Recent advances in small molecule Nav 1.7 inhibitors for cancer pain management.

The dorsal root ganglion (DRG) is the primary neuron responsible for transmitting peripheral pain signals to the central nervous system and plays a crucial role in pain transduction. Modulation of DRG excitability is considered a viable approach for pain management. Neuronal excitability is intricately linked to the ion channels on the neurons. The small and medium-sized DRG neurons are chiefly engaged in pain conduction and have high levels of TTX-S sodium channels, with Nav1.7 accounting for approximately 80% of the current. Voltage-gated sodium channel (VGSC or Nav) blockers are vital targets for the management of central nervous system diseases, particularly chronic pain. VGSCs play a key role in controlling cellular excitability. Clinical research has shown that Nav1.7 plays a crucial role in pain sensation, and there is strong genetic evidence linking Nav1.7 and its encoding gene SCN9A gene to painful disorders in humans. Many studies have shown that Nav1.7 plays an important role in pain management. The role of Nav1.7 in pain signaling pathways makes it an attractive target for the potential development of new pain drugs. Meanwhile, understanding the architecture of Nav1.7 may help to develop the next generation of painkillers. This review provides updates on the recently reported molecular inhibitors targeting the Nav1.7 pathway, summarizes their structure-activity relationships (SARs), and discusses their therapeutic effects on painful diseases. Pharmaceutical chemists are working to improve the therapeutic index of Nav1.7 inhibitors, achieve better analgesic effects, and reduce side effects. We hope that this review will contribute to the development of novel Nav1.7 inhibitors as potential drugs.

Stereoselective Synthesis of Substituted (Z)-N-Allyl Sulfonamides via a Palladium-Catalyzed Three-Component Tandem Reaction.

This paper reports the efficient synthesis of substituted (Z)-N-allyl sulfonamides via a palladium-catalyzed three-component tandem reaction of N-buta-2,3-dienyl sulfonamides with iodides and sulfonyl hydrazide or sulfinic acid sodium salt as nucleophiles. Pd(PPh3)4 (2.5 mol %), K2CO3, and THF were used as the optimal catalyst, base, and solvent, respectively. The substituted (Z)-N-allyl sulfonamides were obtained in a 30-83% overall yield. Mechanistic investigations revealed that the formation of the single (Z)-isomer was controlled by the formation of a six-membered palladacycle intermediate.

A Novel Dose Rate Optimization Method to Maximize Ultrahigh-Dose-Rate Coverage of Critical Organs at Risk Without Compromising Dosimetry Metrics in Proton Pencil Beam Scanning FLASH Radiation Therapy.

PURPOSE: This study aimed to investigate a dose rate optimization framework based on the spot-scanning patterns to improve ultrahigh-dose-rate coverage of critical organs at risk (OARs) for proton pencil beam scanning (PBS) FLASH radiation therapy (ultrahigh dose-rate (often referred to as >40 Gy per second) delivery) and present implementation of a genetic algorithm (GA) method for spot sequence optimization to achieve PBS FLASH dose rate optimization under relatively low nozzle beam currents. METHODS AND MATERIALS: First, a multifield FLASH plan was developed to meet all the dosimetric goals and optimal FLASH dose rate coverage by considering the deliverable minimum monitor unit constraint. Then, a GA method was implemented into the in-house treatment platform to maximize the dose rate by exploring the best spot delivery sequence. A phantom study was performed to evaluate the effectiveness of the dose rate optimization. Then, 10 consecutive plans for patients with lung cancer previously treated using PBS intensity-modulated proton therapy were optimized using 45 GyRBE in 3 fractions for both transmission and Bragg peak FLASH radiation therapy for further validation. The spot delivery sequence of each treatment field was optimized using this GA. The ultrahigh-dose-rate-volume histogram and dose rate coverage V40GyRBE/s were investigated to assess the efficacy of dose rate optimization quantitatively. RESULTS: Using a relatively low monitor unit/spot of 150, corresponding to a nozzle beam current of 65 nA, the FLASH dose rate ratio V40GyRBE/s of the OAR contour of the core was increased from 0% to ∼60% in the phantom study. In the patients with lung cancer, the ultrahigh-dose-rate coverage V40GyRBE/s was improved from 15.2%, 15.5%, 17.6%, and 16.0% before the delivery sequence optimization to 31.8%, 43.5%, 47.6%, and 30.5% after delivery sequence optimization in the lungs-GTV (gross tumor volume), spinal cord, esophagus, and heart (for all, P < .001). When the beam current increased to 130 nA, V40GyRBE/s was improved from 45.1%, 47.1%, 51.2%, and 51.4% to 65.3%, 83.5%, 88.1%, and 69.4% (P < .05). The averaged V40GyRBE/s for the target and OARs increased from 12.9% to 41.6% and 46.3% to 77.5% for 65 and 130 nA, respectively, showing significant improvements based on a clinical proton system. After optimizing the dose rate for the Bragg peak FLASH technique with a beam current of 340 nA, the V40GyRBE/s values for the lung GTV, spinal cord, esophagus, and heart were increased by 8.9%, 15.8%, 22%, and 20.8%, respectively. CONCLUSIONS: An optimal plan quality can be maintained as the spot delivery sequence optimization is a separate independent process after the plan optimization. Both the phantom and patient results demonstrated that novel spot delivery sequence optimization can effectively improve the ultrahigh-dose-rate coverage for critical OARs, which can potentially be applied in clinical practice for better OARs-sparing efficacy.

