Probing the Chemical Space of Guanidino-Carboxylic Acids to Identify the First Blockers of the Creatine-Transporter-1.

The creatine transporter-1 (CRT-1/SLC6A8) maintains the uphill transport of creatine into cells against a steep concentration gradient. Cellular creatine accumulation is required to support the ATP-buffering by phosphocreatine. More than 60 compounds have been explored in the past for their ability to inhibit cellular creatine uptake, but the number of active compounds is very limited. Here, we show that all currently known inhibitors are full alternative substrates. We analyzed their structure-activity relation for inhibition of CRT-1 to guide a rational approach to the synthesis of novel creatine transporter ligands. Measurements of both, inhibition of [3H]creatine uptake and transport associated currents, allowed for differentiating between full and partial substrates and true inhibitors. This combined approach led to a refined understanding of the structural requirements for binding to CRT-1, which translated into the identification of three novel compounds - i.e. compound 1 (2-(N-benzylcarbamimidamido)acetic acid), and MIPA572 (=carbamimidoylphenylalanine) and MIPA573 (=carbamimidoyltryptophane) that blocked CRT-1 transport, albeit with low affinity. In addition, we found two new alternative full substrates, namely MIP574 (carbamimidoylalanine) and GiDi1257 (1-carbamimidoylazetidine-3-carboxylic acid), which was superior in affinity to all known CTR-1 ligands, and one partial substrate, namely GiDi1254 (1-carbamimidoylpiperidine-4-carboxylic acid). Significance Statement The creatine transporter-1 (CRT-1) is required to maintain intracellular creatine levels. Inhibition of CRT-1 has been recently proposed as a therapeutic strategy for cancer, but pharmacological tools are scarce. In fact, all available inhibitors are alternative substrates. We tested existing and newly synthesized guanidinocarboxylic acids for CRT-1 inhibition and identified three blockers, one partial and two full substrates of CRT-1. Our results support a refined structural understanding of ligand binding to CRT-1 and provide a proof-of-principle for blockage of CRT-1.

Targeting NTCP for liver disease treatment: A promising strategy.

The sodium taurocholate co-transporting polypeptide (NTCP), a bile acids transporter, has been identified as a new therapeutic target for the treatment of liver disease. This paper thoroughly investigates the function of NTCP for regulating bile acid regulation, its correlation with hepatitis B and D infections, and its association with various liver diseases. Additionally, in this review we examine recent breakthroughs in creating NTCP inhibitors and their prospective applications in liver disease treatment. While this review emphasizes the promising potential of targeting NTCP, it concurrently underscores the need for broader and more detailed research to fully understand the long-term implications and potential side effects associated with NTCP inhibition.

Novel mechanistic insights - A brand new Era for anti-HBV drugs.

Hepatitis B Virus (HBV) remains a critical global health issue, with substantial morbidity and mortality. Current therapies, including interferons and nucleoside analogs, often fail to achieve complete cure or functional eradication. This review explores recent advances in anti-HBV agents, focusing on their innovative mechanisms of action. HBV entry inhibitors target the sodium taurocholate cotransporting polypeptide (NTCP) receptor, impeding viral entry, while nucleus translocation inhibitors disrupt key viral life cycle steps, preventing replication. Capsid assembly modulators inhibit covalently closed circular DNA (cccDNA) formation, aiming to eradicate the persistent viral reservoir. Transcription inhibitors targeting cccDNA and integrated DNA offer significant potential to suppress HBV replication. Immunomodulatory agents are highlighted for their ability to enhance host immune responses, facil-itating better control and possible eradication of HBV. These novel approaches represent significant advancements in HBV therapy, providing new strategies to overcome current treatment limitations. The development of cccDNA reducers is particularly critical, as they directly target the persistent viral reservoir, offering a promising pathway towards achieving a functional cure or complete viral eradication. Continued research in this area is essential to advance the effectiveness of anti-HBV therapies.

The feasibility of establishing a hamster model for HBV infection: in vitro evidence.

