Multi-centre real-world validation of automated treatment planning for breast radiotherapy.

PURPOSE: To present the results of the first multi-centre real-world validation of autoplanning for whole breast irradiation after breast-sparing surgery, encompassing high complexity cases (e.g. with a boost or regional lymph nodes) and a wide range of clinical practices. METHODS: The 24 participating centers each included 10 IMRT/VMAT/Tomotherapy patients, previously treated with a manually generated plan ('manplan'). There were no restrictions regarding case complexity, planning aims, plan evaluation parameters and criteria, fractionation, treatment planning system or treatment machine/technique. In addition to dosimetric comparisons of autoplans with manplans, blinded plan scoring/ranking was conducted by a clinician from the treating center. Autoplanning was performed using a single configuration for all patients in all centres. Deliverability was verified through measurements at delivery units. RESULTS: Target dosimetry showed comparability, while reductions in OAR dose parameters were 21.4 % for heart Dmean, 16.7 % for ipsilateral lung Dmean, and 101.9 %, 45.5 %, and 35.7 % for contralateral breast D0.03cc, D5% and Dmean, respectively (all p < 0.001). Among the 240 patients included, the clinicians preferred the autoplan for 119 patients, with manplans preferred for 96 cases (p = 0.01). Per centre there were on average 5.0 ± 2.9 (1SD) patients with a preferred autoplan (range [0-10]), compared to 4.0 ± 2.7 with a preferred manplan ([0,9]). No differences were observed regarding deliverability. CONCLUSION: The automation significantly reduced the hands-on planning workload compared to manual planning, while also achieving an overall superiority. However, fine-tuning of the autoplanning configuration prior to clinical implementation may be necessary in some centres to enhance clinicians' satisfaction with the generated autoplans.

Survey on the interest and commitment of AIFM members to scientific activities (SicAS) - The initiative of the FutuRuS working group.

PURPOSE: The "FutuRuS" working group of the Italian Association of Medical Physics and Health Physics (AIFM) designed a survey (SicAS) to get feedback from its members regarding their interests and their experience in taking part in scientific activities and events, with the objective of focusing future efforts of the AIFM towards increasing the scientific activity of the medical physics expert (MPE). METHODS: SicAS was sent out in March 2022 to all AIFM members by newsletter and official communication. SicAS was structured into three sections: personal information and institution of affiliation information, involvement in scientific activities, interest in and commitment to scientific activities. Responses were collected in a fully anonymised mode from the Google Forms platform and analysed with descriptive statistics. RESULTS: Out of 1289 members (active at the end of 2021), 467 responded to the Survey (response rate of 36%). The Survey results highlighted that AIFM members ranked the involvement of the MPE in scientific activities as highly relevant to the profession. However, 34.7% indicated devoting less than 10% of their working time to scientific activities. 67.5% of the respondents were dissatisfied with the time spent on scientific activities. The primary barrier was the lack of time (77%), followed by a lack of mentoring (32%). CONCLUSIONS: SicAS highlighted the need for AIFM initiatives to support members' scientific activities. National societies should help develop and support networks between members, create links among universities, hospitals, research institutions and industries, and provide guidelines and learning platforms for enhancing the MPEs' involvement in scientific activities.

When your MR linac is down: Can an automated pipeline bail you out of trouble?

PURPOSE: The unique treatment delivery technique provided by magnetic resonance guided radiotherapy (MRgRT) can represent a significant drawback when system fail occurs. This retrospective study proposes and evaluates a pipeline to completely automate the workflow necessary to shift a MRgRT treatment to a traditional radiotherapy linac. MATERIAL AND METHODS: Patients undergoing treatment during the last MRgRT system failure were retrospectively included in this study. The core of the proposed pipeline was based on a tool able to mimic the original MR linac dose distribution. The so obtained dose distribution (AUTO) has been compared with the distribution obtained in the conventional radiotherapy linac (MAN). Plan comparison has been performed in terms of time required to obtain the final dose distribution, DVH parameters, dosimetric indices and visual analogue scales scoring by radiation oncologists. RESULTS: AUTO plans generation has been obtained within 10 min for all the considered cases. All AUTO plans were found to be within clinical tolerance, showing a mean target coverage variation of 1.7% with a maximum value of 4.3% and a minimum of 0.6% when compared with MAN plans. The highest OARs mean variation has been found for rectum V60 (6.7%). Dosimetric indices showed no relevant differences, with smaller gradient measure in favour of AUTO plans. Visual analogue scales scoring has confirmed comparable plan quality for AUTO plans. CONCLUSION: The proposed workflow allows a fast and accurate generation of automatic treatment plans. AUTO plans can be considered equivalent to MAN ones, with limited clinical impact in the worst-case scenario.