Pencil Beam Scanning Proton Bragg Peak Conformal FLASH in Prostate Cancer Stereotactic Body Radiotherapy.

Bragg peak FLASH radiotherapy (RT) uses a distal tracking method to eliminate exit doses and can achieve superior OAR sparing. This study explores the application of this novel method in stereotactic body radiotherapy prostate FLASH-RT. An in-house platform was developed to enable intensity-modulated proton therapy (IMPT) planning using a single-energy Bragg peak distal tracking method. The patients involved in the study were previously treated with proton stereotactic body radiotherapy (SBRT) using the pencil beam scanning (PBS) technique to 40 Gy in five fractions. FLASH plans were optimized using a four-beam arrangement to generate a dose distribution similar to the conventional opposing beams. All of the beams had a small angle of two degrees from the lateral direction to increase the dosimetry quality. Dose metrics were compared between the conventional PBS and the Bragg peak FLASH plans. The dose rate histogram (DRVH) and FLASH metrics of 40 Gy/s coverage (V40Gy/s) were investigated for the Bragg peak plans. There was no significant difference between the clinical and Bragg peak plans in rectum, bladder, femur heads, large bowel, and penile bulb dose metrics, except for Dmax. For the CTV, the FLASH plans resulted in a higher Dmax than the clinical plans (116.9% vs. 103.3%). For the rectum, the V40Gy/s reached 94% and 93% for 1 Gy dose thresholds in composite and single-field evaluations, respectively. Additionally, the FLASH ratio reached close to 100% after the application of the 5 Gy threshold in composite dose rate assessment. In conclusion, the Bragg peak distal tracking method can yield comparable plan quality in most OARs while preserving sufficient FLASH dose rate coverage, demonstrating that the ultra-high dose technique can be applied in prostate FLASH SBRT.

Validation and reproducibility of in vivo dosimetry for pencil beam scanned FLASH proton treatment in mice.

PURPOSE: To investigate quality assurance (QA) techniques for in vivo dosimetry and establish its routine uses for proton FLASH small animal experiments with a saturated monitor chamber. METHODS AND MATERIALS: 227 mice were irradiated at FLASH or conventional (CONV) dose rates with a 250 MeV FLASH-capable proton beamline using pencil beam scanning to characterize the proton FLASH effect on abdominal irradiation and examining various endpoints. A 2D strip ionization chamber array (SICA) detector was positioned upstream of collimation and used for in vivo dose monitoring during irradiation. Before each irradiation series, SICA signal was correlated with the isocenter dose at each delivered dose rate. Dose, dose rate, and 2D dose distribution for each mouse were monitored with the SICA detector. RESULTS: Calibration curves between the upstream SICA detector signal and the delivered dose at isocenter had good linearity with minimal R2 values of 0.991 (FLASH) and 0.985 (CONV), and slopes were consistent for each modality. After reassigning mice, standard deviations were less than 1.85 % (FLASH) and 0.83 % (CONV) for all dose levels, with no individual subject dose falling outside a ± 3.6 % range of the designated dose. FLASH fields had a field-averaged dose rate of 79.0 ± 0.8 Gy/s and mean local average dose rate of 160.6 ± 3.0 Gy/s. In vivo dosimetry allowed for the accurate detection of variation between the delivered and the planned dose. CONCLUSION: In vivo dosimetry benefits FLASH experiments through enabling real-time dose and dose rate monitoring allowing mouse cohort regrouping when beam fluctuation causes delivered dose to vary from planned dose.