Chronic hepatitis B virus (HBV) infection remains a significant public health burden with no cure currently available. The research to cure HBV has long been hampered by the lack of immunocompetent small animal models capable of supporting HBV infection. Here, we set out to explore the feasibility of the golden Syrian hamster as an immunocompetent small rodent model for HBV infection. We first started with in vitro assessments of the HBV replication cycle in primary hamster hepatocytes (PHaHs) by adenoviral HBV (Ad-HBV) transduction. Our results demonstrated that PHaHs support HBV reverse transcription and subsequent cccDNA formation via the intracellular recycling pathway. Next, with luciferase reporter assays, we confirmed that PHaHs support the activities of all HBV major promoters. Then, we transduced PHaHs with an adenoviral vector expressing HBV receptor human Na+/taurocholate cotransporting polypeptide NTCP (Ad-huNTCP), followed by HBV inoculation. While the untransduced PHaHs did not support HBV infection, Ad-huNTCP-transduced PHaHs supported de novo cccDNA formation, viral mRNA transcription, and expression of viral antigens. We then humanized the amino acid (aa) residues of hamster NTCP (haNTCP) critical for HBV entry, aa84-87 and aa157-165, and transfected HepG2 cells with constructs expressing wild-type haNTCP and humanized-haNTCP, H84R/P87N and H84R/P87N/G157K/M160V/M165L, respectively, followed by HBV inoculation. The results showed that the humanization of H84R/P87N alone was sufficient to support HBV infection at a level comparable to that supported by huNTCP. Taken together, the above in vitro evidence supports the future direction of humanizing haNTCP for HBV infection in vivo.IMPORTANCEOne of the biggest challenges in developing an HBV cure is the lack of immunocompetent animal models susceptible to HBV infection. Developing such models in mice has been unsuccessful due to the absence of a functional HBV receptor, human NTCP (huNTCP), and the defect in supporting viral cccDNA formation. In search of alternative models, we report herein multiple lines of in vitro evidence for developing a golden Syrian hamster model for HBV infection. We demonstrate that the primary hamster hepatocytes (PHaHs) support HBV replication, transcription, and cccDNA formation, and PHaHs are susceptible to de novo HBV infection in the presence of huNTCP. Furthermore, expressing hamster NTCP with two humanized residues critical for HBV entry renders HepG2 cells permissive to HBV infection. Thus, our work lays a solid foundation for establishing a gene-edited hamster model that expresses humanized NTCP for HBV infection in vivo.

The loss of hepatitis B virus receptor NTCP/SLC10A1 in human liver cancer cells is due to epigenetic silencing.

Human Na+-taurocholate cotransporting polypeptide (hNTCP) is predominantly expressed in hepatocytes, maintaining bile salt homeostasis and serving as a receptor for hepatitis B virus (HBV). hNTCP expression is downregulated during hepatocellular carcinoma (HCC) development. In this study, we investigated the molecular mechanisms underlying hNTCP dysregulation using HCC tissues and cell lines, and primary human hepatocytes (PHHs). Firstly, we observed a significant reduction of hNTCP in HCC tumors compared to adjacent and normal tissues. Additionally, hNTCP mRNA levels were markedly lower in HepG2 cells compared to PHHs, which was corroborated at the protein level by immunoblotting. Sanger sequencing confirmed identical sequences for hNTCP promoter, exons, and mRNA coding sequences between PHH and HepG2 cells, indicating no mutations or splicing alterations. We then assessed the epigenetic status of hNTCP. The hNTCP promoter, with low CG content, showed no significant methylation differences between PHH and HepG2 cells. Chromatin immunoprecipitation coupled with qPCR (ChIP-qPCR) revealed a loss of activating histone posttranslational modification (PTM) H3K27ac near the hNTCP transcription start site (TSS) in HepG2 cells. This loss was also confirmed in HCC tumor cells compared to adjacent and background cells. Treating HepG2 cells with histone deacetylase inhibitors enhanced H3K27ac accumulation and glucocorticoid receptor (GR) binding at the hNTCP TSS, significantly increasing hNTCP mRNA and protein levels, and rendering the cells susceptible to HBV infection. In summary, histone PTM-related epigenetic mechanisms play a critical role in hNTCP dysregulation in liver cancer cells, providing insights into hepatocarcinogenesis and its impact on chronic HBV infection. IMPORTANCE: HBV is a hepatotropic virus that infects human hepatocytes expressing the viral receptor hNTCP. Without effective antiviral therapy, chronic HBV infection poses a high risk of liver cancer. However, most liver cancer cell lines, including HepG2 and Huh7, do not support HBV infection due to the absence of hNTCP expression, and the mechanism underlying this defect remains unclear. This study demonstrates a significant reduction of hNTCP in hepatocellular carcinoma samples and HepG2 cells compared to normal liver tissues and primary human hepatocytes. Despite identical hNTCP genetic sequences, epigenetic analyses revealed a loss of the activating histone modification H3K27ac near the hNTCP transcription start site in cancer cells. Treatment with histone deacetylase inhibitors restored H3K27ac levels, reactivated hNTCP expression, and rendered HepG2 cells susceptible to HBV infection. These findings highlight the role of epigenetic modulation in hNTCP dysregulation, offering insights into hepatocarcinogenesis and its implications for chronic HBV infection.

Identification of pyrimidine structure-based compounds as allosteric ligands of the dopamine transporter as therapeutic agents for NeuroHIV.