On dose cube pixel spacing pre-processing for features extraction stability in dosiomic studies.

PURPOSE: Dosiomics allows to parameterize regions of interest (ROIs) and to produce quantitative dose features encoding the spatial and statistical distribution of radiotherapy dose. The stability of dosiomics features extraction on dose cube pixel spacing variation has been investigated in this study. MATERIAL AND METHODS: Based on 17 clinical delivered dose distributions (Pn), dataset has been generated considering all the possible combinations of four dose grid resolutions and two calculation algorithms. Each dose voxel cube has been post-processed considering 4 different dose cube pixel spacing values: 1x1x1, 2x2x2, 3x3x3 mm3 and the one equal to the planning CT. Dosiomics features extraction has been performed from four different ROIs. The stability of each extracted dosiomic feature has been analyzed in terms of coefficient of variation (CV) intraclass correlation coefficient (ICC). RESULTS: The highest CV mean values were observed for PTV ROI and for the grey level size zone matrix features family. On the other hand, the lowest CV mean values have been found for RING ROI for the grey level co-occurrence matrix features family. P3 showed the highest percentage of CV >1 (1.14%) followed by P15 (0.41%), P1 (0.29%) and P13 (0.19%). ICC analysis leads to identify features with an ICC >0.95 that could be considered stable to use in dosiomic studies when different dose cube pixel spacing are considered, especially the features in common among the seventeen plans. CONCLUSION: Considering the observed variability, dosiomic studies should always provide a report not only on grid resolution and algorithm dose calculation, but also on dose cube pixel spacing.

VMAT-like plans for magnetic resonance guided radiotherapy: Addressing unmet needs.

PURPOSE: VMAT delivery technique is currently not applicable to Magnetic Resonance-guided radiotherapy (MRgRT) hybrid systems. Aim of this study is to evaluate an innovative VMAT-like (VML) delivery technique. MATERIAL AND METHODS: First, planning and dosimetric evaluation of the MRgRT VML treatment have been performed on 10 different disease sites and the results have been compared with the corresponding IMRT plans. Then, in the second phase, 10 of the most dosimetrically challenging locally advanced pancreas treatment plans have been retrospectively re-planned using the VML approach to explore the potentiality of this new delivery technique. Finally, VML robustness was evaluated and compared with the IMRT plans, considering a lateral positioning error of ± 5 mm. RESULTS: In phase one, all VML plans were within constraint for all OARs. When PTV coverage is considered, in the 50% of the cases VML PTV coverage is equal or higher than in IMRT plan. In the remaining 50%, the highest target under coverage difference in comparison with IMRT plan is -1.71%. The mean and maximum treatment time differences (VML-IMRT) is 0.2 min and 3.1 min respectively. In phase two, the treatment time variation (VML-IMRT), shows a mean, maximum and minimum variations of 1.3, 4.6 and -0.6 min respectively. All VML plans have a better target coverage if compared with IMRT plans, keeping in any case the OARs constraints within tolerance. VML doesn't increase plan robustness. CONCLUSION: VMAT-like treatment approach appeared to be an efficient planning solution and it was decided to clinically implement it in daily practice, especially in the frame of hypo fractionated treatments.

Dosimetric accuracy of dual isocenter irradiation in low magnetic field resonance guided radiotherapy system for extended abdominal tumours.

