 °C). We believe this work opens the door to the facile and low-cost synthesis of many new chelator-linker-peptide conjugates that were previously cumbersome or cost-prohibitive to produce with manual SPPS.

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.

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.

GABA allosteric modulators: An overview of recent developments in non-benzodiazepine modulators.

γ-Aminobutyric acid (GABA) is the major inhibitory transmitter controlling synaptic transmission and neuronal excitability. It is present in a high percentage of neurons in the central nervous system (CNS) and also present in the peripheral nervous system, and acts to maintain a balance between excitation and inhibition. GABA acts via three subclasses of receptors termed GABAA, GABAB, and GABAC. GABAA and GABAC receptors are ligand-gated ion channels, while GABAB receptors are G-protein coupled receptors. Each class of GABA receptor has distinct pharmacology and physiology. GABAA receptors are heteropentameric transmembrane protein complexes made up of α1-6, ß1-3, γ1-3, δ, ε, θ, π subunits, giving rise to numerous allosteric binding sites and have thus attracted much attention targets for the treatment of conditions such as epilepsy, anxiety and sleep disorders. The development of ligands for these binding sites has also led to an improved understanding of the different physiological functions and pathological processes and offers the opportunity for the development of novel therapeutics. This review focuses on the medicinal chemistry aspects including drug design, structure-activity relationships (SAR), and mechanism of actions of GABA modulators, including non-benzodiazepine ligands at the benzodiazepine binding site and modulators acting at sites other than the high-affinity benzodiazepine binding site. Recent advances in this area their future applications and potential therapeutic effects are also highlighted.

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.

A 89Zr-labeled lipoplex nanosystem for image-guided gene delivery: design, evaluation of stability and in vivo behavior.

BACKGROUND: With the advances in radiopharmaceutical research, the development of image-guided therapy has become a major interest. While the development of theranostic nanotherapeutics is frequently associated with cancer chemotherapy, phototherapy and radiotherapy, there is little information available on the in vivo monitoring of gene delivery systems and the application of image-guided approach in gene therapy. The goal of this work was to determine the in vivo behavior of DNA delivery nanosystems - based on cationic gemini surfactants - designed for image-guided gene therapy. We tested the feasibility of monitoring tumor accumulation of gene delivery nanoparticles by positron emission tomography. METHODS: To be able to conjugate radiotracers to the nanoparticles, a deferoxamine-modified gemini surfactant was synthesized, DNA-containing lipoplex nanoparticles were formulated, and radiolabeled with Zirconium-89 (89Zr). The pharmacokinetics and biodistribution of 89Zr labeled surfactant and 89Zr labeled nanoparticles were monitored in mice by microPET/CT imaging and ex vivo gamma counting. RESULTS: Modification of the nanoparticles with deferoxamine did not alter their physicochemical properties. The radiolabeled nanoparticles (labeling efficiency of 95±3%) were stable in PBS and serum. The biological half-life of the 89Zr labeled nanoparticles was significantly higher compared to 89Zr labeled surfactant. As expected, the nanoparticles had significantly higher liver accumulation than the radiolabeled surfactant alone and lower kidney accumulation. Tumor uptake was detected at 2 hours post injection and decreased throughout the 3-day monitoring. CONCLUSION: We propose that radiolabeling DNA delivery lipoplex nanosystems is a promising approach for the design and optimization of image-guided nanomedicines, especially in the context of cancer gene therapy.

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.

Identification of SNAIL1 Peptide-Based Irreversible Lysine-Specific Demethylase 1-Selective Inactivators.

Inhibition of lysine-specific demethylase 1 (LSD1), a flavin-dependent histone demethylase, has recently emerged as a new strategy for treating cancer and other diseases. LSD1 interacts physically with SNAIL1, a member of the SNAIL/SCRATCH family of transcription factors. This study describes the discovery of SNAIL1 peptide-based inactivators of LSD1. We designed and prepared SNAIL1 peptides bearing a propargyl amine, hydrazine, or phenylcyclopropane moiety. Among them, peptide 3, bearing hydrazine, displayed the most potent LSD1-inhibitory activity in enzyme assays. Kinetic study and mass spectrometric analysis indicated that peptide 3 is a mechanism-based LSD1 inhibitor. Furthermore, peptides 37 and 38, which consist of cell-membrane-permeable oligoarginine conjugated with peptide 3, induced a dose-dependent increase of dimethylated Lys4 of histone H3 in HeLa cells, suggesting that they are likely to exhibit LSD1-inhibitory activity intracellularly. In addition, peptide 37 decreased the viability of HeLa cells. We believe this new approach for targeting LSD1 provides a basis for development of potent selective inhibitors and biological probes for LSD1.