The disruption of dopamine neurotransmission by the HIV-1 Transactivator of transcription (Tat) during HIV-1 infection has been linked to the development of neurocognitive disorders, even under combined antiretroviral therapy (cART) treatment. We have demonstrated that SRI-32742, a novel allosteric modulator of dopamine (DA) transporter (DAT), attenuates cocaine- and Tat-binding to DAT, alleviates Tat-induced cognitive deficits and potentiation of cocaine reward in inducible Tat transgenic mice. The current study determined the in vitro pharmacological profile of SRI-32743 and its optimized second-generation analogs and their effects as allosteric modulators. Through structure-activity relationship studies of SRI-32743, 170 compounds were synthesized and evaluated for their ability to modulate DAT function. We identified 21 analogs as atypical competitors of DAT (Emax {less than or equal to}60%). Four compounds, SRI-46564, SRI-47056, SRI-46286 and SRI-47867, displayed IC50 values for [3H]DA uptake inhibition from 9.33 {plus minus} 0.50 to 0.96 {plus minus} 0.05 µM and from 3.96 {plus minus} 1.36 to 1.29 {plus minus} 0.19 for DAT binding, respectively. The four analogs also displayed high potency at two different concentrations (0.5 nM and 0.05 nM) to attenuate Tat-induced inhibition of [3H]DA uptake and cocaine-mediated dissociation of [3H]WIN35,428 binding in CHO cells expressing hDAT, suggesting that the effects occur through an allosteric mechanism. In further ex vivo studies using Fast-Scan Cyclic Voltammetry, we demonstrated that the analogs do not disrupt the baseline phasic-like DA release. These findings provide a new insight into the potential for development of novel therapeutic agents to attenuate DAT-Tat interactions to normalize DA neurotransmission in NeuroHIV. Significance Statement The allosteric inhibition of the dopamine (DA) transporter by the HIV-1 Transactivator of transcription (Tat) disrupts dopamine homeostasis, leading to HIV-associated neurocognitive disorders (HANDs). Analogs of SRI-32743, a novel allosteric modulator of the Tat-DAT interaction, were evaluated in the current study and characterized as atypical ligands of DA uptake. Four novel lead compounds demonstrated high potency to attenuate Tat-induced inhibition of hDAT-mediated DA uptake in an allosteric modulatory manner with no effects on the dynamics of DA uptake-release in DAT.

Exploring the tolerable region for HiBiT tag insertion in the hepatitis B virus genome.

A cell culture system that allows the reproduction of the hepatitis B virus (HBV) life cycle is indispensable to exploring novel anti-HBV agents. To establish the screening system for anti-HBV agents, we exploited the high affinity and bright luminescence (HiBiT) tag and comprehensively explored the regions in the HBV genome where the HiBiT tag could be inserted. The plasmids for the HiBiT-tagged HBV molecular clones with a 1.38-fold HBV genome length were prepared. The HiBiT tag was inserted into five regions: preS1, preS2, hepatitis B e (HBe), hepatitis B X (HBx), and hepatitis B polymerase (HB pol). HiBiT-tagged HBVs were obtained by transfecting the prepared plasmids into sodium taurocholate cotransporting polypeptide-transduced HepG2 (HepG2/NTCP) cells, and their infectivity was evaluated in human primary hepatocytes and HepG2/NTCP cells. Among the evaluated viruses, the infection of HiBiT-tagged HBVs in the preS1 or the HB pol regions exhibited a time-dependent increase of the hepatitis B surface antigen (HBsAg) level after infection to HepG2/NTCP cells as well as human primary hepatocytes. Immunostaining of the hepatitis B core (HBc) antigen in infected cells confirmed these viruses are infectious to those cells. However, the time-dependent increase of the HiBiT signal was only detected after infection with the HiBiT-tagged HBV in the preS1 region. The inhibition of this HiBiT-tagged HBV infection in human primary hepatocytes and HepG2/NTCP cells by the preS1 peptide could be detected by measuring the HiBiT signal. The infection system with the HiBiT-tagged HBV in HepG2/NTCP cells facilitates easy, sensitive, and high-throughput screening of anti-HBV agents and will be a useful tool for assessing the viral life cycle and exploring antiviral agents. IMPORTANCE: Hepatitis B virus (HBV) is the principal causative agent of chronic hepatitis. Despite the availability of vaccines in many countries, HBV infection has spread worldwide and caused chronic infection. In chronic hepatitis B patients, liver inflammation leads to cirrhosis, and the accumulation of viral genome integration into host chromosomes leads to the development of hepatocellular carcinoma. The currently available treatment strategy cannot expect the eradication of HBV. To explore novel anti-HBV agents, a cell culture system that can detect HBV infection easily is indispensable. In this study, we examined the regions in the HBV genome where the high affinity and bright luminescence (HiBiT) tag could be inserted and established an HBV infection system to monitor infection by measuring the HiBiT signal by infecting the HiBiT-tagged HBV in sodium taurocholate cotransporting polypeptide-transduced HepG2 (HepG2/NTCP) cells. This system can contribute to screening for novel anti-HBV agents.