PURPOSE: Due to limited field size of Magnetic Resonance Linear Accelerators (MR-Linac), some treatments could require a dual-isocenter planning approach to achieve a complete target coverage and thus exploit the benefits of the online adaptation. This study evaluates the dosimetric accuracy of the dual-isocenter intensity modulated radiation therapy (IMRT) delivery technique for MR-Linac. MATERIAL AND METHODS: Dual-isocenter multi leaf collimator (MLC) and couch accuracy tests have been performed to evaluate the delivery accuracy of the system. A mono-isocenter plan delivered in clinical practice has then been retrospectively re-planned with dual-isocenter technique. The dual-isocenter plan has been re-calculated and delivered on a 3-dimensional (3D) ArcCHECK phantom and 2-dimensional (2D) films to assess its dosimetric accuracy in terms of gamma analysis. Clinical and planning target volume (CTV and PTV respectively) coverage robustness was then investigated after the introduction of ± 2 mm and ± 5 mm positioning errors by shifting the couch. RESULTS: MLC and couch accuracy tests confirmed the system accuracy in delivering a dual-isocenter irradiation. 2D/3D gamma analysis results occurred always to be above 95% if considered a gamma criteria 1%/2 mm and 1%/1 mm respectively for the 2D and 3D analysis. The mean variations for CTV D98% and PTV V95% were 0.2% and 1.1% respectively when positioning error was introduced separately in each direction, while the maximum observed variations were 0.9% (CTV) and 3.7% (PTV). CONCLUSION: The dosimetric accuracy of dual-isocenter irradiation has been verified for MR-Linac, achieving accurate and robust treatment strategy and improving dose conformality also in presence of targets whose extension exceeds the nominal maximum field size.

Process mining to optimize palliative patient flow in a high-volume radiotherapy department.

INTRODUCTION: In radiotherapy, palliative patients are often suboptimal managed and patients experience long waiting times. Event-logs (recorded local files) of palliative patients, could provide a continuative decision-making system by means of shared guidelines to improve patient flow. Based on an event-log analysis, we aimed to accurately understand how to successively optimize patient flow in palliative care. METHODS: A process mining methodology was applied on palliative patient flow in a high-volume radiotherapy department. Five hundred palliative radiation treatment plans of patients with bone and brain metastases were included in the study, corresponding to 290 patients treated in our department in 2018. Event-logs and the relative attributes were extracted and organized. A process discovery algorithm was applied to describe the real process model, which produced the event-log. Finally, conformance checking was performed to analyze how the acquired event-log database works in a predefined theoretical process model. RESULTS: Based on the process discovery algorithm, 53 (10%) plans had a dose prescription of 8 Gy, 249 (49.8%) plans had a dose prescription of 20 Gy and 159 (31.8%) plans had a dose prescription of 30 Gy. The remaining 39 (7.8%) plans had different dose prescriptions. Considering a median value, conformance checking demonstrated that event-logs work in the theoretical model. CONCLUSIONS: The obtained results partially validate and support the palliative patient care guideline implemented in our department. Process mining can be used to provide new insights, which facilitate the improvement of existing palliative patient care flows.

Stability of dosomics features extraction on grid resolution and algorithm for radiotherapy dose calculation.

PURPOSE: Dosomics is a novel texture analysis method to parameterize regions of interest and to produce dose features that encode the spatial and statistical distribution of radiotherapy dose at higher resolution than organ-level dose-volume histograms. This study investigates the stability of dosomics features extraction, as their variation due to changes of grid resolution and algorithm dose calculation. MATERIAL AND METHODS: Dataset has been generated considering all the possible combinations of four grid resolutions and two algorithms dose calculation of 18 clinical delivered dose distributions, leading to a 144 3D dose distributions dataset. Dosomics features extraction has been performed with an in-house developed software. A total number of 214 dosomics features has been extracted from four different region of interest: PTV, the two closest OARs and a RING structure. Reproducibility and stability of each extracted dosomic feature (Rfe, Sfe), have been analyzed in terms of intraclass correlation coefficient (ICC) and coefficient of variation. RESULTS: Dosomics features extraction was found reproducible (ICC > 0.99). Dosomic features, across the combination of grid resolutions and algorithms dose calculation, are more stable in the RING for all the considered feature's families. Sfe is higher in OARs, in particular for GLSZM features' families. Highest Sfe have been found in the PTV, in particular in the GLCM features' family. CONCLUSION: Stability and reproducibility of dosomics features have been evaluated for a representative clinical dose distribution case mix. These results suggest that, in terms of stability, dosomic studies should always perform a reporting of grid resolution and algorithm dose calculation.

Hybrid MRI guided radiotherapy in locally advanced cervical cancer: Case report of an innovative personalized therapeutic approach.