Synthesis and pharmacological evaluation of 3-cyclohexyl-2-substituted hydrazino-3H-quinazolin-4-ones as analgesic and anti-inflammatory agents.

A series of novel 3-cyclohexyl-2-substituted hydrazino-quinazolin-4(3H)-ones were synthesized by reacting the amino group of 3-cyclohexyl-2-hydrazino quinazolin-4(3H)-one with a variety of aldehydes and ketones. The starting material, 3-cyclohexyl-2-hydrazino quinazolin-4(3H)-one, was synthesized from cyclohexyl amine. Title compounds were investigated for analgesic, anti-inflammatory and ulcerogenic behavior. The compound 3-cyclohexyl-2-(1-methylbutylidene-hydrazino)-3H-quinazolin-4-one (4c) emerged as the most active compound of the series and is moderately more potent in its analgesic and anti-inflammatory activities compared to the reference standard diclofenac sodium. Interestingly, test compounds showed only mild ulcerogenic potential when compared to acetylsalicylic acid.

Synthesis and pharmacological investigation of novel 4-(3-ethylphenyl)-1-substituted-4H-[1,2,4]triazolo[4,3-a] quinazolin-5-ones as a new class of H1-antihistaminic agents.

A series of novel 4-(3-ethylphenyl)-1-substituted-4H-[1,2,4] triazolo[4,3-a]quinazolin-5-ones (4a-j) were synthesized by the cyclization of 3-(3-ethylphenyl)-2-hydrazino-3H-quinazolin-4-one (3) with various one-carbon donors. The starting material, compound 3, was synthesized from 3-ethyl aniline by a new innovative route with improved yield. When tested for their in vivo H1-antihistaminic activity on conscious guinea pigs, all test compounds protected the animals from histamine induced bronchospasm significantly. Compound 4-(3-ethylphenyl)-1-methyl-4H - [1,2,4]triazolo[4,3-a]quinazolin-5-one (4b) emerged as the most active compound of the series and it is more potent (74.6 % protection) compared to the reference standard chlorpheniramine maleate (71 % protection). Compound 4b shows negligible sedation (10 %) compared to chlorpheniramine maleate (30 %). Therefore compound 4b can serve as the leading compound for further development of a new class of H1-antihistamines.

Synthesis and pharmacological evaluation of some 3-(2-methylphenyl)-2-substituted amino-quinazolin-4(3H)-ones as analgesic and antiinflammatory agents.

A variety of novel 3-(2-methylphenyl)-2-substituted amino-quinazolin-4(3H)-ones were synthesized by reacting the amino group of 2-hydrazino-3-(2-methylphenyl)-quinazolin-4(3H)-one with a variety of aldehydes and ketones. The starting material 2-hydrazino-3-(2-methylphenyl)-quinazolin-4(3H)-one was synthesized from 2-methyl aniline. The title compounds were investigated for analgesic, anti-inflammatory and ulcerogenic index activities. Among these, the compound 2-(1-ethylpropylidene)-hydrazino-3-(2-methylphenyl)-quinazolin-4(3H)-one emerged as the most active compound for analgesic activity, while the compound 2-(1-methylbutylidene)-hydrazino-3-(2-methylphenyl)-quinazolin-4(3H)-one showed most potent anti-inflamma-tory activity of the series and was moderately more potent in its anti-inflammatory activity when compared to the reference standard diclofenac sodium (CAS 15307-86-5). Interestingly, the test compounds showed only mild ulcerogenic potential when compared to acetylsalicylic acid (CAS No: 50-78-2).