Incidence of alopecia in brain tumour patients treated with pencil scanning proton therapy and validation of existing NTCP models.

BACKGROUND AND PURPOSE: Radiation-induced alopecia (RIA) is one of the most frequent and upsetting cosmetic side effects after radiotherapy (RT) for brain cancer. We report the incidence of RIA in a cohort of brain tumours patients treated with Proton Therapy (PT) and externally validate published NTCP models of grade 2 (G2) RIA for their implementation in clinical practice. METHODS: Data for patients treated for brain tumours with scanning beam PT between 2018 and 2022 were extracted. Acute, late and permanent RIA events were evaluated according to CTCAE 5.0. Lyman-Kutcher-Burman (LKB) and multivariable logistic regression (MLR) published models were computed from the relative dose-surface histogram of the scalp. External validity of models was assessed in terms of discrimination and calibration. RESULTS: In the 264 patients analysed, rates of any grade acute (≤90 days after PT completion), late (>90 days) and permanent RIA (persisting for> 12 months) were 61.8 %, 24.7 % and 14.4 %, respectively. In our independent cohort, LKB- and MLR-NTCP showed a good discrimination for G2 RIA (0.71≤ROC-AUC≤0.83) while model calibration was unsatisfactory possibly due to a different outcome evaluation between training and validation cohorts, as well as differences in clinical and treatment related variables between the two groups. CONCLUSIONS: Despite the reasonable sensitivity and specificity of the NTCP models for RIA in the validation cohort, our study emphasizes the significance of differences between the cohorts utilized for model development and validation. Specifically, variations in the reporting of clinical outcomes inevitably jeopardize the validation of NTCP models. A standardize and objective RIA scoring system is essential.

RBE-based dose planning, and calculation of TCP and NTCP with gold nanoparticles for intermediate photon energy in pancreatic cancer.

Objective.This study simulated the potential of gold nanoparticles (GNPs) to improve the effectiveness of radiation therapy in pancreatic cancer cases. The purpose of this study was to assess the impact of GNPs on tumor control probability (TCP) and normal tissue complication probability (NTCP) in pancreatic cancer cases undergoing radiation therapy. The work aimed to compare treatment plans generated with a novel 2.5 MV beam using GNPs to conventional 6 MV plans and evaluate the dose-volume histogram (DVH), TCP, and NTCP.Approach.Treatment planning for five pancreatic computed tomography (CT) images was performed using the open-source MATLAB-based treatment planning program matRad. MATLAB codes were developed to calculate the relative biological effectiveness (RBE) of GNPs and apply the corresponding dose and RBE values to each voxel. TCP and NTCP were calculated based on the applied RBE values.Main results.Adding GNPs to the 2.5 MV treatment plan resulted in a significant increase in TCP, from around 59% to 93.5%, indicating that the inclusion of GNPs improved the effectiveness of the radiation treatment. The range in NTCP without GNPs was relatively larger compared to that with GNPs.Significance.The results indicated that the addition of GNPs to a 2.5 MV plan can increase TCP while maintaining a relatively low NTCP value (<1%). The use of GNPs may also reduce NTCP values by decreasing the dose to normal tissues while maintaining the same prescribed dose to the tumor. Hence, the addition of GNPs can improve the balance between TCP and NTCP.

Rapid and Reliable Protein-Free HBV DNA Extraction and Sensitive Branched DNA Southern Blot Assay.

HBV covalently closed circular DNA (cccDNA) plays an important role in the persistence of hepatitis B virus (HBV) infection by serving as the template for transcription of viral RNAs. To cure HBV infection, it is expected that cccDNA needs either to be eliminated or silenced. Hence, precise cccDNA quantification is essential. Sample preparation is crucial to specifically detect cccDNA. Southern blot is regarded as the "gold standard" for specific cccDNA detection but lacks sensitivity. Here, we describe a rapid and reliable modified kit-based, HBV protein-free DNA extraction method as well as a novel enhanced sensitivity Southern blot that uses branched DNA technology to detect HBV DNA in cell culture and liver tissue samples. It is useful for both HBV molecular biology and antiviral research.

Dosimetric comparison of IMPT vs VMAT for multiple lung lesions: an NTCP model-based decision-making strategy.