•The case report of a patient affected by locally advanced cervical MRgRT is described.•MRgRT appears to be feasible for cervical cancer and may improve treatment quality.•MRgRT insights are discussed focusing on adaptive response and toxicity monitoring.

The dependence of interplay effects on the field scan direction in PBS proton therapy.

The literature is controversial about the scan direction dependency of interplay effects in pencil beam scanning (PBS) treatment of moving targets. A directional effect is supported by many simulation studies, whereas the experimental data are mostly limited to simple geometries, not reflecting realistically clinical treatment plans. We have compared increasingly complex treatment fields, from a homogeneous single energy layer to a more modulated lung plan, under identical experimental settings, seeking evidence for differences in motion mitigation due to the selection of primary scanning direction. In total, 120 experimental samples were taken, combining two primary scan directions and three rescanning regimes with different motion scenarios. 4D dose distributions were measured in water with a moving ionisation chamber array and compared to those of a stationary delivery using 2D gamma analysis. Each plan has been verified twice for the same rescanning regime and motion scenario, changing the meandering direction in between to scan perpendicularly to, or along, the target motion. Additionally, machine log files of the lung plan, together with 4DCT data, were used to calculate the dose distribution that such deliveries would have produced in the patient. The primary meandering direction has a clear influence on measured dose distributions when considering a single energy layer. Introducing spot weight modulation and multiple energy layers however, makes the dynamic of interplay more complex and difficult to predict. Overall, gamma (3%/3 mm) differences between scanning along or orthogonal to the target motion follow a normal distribution [Formula: see text] when considering multiple motion scenarios and rescanning regimes. Nevertheless, data spread [Formula: see text] is significant enough such that, for individual experiments and set-ups, a dependency may be observed even if this is not a general result. Patient reconstructed doses follow the same trend, the two primary scan directions producing statistically insignificant differences in dose distributions in terms of conformity or homogeneity. Except for extremely simplified cases of mono-energetic and homogeneous treatment fields, the interplay effect has been found to be only marginally influenced by the choice of the primary scanning direction.

Positioning of head and neck patients for proton therapy using proton range probes: a proof of concept study.

To exploit the full potential of proton therapy, accurate and on-line methods to verify the patient positioning and the proton range during the treatment are desirable. Here we propose and validate an innovative technique for determining patient misalignment uncertainties through the use of a small number of low dose, carefully selected proton pencil beams ('range probes') (RP) with sufficient energy that their residual Bragg peak (BP) position and shape can be measured on exit. Since any change of the patient orientation in relation to these beams will result in changes of the density heterogeneities through which they pass, our hypothesis is that patient misalignments can be deduced from measured changes in Bragg curve (BC) shape and range. As such, a simple and robust methodology has been developed that estimates average proton range and range dilution of the detected residual BC, in order to locate range probe positions with optimal prediction power for detecting misalignments. The validation of this RP based approach has been split into two phases. First we retrospectively investigate its potential to detect translational patient misalignments under real clinical conditions. Second, we test it for determining rotational errors of an anthropomorphic phantom that was systematically rotated using an in-house developed high precision motion stage. Simulations of RPs in these two scenarios show that this approach could potentially predict translational errors to lower than1.5 mm and rotational errors to smaller than 1° using only three or five RPs positions respectively.

Pharmacology of beta-L-thymidine and beta-L-2'-deoxycytidine in HepG2 cells and primary human hepatocytes: relevance to chemotherapeutic efficacy against hepatitis B virus.