Pencil Beam Scanning Bragg Peak FLASH Technique for Ultra-High Dose Rate Intensity-Modulated Proton Therapy in Early-Stage Breast Cancer Treatment.

Bragg peak FLASH-RT can deliver highly conformal treatment and potentially offer improved normal tissue protection for radiotherapy patients. This study focused on developing ultra-high dose rate (≥40 Gy × RBE/s) intensity-modulated proton therapy (IMPT) for hypofractionated treatment of early-stage breast cancer. A novel tracking technique was developed to enable pencil beaming scanning (PBS) of single-energy protons to adapt the Bragg peak (BP) to the target distally. Standard-of-care PBS treatment plans of consecutively treated early-stage breast cancer patients using multiple energy layers were reoptimized using this technique, and dose metrics were compared between single-energy layer BP FLASH and conventional IMPT plans. FLASH dose rate coverage by volume (V40Gy/s) was also evaluated for the FLASH sparing effect. Distal tracking can precisely stop BP at the target distal edge. All plans (n = 10) achieved conformal IMPT-like dose distributions under clinical machine parameters. No statistically significant differences were observed in any dose metrics for heart, ipsilateral lung, most ipsilateral breast, and CTV metrics (p > 0.05 for all). Conventional plans yielded slightly superior target and skin dose uniformities with 4.5% and 12.9% lower dose maxes, respectively. FLASH-RT plans reached 46.7% and 61.9% average-dose rate FLASH coverage for tissues receiving more than 1 and 5 Gy plan dose total under the 250 minimum MU condition. Bragg peak FLASH-RT techniques achieved comparable plan quality to conventional IMPT while reaching adequate dose rate ratios, demonstrating the feasibility of early-stage breast cancer clinical applications.

Pubertal Bisphenol A exposure increases adult rat serum testosterone by resetting pituitary homeostasis.

Bisphenol A (BPA) is widely used by manufacturers and in consumer products. Its release in the environment may affect male reproductive function. In this study, we examined the effect of low dose (0.1 mg/kg BW), short term exposure during puberty (PD21-35) on adult rat male reproduction. The results indicated that such exposure reset growth hormone (GH) and follicular stimulating hormone (FSH) homeostasis and resulted in a significantly higher level of serum testosterone without affecting serum luteinizing hormone level. QPCR and Western blot results showed that BPA significantly up-regulated selective genes/proteins in the Leydig cell steroidogenic pathway, including steroidogenic acute regulatory protein, cytochrome P450 11A1, cytochrome P450 17A, and low-density lipoprotein receptor. RNA-Seq analysis of testicular RNAs showed that BPA significantly affected the gene profiles of multiple testicular interstitial populations without affecting germ cells. Also, GO- and KEGG-analysis suggested that IGF1-related PI3K/AKT signaling was activated, which was confirmed by the increased phosphorylation of IRS1, AKT1 and CREB. The results indicated that a low-dose, short-term BPA exposure during puberty affected the adult male rat pituitary (GH and FSH) and testis (testosterone) homeostasis.

Isolation of Leydig cells from adult rat testes by magnetic-activated cell sorting protocol based on prolactin receptor expression.

BACKGROUND: The primary function of testicular Leydig cells (LCs) is to produce testosterone (T). In vitro culture of the cells represents a very important approach to study androgen production and its regulations. Various methods have been developed for the enrichment of the cells from the testes. However, getting cells in large numbers with high purity and viability is still challenging. Here, we describe a new way to isolate LCs from rat testes in large quantity with high purity and viability. METHODS: Enzymatic digested testicular cells from adult rats were labelled with prolactin receptor (PRLR) antibody. The positive cells were isolated by magnetic-activated cell sorting (MACS) protocol. Purified LCs were tested in vitro for their steroidogenic (T production) and non-steroidogenic (25-OH-vitamin D production and Insl3 and Cyp2r1expressions) functions in the presence of Luteinizing Hormone (LH) for up to 24 h. RESULTS: Reanalysis of scRNA-seq data indicates that Prlr expression is highly specific in LCs of adult rat testis. MACS procedure based on PRLR expression was able to isolate LCs with very high yield (about 106 cells/testis), high purity (about 95%) and viability (> 93%). Purified LCs retained high steroidogenic and non-steroidogenic functions in responding to maximal LH stimulations, with more than 10-fold increases in T production in 3 h and 42% and 103% increases in Insl3 and Cyp2r1 expressions in 24 h. DISCUSSION AND CONCLUSION: We have established an excellent way to purify high quality LCs from adult rat testis that can serve as a useful tool to study the physiology, pharmacology and toxicology of the cells in vitro.