To compare the dosimetric differences in volumetric modulated arc therapy (VMAT) and intensity modulated proton therapy (IMPT) in stereotactic body radiation therapy (SBRT) of multiple lung lesions and determine a normal tissue complication probability (NTCP) model-based decision strategy that determines which treatment modality the patient will use. A total of 41 patients were retrospectively selected for this study. The number of patients with 1-6 lesions was 5, 16, 7, 6, 3, and 4, respectively. A prescription dose of 70 GyRBE in 10 fractions was given to each lesion. SBRT plans were generated using VMAT and IMPT. All the IMPT plans used robustness optimization with ± 3.5% range uncertainties and 5 mm setup uncertainties. Dosimetric metrics and the predicted NTCP value of radiation pneumonitis (RP), esophagitis, and pericarditis were analyzed to evaluate the potential clinical benefits between different planning groups. In addition, a threshold for the ratio of PTV to lungs (%) to determine whether a patient would benefit highly from IMPT was determined using receiver operating characteristic curves. All plans reached target coverage (V70GyRBE ≥ 95%). Compared with VMAT, IMPT resulted in a significantly lower dose of most thoracic normal tissues. For the 1-2, 3-4 and 5-6 lesion groups, the lung V5 was 29.90 ± 9.44%, 58.33 ± 13.35%, and 81.02 ± 5.91% for VMAT and 11.34 ± 3.11% (p < 0.001), 21.45 ± 3.80% (p < 0.001), and 32.48 ± 4.90% (p < 0.001) for IMPT, respectively. The lung V20 was 12.07 ± 4.94%, 25.57 ± 6.54%, and 43.99 ± 11.83% for VMAT and 6.76 ± 1.80% (p < 0.001), 13.14 ± 2.27% (p < 0.01), and 19.62 ± 3.48% (p < 0.01) for IMPT. The Dmean of the total lung was 7.65 ± 2.47 GyRBE, 14.78 ± 2.75 GyRBE, and 21.64 ± 4.07 GyRBE for VMAT and 3.69 ± 1.04 GyRBE (p < 0.001), 7.13 ± 1.41 GyRBE (p < 0.001), and 10.69 ± 1.81 GyRBE (p < 0.001) for IMPT. Additionally, in the VMAT group, the maximum NTCP value of radiation pneumonitis was 73.91%, whereas it was significantly lower in the IMPT group at 10.73%. The accuracy of our NTCP model-based decision model, which combines the number of lesions and PTV/Lungs (%), was 97.6%. The study demonstrated that the IMPT SBRT for multiple lung lesions had satisfactory dosimetry results, even when the number of lesions reached 6. The NTCP model-based decision strategy presented in our study could serve as an effective tool in clinical practice, aiding in the selection of the optimal treatment modality between VMAT and IMPT.

Transporter Proteins as Therapeutic Drug Targets-With a Focus on SGLT2 Inhibitors.

Membrane transporters interact not only with endogenous substrates but are also engaged in the transport of xenobiotics, including drugs. While the coordinated function of uptake (solute carrier family-SLC and SLCO) and efflux (ATP-binding cassette family-ABC, multidrug and toxic compound extrusion family-MATE) transporter system allows vectorial drug transport, efflux carriers alone achieve barrier functions. The modulation of transport functions was proved to be effective in the treatment strategies of various pathological states. Sodium-glucose cotransporter-2 (SGLT2) inhibitors are the drugs most widely applied in clinical practice, especially in the treatment of diabetes mellitus and heart failure. Sodium taurocholate co-transporting polypeptide (NTCP) serves as virus particles (HBV/HDV) carrier, and inhibition of its function is applied in the treatment of hepatitis B and hepatitis D by myrcludex B. Inherited cholestatic diseases, such as Alagille syndrome (ALGS) and progressive familial intrahepatic cholestasis (PFIC) can be treated by odevixibat and maralixibat, which inhibit activity of apical sodium-dependent bile salt transporter (ASBT). Probenecid can be considered to increase uric acid excretion in the urine mainly via the inhibition of urate transporter 1 (URAT1), and due to pharmacokinetic interactions involving organic anion transporters 1 and 3 (OAT1 and OAT3), it modifies renal excretion of penicillins or ciprofloxacin as well as nephrotoxicity of cidofovir. This review discusses clinically approved drugs that affect membrane/drug transporter function.

Improvement of accumulated dose distribution in combined cervical cancer radiotherapy with deep learning-based dose prediction.