beta-L-Thymidine (L-dT) and beta-L-2'-deoxycytidine (L-dC) are potent and highly specific inhibitors of hepatitis B virus (HBV) replication both in vivo and in vitro (50% effective concentrations, 0.19 to 0.24 microM in 2.2.15 cells). The intracellular metabolisms of L-dT and L-dC were investigated in HepG2 cells and primary cultured human hepatocytes. L-dT and L-dC were extensively phosphorylated in both cell types, with the 5'-triphosphate derivative being the predominant metabolite. In HepG2 cells, the 5'-triphosphate levels were 27.7 +/- 12.1 and 72.4 +/- 1.8 pmol/10(6) cells for L-dT and L-dC, respectively. In primary human hepatocytes, the 5'-triphosphate levels were 16.5 +/- 9.8 and 90.1 +/- 36.4 pmol/10(6) cells for L-dT and L-dC, respectively. Furthermore, a choline derivative of L-dCDP was detected at concentrations of 15.8 +/- 1.8 and 25.6 +/- 0.1 pmol/10(6) cells in human hepatocytes and HepG2 cells, respectively. In HepG2 cells exposed to L-dC, the 5'-monophosphate and 5'-triphosphate derivatives of beta-L-2'-deoxyuridine (L-dUMP and L-dUTP, respectively) were also observed, reaching intracellular concentrations of 6.7 +/- 0.4 and 18.2 +/- 1.0 pmol/10(6) cells, respectively. In human hepatocytes, L-dUMP and L-dUTP were detected at concentrations of 5.7 +/- 2.4 and 43.5 +/- 26.8 pmol/10(6) cells, respectively. It is likely that deamination of L-dCMP by deoxycytidylate deaminase leads to the formation of L-dUMP, as the parent compound, L-dC, was not a substrate for deoxycytidine deaminase. The intracellular half-lives of L-dTTP, L-dCTP, and L-dUTP were at least 15 h, with intracellular concentrations of each metabolite remaining above their respective 50% inhibitory concentrations for the woodchuck hepatitis virus DNA polymerase for as long as 24 h after removal of the drug from cell cultures. Exposure of HepG2 cells to L-dT in combination with L-dC led to concentrations of the activated metabolites similar to those achieved with either agent alone. These results suggest that the potent anti-HBV activities of L-dT and L-dC are associated with their extensive phosphorylation.

Antiviral beta-L-nucleosides specific for hepatitis B virus infection.

Three simple, related nucleosides, beta-L-2'-deoxycytidine (LdC), beta-Lthymidine (LdT), and beta-L-2'-deoxyadenosine (LdA), have been discovered to be potent, specific and selective inhibitors of the replication hepatitis B virus (HBV), as well as the closely related duck and woodchuck hepatitis viruses (WHV). Structure-activity relationship analysis indicates that the 3'-OH group of the beta-L-2'-deoxyribose of the beta-L-2'-deoxynucleoside confers specific anti-hepadnavirus activity. The simple nucleosides had no effect on the replication of 15 other RNA and DNA viruses, and did not inhibit human DNA polymerases (alpha, beta and gamma) or compromise mitochondrial function. The nucleosides are efficiently converted intracellularly into active triphosphate metabolites that have a long half-life. Once-daily oral administration of these compounds in the woodchuck efficacy model of chronic HBV infection reduced viral load by as much as 10(8) genome equivalents/ml serum and there was no drug-related toxicity. In addition, a decline in WHV surface antigen (WHsAg) paralleled the decrease in viral load. This class of nucleosides displays an excellent overall safety profile. The first compound, LdT, has already entered clinical trials and LdC, currently being developed as a prodrug, is expected to enter the clinic in the near future. These compounds have the potential for use in combination therapy with the goal of achieving superior viral suppression and diminishing the onset of resistance.

Anti-HBV specific beta-L-2'-deoxynucleosides.

A unique series of simple unnatural L-nucleosides that specifically inhibit hepatitis B virus (HBV) replication has been discovered. These molecules have in common a hydroxyl group in the 3'-position (3'-OH) of the beta-L-2'-deoxyribose sugar that confers antiviral activity specifically against hepadnaviruses. Replacement of the 3'-OH broadens activity to other viruses. Substitution in the base decreases antiviral potency and selectivity. Human DNA polymerases and mitochondrial function are not effected. Plasma viremia is reduced up to 8 logs in a woodchuck model of chronic HBV infection. These investigational drugs, used alone or in combination, are expected to offer new therapeutic options for patients with chronic HBV infection.

Antiviral activity and intracellular metabolism of bis(tButylSATE) phosphotriester of beta-L-2',3'dideoxyadenosine, a potent inhibitor of HIV and HBV replication.