Single-cell RNA sequencing of adult rat testes after Leydig cell elimination and restoration.

Spermatogenesis is an efficient, complex, and highly organized proliferation and differentiation process that relies on multiple factors including testosterone produced by the Leydig cells. Although the critical role played by testosterone in spermatogenesis is well recognized, the mechanism by which it works is still not completely understood, partially due to the inability to specifically and precisely monitor testosterone-dependent changes within developing germ cells. Here we present single-cell RNA sequencing data from10,983 adult rat testicular cells after the rats were treated with ethanedimethanesulfonate, which temporarily eliminates Leydig cells. The elimination and recovery of Leydig cells represented a complete testosterone depletion and restoration cycle. The dataset, which includes all developing germ cells from spermatogonia to spermatozoa, should prove useful for characterizing developing germ cells, their regulatory networks, and novel cell-specific markers. The dataset should be particularly useful for exploring the effects of the androgen environment on the regulation of spermatogenesis. As this is the first single-cell RNA-Seq dataset for rat testes, it can also serve as a reference for future studies.

Identification of Rat Testicular Leydig Precursor Cells by Single-Cell-RNA-Sequence Analysis.

Stem Leydig cells (SLCs) play a critical role in the development and maintenance of the adult Leydig cell (ALC) population. SLCs also are present in the adult testis. Their identification, characteristics, and regulation in the adult testis remain uncertain. Using single-cell RNA-seq, we found that the mesenchymal stromal population may be involved in ALC regeneration. Upon ALC elimination, a fraction of stromal cells begins to proliferate while a different fraction begins to differentiate to ALCs. Transcriptomic analysis identified five stromal clusters that can be classified into two major groups representing proliferation and differentiation populations. The proliferating group represents stem cells expressing high levels of CD90, Nes, Lum, Fn and Gap43. The differentiating group represents a progenitor stage that is ready to form ALCs, and specifically expresses Vtn, Rasl11a, Id1 and Egr2. The observation that the actively dividing cells after ALC loss were not those that formed ALCs suggests that stem cell proliferation and differentiation are regulated separately, and that the maintenance of the stromal stem cell pool occurs at the population level. The study also identified specific markers for the major interstitial cell groups and potential paracrine factors involved in the regulation of SLCs. Our data suggest a new theory about SLC identity, proliferation, differentiation, and regulation.

Effects of Midazolam on the Development of Adult Leydig Cells From Stem Cells In Vitro.

Background: Midazolam is a neurological drug with diverse functions, including sedation, hypnosis, decreased anxiety, anterograde amnesia, brain-mediated muscle relaxation, and anticonvulsant activity. Since it is frequently used in children and adolescents for extended periods of time, there is a risk that it may affect their pubertal development. Here, we report a potential effect of the drug on the development of Leydig cells (LCs), the testosterone (T)-producing cells in the testis. Methods: Stem LCs (SLCs), isolated from adult rat testes by a magnetic-activated cell sorting technique, were induced to differentiate into LCs in vitro for 3 weeks. Midazolam (0.1-30 µM) was added to the culture medium, and the effects on LC development were assayed. Results: Midazolam has dose-dependent effects on SLC differentiation. At low concentrations (0.1-5 µM), the drug can mildly increase SLC differentiation (increased T production), while at higher concentrations (15-30 µM), it inhibits LC development (decreased T production). T increases at lower levels may be due to upregulations of scavenger receptor class b Member 1 (SCARB1) and cytochrome P450 17A1 (CYP17A1), while T reductions at higher levels of midazolam could be due to changes in multiple steroidogenic proteins. The uneven changes in steroidogenic pathway proteins, especially reductions in CYP17A1 at high midazolam levels, also result in an accumulation of progesterone. In addition to changes in T, increases in progesterone could have additional impacts on male reproduction. The loss in steroidogenic proteins at high midazolam levels may be mediated in part by the inactivation of protein kinase B/cAMP response element-binding protein (AKT/CREB) signaling pathway. Conclusion: Midazolam has the potential to affect adult Leydig cell (ALC) development at concentrations comparable with the blood serum levels in human patients. Further studies are needed to test the effects on human cells.