Purpose: Difficulties remain in dose optimization and evaluation of cervical cancer radiotherapy that combines external beam radiotherapy (EBRT) and brachytherapy (BT). This study estimates and improves the accumulated dose distribution of EBRT and BT with deep learning-based dose prediction. Materials and methods: A total of 30 patients treated with combined cervical cancer radiotherapy were enrolled in this study. The dose distributions of EBRT and BT plans were accumulated using commercial deformable image registration. A ResNet-101-based deep learning model was trained to predict pixel-wise dose distributions. To test the role of the predicted accumulated dose in clinic, each EBRT plan was designed using conventional method and then redesigned referencing the predicted accumulated dose distribution. Bladder and rectum dosimetric parameters and normal tissue complication probability (NTCP) values were calculated and compared between the conventional and redesigned accumulated doses. Results: The redesigned accumulated doses showed a decrease in mean values of V50, V60, and D2cc for the bladder (-3.02%, -1.71%, and -1.19 Gy, respectively) and rectum (-4.82%, -1.97%, and -4.13 Gy, respectively). The mean NTCP values for the bladder and rectum were also decreased by 0.02‰ and 0.98%, respectively. All values had statistically significant differences (p < 0.01), except for the bladder D2cc (p = 0.112). Conclusion: This study realized accumulated dose prediction for combined cervical cancer radiotherapy without knowing the BT dose. The predicted dose served as a reference for EBRT treatment planning, leading to a superior accumulated dose distribution and lower NTCP values.

A deep-learning-based surrogate model for Monte-Carlo simulations of the linear energy transfer in primary brain tumor patients treated with proton-beam radiotherapy.

Objective.This study explores the use of neural networks (NNs) as surrogate models for Monte-Carlo (MC) simulations in predicting the dose-averaged linear energy transfer (LETd) of protons in proton-beam therapy based on the planned dose distribution and patient anatomy in the form of computed tomography (CT) images. As LETdis associated with variability in the relative biological effectiveness (RBE) of protons, we also evaluate the implications of using NN predictions for normal tissue complication probability (NTCP) models within a variable-RBE context.Approach.The predictive performance of three-dimensional NN architectures was evaluated using five-fold cross-validation on a cohort of brain tumor patients (n= 151). The best-performing model was identified and externally validated on patients from a different center (n= 107). LETdpredictions were compared to MC-simulated results in clinically relevant regions of interest. We assessed the impact on NTCP models by leveraging LETdpredictions to derive RBE-weighted doses, using the Wedenberg RBE model.Main results.We found NNs based solely on the planned dose distribution, i.e. without additional usage of CT images, can approximate MC-based LETddistributions. Root mean squared errors (RMSE) for the median LETdwithin the brain, brainstem, CTV, chiasm, lacrimal glands (ipsilateral/contralateral) and optic nerves (ipsilateral/contralateral) were 0.36, 0.87, 0.31, 0.73, 0.68, 1.04, 0.69 and 1.24 keV µm-1, respectively. Although model predictions showed statistically significant differences from MC outputs, these did not result in substantial changes in NTCP predictions, with RMSEs of at most 3.2 percentage points.Significance.The ability of NNs to predict LETdbased solely on planned dose distributions suggests a viable alternative to compute-intensive MC simulations in a variable-RBE setting. This is particularly useful in scenarios where MC simulation data are unavailable, facilitating resource-constrained proton therapy treatment planning, retrospective patient data analysis and further investigations on the variability of proton RBE.

Comparison of deep inspiration breath hold and free breathing intensity modulated proton therapy of locally advanced lung cancer.

Background and purpose: For locally advanced non-small cell lung cancer (LA-NSCLC), intensity-modulated proton therapy (IMPT) can reduce organ at risk (OAR) doses compared to intensity-modulated radiotherapy (IMRT). Deep inspiration breath hold (DIBH) reduces OAR doses compared to free breathing (FB) in IMRT. In IMPT, differences in dose distributions and robustness between DIBH and FB are unclear. In this study, we compare DIBH to FB in IMPT, and IMPT to IMRT. Materials and methods: Fortyone LA-NSCLC patients were prospectively included. 4D computed tomography images (4DCTs) and DIBH CTs were acquired for treatment planning and during weeks 1 and 3 of treatment. A new system for automated robust planning was developed and used to generate a FB and a DIBH IMPT plan for each patient. Plans were compared in terms of dose-volume parameters and normal tissue complication probabilities (NTCPs). Dose recalculations on repeat CTs were used to compare inter-fraction plan robustness. Results: In IMPT, DIBH reduced median lungs Dmean from 9.3 Gy(RBE) to 8.0 Gy(RBE) compared to FB, and radiation pneumonitis NTCP from 10.9 % to 9.4 % (p < 0.001). Inter-fraction plan robustness for DIBH and FB was similar. Median NTCPs for radiation pneumonitis and mortality were around 9 percentage points lower with IMPT than IMRT (p < 0.001). These differences were much larger than between FB and DIBH within each modality. Conclusion: DIBH IMPT resulted in reduced lung dose and radiation pneumonitis NTCP compared to FB IMPT. Inter-fraction robustness was comparable. OAR doses were far lower in IMPT than IMRT.