The beta-L-nucleoside analogue beta-L-2',3'-dideoxy adenosine (beta-L-ddA) has been shown to exhibit limited antiviral activities. This was attributed to its rapid catabolism through cleavage of the glycosidic bond and poor phosphorylation to the nucleotide beta-L-2',3'-dideoxyadenosine-5'-mono phosphate (beta-L-ddAMP) (Placidi et al., 2000). However, the nucleotide beta-L-2',3'-dideoxyadenosine-5'-triphosphate (beta-L-ddATP) inhibited the activity of both HIV-1 reverse transcriptase (RT) and viral DNA polymerase isolated from woodchuck hepatitis virus-infected serum (a model of hepatitis B) with an inhibitory concentration (IC50) of 2.0 microM without inhibiting human DNA polymerases alpha, beta, or gamma up to a concentration of 100 microM. These results suggested that prodrugs of beta-L-ddAMP may bypass the poor metabolic activation of beta-L-ddA and lead to more potent and selective antiviral activity. Therefore, the mononucleoside phosphotriester derivative of beta-L-ddAMP incorporating the S-pivaloyl-2-thioethyl (tButylSATE) groups, beta-L-ddAMP-bis(tButylSATE) was synthesized. Beta-L-ddAMP-bis(tButylSATE) inhibited HIV replication in human peripheral blood mononuclear cells (PBMCs) and HBV replication in 2.2.15 cells with effective concentrations (EC50s) of 2 and 80 nM, respectively. Intracellular metabolism of beta-L-ddAMP-bis(tButylSATE) demonstrated that beta-L-ddATP was the predominant intracellular metabolite in PBMC and liver cells. The intracellular half-life of beta-L-ddATP was 5.4 and 9.2 h in HepG2 and PBMCs, respectively. The intracellular concentrations of beta-L-ddATP were maintained above the EC50 for the inhibition of HIV RT and hepatitis B virus (HBV) for as long as 24 h after removal of the drug.

Intravenous infusion of cereport increases uptake and efficacy of acyclovir in herpes simplex virus-infected rat brains.

The outcome of herpes simplex virus (HSV) infections manifesting as encephalitis in healthy or immunocompromised individuals is generally very poor with mortality rates of about 8 to 28% with treatment. The long-term prognosis of survivors is often problematic, posing the need for alternative treatments that may decrease the mortality and morbidity associated with herpes encephalitis. This study addresses one such approach that includes a temporary permeabilization of the blood-brain barrier during treatment with acyclovir (ACV). In these studies we utilized a synthetic bradykinin analog, Cereport (RMP-7), in conjunction with ACV to treat HSV infection of the brain in a rat model. Cereport, infused intravenously via the jugular vein, was shown to increase [(14)C]ACV uptake in both the HSV-1-infected and -uninfected rat brain by approximately two- to threefold, correlating with enhanced efficacy of ACV in various brain compartments. In another series of experiments to determine efficacy, various doses of unlabeled ACV were administered during infusion with RMP-7. The decrease in viral titers in the temporal regions of the brain after 5 days of treatment suggested that this approach enhanced the efficacy of ACV treatment. These data indicated that Cereport infused with ACV enhances both the penetration and efficacy of this drug in the treatment of an experimental HSV-1 infection of the rat brain.

Evaluation of the mutagenic and genotoxic activities of anti-hepatitis B analogs of beta-L-adenosine by the Ames test and the Comet assay.

beta-L-2'-deoxyadenosine (beta-L-dA), beta-L-2',3'-dideoxyadenosine (beta-L-ddA) and its two bis (S-acyl-2-thioethyl; SATE) phosphotriester derivatives, beta-L-2',3'-dideoxyadenosine-5'-monophosphate-bis(MeSATE) and beta-L-2',3'-dideoxyadenosine-5'-monophosphate-bis(tButylSATE) have been previously shown to exhibit potent and selective anti-hepatitis B activity in vitro. None of the four compounds was mutagenic up to 100 microg in the Ames test (microtechnique) using Salmonella typhimurium strains TA 97a, TA 98, TA 100 and TA 102, with and without metabolic activation. In addition, the genotoxicity of beta-LdA and the three other compounds was evaluated in human lymphocytes using the Comet assay, at doses up to 5 microg with or without the addition of a microsomal S9 fraction. None of the four compounds induced DNA strand breakage with and without metabolic activation. In summary, the data clearly demonstrate that the purine nucleoside beta-L-dA, beta-L-ddA and the two prodrugs, beta-L-ddAMP-bis(MeSATE) and beta-L-ddAMP-bis(tButylSATE) are not mutagenic in the Ames test and do not induce DNA damage in human lymphocytes, as assessed by the Comet assay.