Comparative dosimetric study of h-IMRT and VMAT plans for breast cancer after breast-conserving surgery.

AIM: To compare the dosimetric advantages and disadvantages between hybrid intensity-modulated radiation therapy (h-IMRT) and the volumetric modulated arc therapy (VMAT) technique in hypofractionated whole-breast irradiation (HF-WBI) for early-stage breast cancer (BC). METHODS: The dose distribution of h-IMRT and VMAT plans was compared in 20 breast cancer patients. This comparison included evaluation of dosimetric parameters using dose volume histograms (DVHs) for the planning target volume (PTV) and organs-at-risk (OARs). Additionally, the study examined the normal tissue complication probability (NTCP), the second cancer complication probability (SCCP) and the tumor control probability (TCP) based on different models. RESULTS: Significant differences were detected between the two plans, in terms of Machine units (MUs), the control points, 95 % volume (V95 %), dose homogeneity index (DHI) and conformity index (CI). The endpoint of grade II radiation pneumonitis and cardiac death due to ischemic heart disease were assessed. In h-IMRT plan, the NTCP values were marginally lower for radiation pneumonitis and slightly higher for cardiac death compared to VMAT plan, as determined by the Lyman-Kutcher-Burman model. The Schneider model was employed to predict the SCCP for both the bilateral lungs and contralateral breast, the results demonstrate that the h-IMRT plan outperforms the VMAT plan, with statistical significance. Additionally, the LQ-Poisson model was employed to forecast the TCP of the PTV, showing that the h-IMRT plan outperformed the VMAT plan (P > 0.05). CONCLUSION: The h-IMRT technique, offering superior dose coverage and better therapeutic efficacy with fewer side effects as calculated by models, is more suitable for HF-WBI compared to the VMAT technique.

Helium Ion Therapy for Advanced Juvenile Nasopharyngeal Angiofibroma.

Helium ion therapy (HRT) is a promising modality for the treatment of pediatric tumors and those located close to critical structures due to the favorable biophysical properties of helium ions. This in silico study aimed to explore the potential benefits of HRT in advanced juvenile nasopharyngeal angiofibroma (JNA) compared to proton therapy (PRT). We assessed 11 consecutive patients previously treated with PRT for JNA in a definitive or postoperative setting with a relative biological effectiveness (RBE) weighted dose of 45 Gy (RBE) in 25 fractions at the Heidelberg Ion-Beam Therapy Center. HRT plans were designed retrospectively for dosimetric comparisons and risk assessments of radiation-induced complications. HRT led to enhanced target coverage in all patients, along with sparing of critical organs at risk, including a reduction in the brain integral dose by approximately 27%. In terms of estimated risks of radiation-induced complications, HRT led to a reduction in ocular toxicity, cataract development, xerostomia, tinnitus, alopecia and delayed recall. Similarly, HRT led to reduced estimated risks of radiation-induced secondary neoplasms, with a mean excess absolute risk reduction of approximately 30% for secondary CNS malignancies. HRT is a promising modality for advanced JNA, with the potential for enhanced sparing of healthy tissue and thus reduced radiation-induced acute and long-term complications.

Dosimetric and NTCP advantages of robust proton therapy over robust VMAT for Stage III NSCLC in the immunotherapy era.

AIMS: To assess the robustness and to define the dosimetric and NTCP advantages of pencil-beam-scanning proton therapy (PBSPT) compared with VMAT for unresectable Stage III non-small lung cancer (NSCLC) in the immunotherapy era. MATERIAL AND METHODS: 10 patients were re-planned with VMAT and PBSPT using: 1) ITV-based robust optimization with 0.5 cm setup uncertainties and (for PBSPT) 3.5 % range uncertainties on free-breathing CT 2) CTV-based RO including all 4DCTs anatomies. Target coverage (TC), organs at risk dose and TC robustness (TCR), set at V95%, were compared. The NTCP risk for radiation pneumonitis (RP), 24-month mortality (24MM), G2 + acute esophageal toxicity (ET), the dose to the immune system (EDIC) and the left anterior descending (LAD) coronary artery V15 < 10 % were registered. Wilcoxon test was used. RESULTS: Both PBSPT methods improved TC and TCR (p < 0.01). The mean lung dose and lung V20 were lower with PBSPT (p < 0.01). Median mean heart dose reduction with PBSPT was 8 Gy (p < 0.001). PT lowered median LAD V15 (p = 0.004). ΔNTCP > 5 % with PBSPT was observed for two patients for RP and for five patients for 24 MM. ΔNTCP for ≥ G2 ET was not in favor of PBSPT for all patients. PBSPT halved median EDIC (4.9/5.1 Gy for ITV/CTV-based VMAT vs 2.3 Gy for both ITV/CTV-based PBSPT, p < 0.01). CONCLUSIONS: PBSPT is a robust approach with significant dosimetric and NTCP advantages over VMAT; the EDIC reduction could allow for a better integration with immunotherapy. A clinical benefit for a subset of NSCLC patients is expected.

The potential of integrating stereotactic ablative radiotherapy techniques with hyperfractionation for lung cancer.

BACKGROUND: Limited literature exists on the feasibility and effectiveness of integrating stereotactic ablative radiotherapy (SABR) techniques with hyperfractionated regimens for patients with lung cancer. This study aims to assess whether the SABR technique with hyperfractionation can potentially reduce lung toxicity. METHODS: We utilized the linear-quadratic model to find the optimal fraction to maximize the tumor biological equivalent dose (BED) to normal-tissue BED ratio. Validation was performed by comparing the SABR plans with 50 Gy/5 fractions and hyperfractionationed plans with 88.8 Gy/74 fractions with the same tumor BED and planning criteria for 10 patients with early-stage lung cancer. Mean lung BED, Lyman-Kutcher-Burman (LKB) normal tissue complication probability (NTCP), critical volume (CV) criteria (volume below BED of 22.92 and 25.65 Gy, and mean BED for lowest 1000 and 1500 cc) and the percentage of the lung receiving 20Gy or more (V20) were compared using the Wilcoxon signed-rank test. RESULTS: The transition point occurs when the tumor-to-normal tissue ratio (TNR) of the physical dose equals the TNR of α/ß in the BED dose-volume histogram of the lung. Compared with the hypofractionated regimen, the hyperfractionated regimen is superior in the dose range above but inferior below the transition point. The hyperfractionated regimen showed a lower mean lung BED (6.40 Gy vs. 7.73 Gy) and NTCP (3.50% vs. 4.21%), with inferior results concerning CV criteria and higher V20 (7.37% vs. 7.03%) in comparison with the hypofractionated regimen (p < 0.01 for all). CONCLUSIONS: The hyperfractionated regimen has an advantage in the high-dose region of the lung but a disadvantage in the low-dose region. Further research is needed to determine the superiority between hypo- and hyperfractionation.

Bulevirtide monotherapy in patients with chronic HDV: Efficacy and safety results through week 96 from a phase III randomized trial.

BACKGROUND & AIMS: Bulevirtide (BLV), a first-in-class entry inhibitor, is approved in Europe for the treatment of chronic hepatitis delta (CHD). BLV monotherapy was superior to delayed treatment at week (W) 48, the primary efficacy endpoint, in the MYR301 study (NCT03852719). Here, we assessed if continued BLV therapy until W96 would improve virologic and biochemical response rates, particularly among patients who did not achieve virologic response at W24. METHODS: In this ongoing, open-label, randomized phase III study, patients with CHD (N = 150) were randomized (1:1:1) to treatment with BLV 2 mg/day (n = 49) or 10 mg/day (n = 50), each for 144 weeks, or to delayed treatment for 48 weeks followed by BLV 10 mg/day for 96 weeks (n = 51). Combined response was defined as undetectable hepatitis delta virus (HDV) RNA or a decrease in HDV RNA by ≥2 log10 IU/ml from baseline and alanine aminotransferase (ALT) normalization. Other endpoints included virologic response, ALT normalization, and change in HDV RNA. RESULTS: Of 150 patients, 143 (95%) completed 96 weeks of the study. Efficacy responses were maintained and/or improved between W48 and W96, with similar combined, virologic, and biochemical response rates between BLV 2 and 10 mg. Of the patients with a suboptimal early virologic response at W24, 43% of non-responders and 82% of partial responders achieved virologic response at W96. Biochemical improvement often occurred independently of virologic response. Adverse events were mostly mild, with no serious adverse events related to BLV. CONCLUSIONS: Virologic and biochemical responses were maintained and/or increased with longer term BLV therapy, including in those with suboptimal early virologic response. BLV monotherapy for CHD was safe and well tolerated through W96. IMPACT AND IMPLICATIONS: In July 2023, bulevirtide was fully approved for the treatment of chronic hepatitis delta (CHD) in Europe based on clinical study results from up to 48 weeks of treatment. Understanding the efficacy and safety of bulevirtide over the longer term is important for healthcare providers. In this analysis, we demonstrate that bulevirtide monotherapy for 96 weeks in patients with CHD was associated with continued improvements in combined, virologic, and biochemical responses as well as liver stiffness from week 48 at both the 2 mg and 10 mg doses. Patients with suboptimal virologic responses to bulevirtide at week 24 also benefited from continued therapy, with the majority achieving virologic response or biochemical improvement by week 96. GOV IDENTIFIER: NCT03852719.