Antiviral L-nucleosides specific for hepatitis B virus infection.

A unique series of simple "unnatural" nucleosides has been discovered to inhibit hepatitis B virus (HBV) replication. Through structure-activity analysis it was found that the 3'-OH group of the beta-L-2'-deoxyribose of the beta-L-2'-deoxynucleoside confers specific antihepadnavirus activity. The unsubstituted nucleosides beta-L-2'-deoxycytidine, beta-L-thymidine, and beta-L-2'-deoxyadenosine had the most potent, selective, and specific antiviral activity against HBV replication. Human DNA polymerases (alpha, beta, and gamma) and mitochondrial function were not affected. In the woodchuck model of chronic HBV infection, viral load was reduced by as much as 10(8) genome equivalents/ml of serum and there was no drug-related toxicity. In addition, the decline in woodchuck hepatitis virus surface antigen paralleled the decrease in viral load. These investigational drugs, used alone or in combination, are expected to offer new therapeutic options for patients with chronic HBV infection.

In vitro and in vivo metabolism and pharmacokinetics of bis [(t-butyl)-S-acyl-2-thioethyl]-beta-L-2',3'-dideoxy-5-fluorocytidine monophosphate.

Exposure to 10 &M L-FddCMP-bisSATE led to formation of intracellular L-FddCTP levels of 410.1(+/-) +/- 46.2 and 242.1 +/- 13.2 pmol/10(6) cells in unstimulated and PHAstimulated PBM cells, respectively; whereas, exposure of cells to the parent nucleoside, L-FddC, generated 5-10-fold less L-FddCTP. In Hep-G2 cells and EGF/HGF stimulated and unstimulated primary cultured hepatocytes, the active metabolite reached 113 +/- 29, 23.9 +/- 15.6, and 20.6 +/- 10.5 pmol/10(6) cells. Three other metabolites, L-FddCMP-monoSATE, L-FddCMP-SH, and M I, were detected intracellularly and extracellularly in all cell types examined. Intravenous administered dose of 3 mg/kg L-FddCMP-bisSATE to rhesus monkeys resulted in plasma concentration levels of 2.06 +/- 1.00 and 0.39 +/- 0.15 &M of L-FddCMP-monoSATE and L-FddC, respectively, while the prodrug was completely cleared metabolically within 15 min. Following oral administration of an equivalent dose, the absolute oral bioavailability of L-FddC derived from L-FddCMP-bisSATE administration was 65%.

Intracellular metabolism of beta-L-2',3'-dideoxyadenosine: relevance to its limited antiviral activity.

The intracellular metabolism of the beta-L- enantiomer of 2', 3'-dideoxyadenosine (beta-L-ddA) was investigated in HepG2 cells, human peripheral blood mononuclear cells (PBMC), and primary cultured human hepatocytes in an effort to understand the metabolic basis of its limited activity on the replication of human immunodeficiency virus and hepatitis B virus. Incubation of cells with 10 microM [2',3',8-(3)H]-beta-L-ddA resulted in an increased intracellular concentration of beta-L-ddA with time, demonstrating that these cells were able to transport beta-L-ddA. However, it did not result in the phosphorylation of beta-L-ddA to its pharmacologically active 5'-triphosphate (beta-L-ddATP). Five other intracellular metabolites were detected and identified as beta-L-2', 3'-dideoxyribonolactone, hypoxanthine, inosine, ADP, and ATP, with the last being the predominant metabolite, reaching levels as high as 5.14 +/- 0.95, 8.15 +/- 2.64, and 15.60 +/- 1.74 pmol/10(6) cells at 8, 4, and 2 h in HepG2 cells, PBMC, and hepatocytes, respectively. In addition, a beta-glucuronic derivative of beta-L-ddA was detected in cultured hepatocytes, accounting for 12.5% of the total metabolite pool. Coincubation of hepatocytes in primary culture with beta-L-ddA in the presence of increasing concentrations of 5'-methylthioadenosine resulted in decreased phosphorolysis of beta-L-ddA and formation of associated metabolites. These results indicate that the limited antiviral activity of beta-L-ddA is the result of its inadequate phosphorylation to the nucleotide level due to phosphorolysis and catabolism of beta-L-ddA by methylthioadenosine phosphorylase (EC 2.4.2.28).