This study explores a novel strategy called clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9-mediated Epstein-Barr virus (EBV) reactivation (CMER) to reactivate the Epstein-Bar...

The focus of the Antiviral Pharmacology group led by Anne-Grete Märtson is optimizing antiviral therapy for different viral infections by conducting clinical trials and using innovative computational ...

Ganciclovir remains the primary therapeutic for cytomegalovirus (CMV) infections in early infancy, but its pharmacokinetics and dosing in very preterm infants with end-organ CMV disease have not been fully evaluated.MethodsPremature infants with confirmed CMV infection and receiving ganciclovir as standard of care were enrolled into a pharmacokinetic sampling study. All were <32 weeks gestational age and >500 grams at enrollment. Plasma for ganciclovir quantitation was collected at steady-state at 0, 1, and between 2-3, 5-7, and 10-12 hours post-dose. Specimens were shipped, analyzed, and pharmacokinetic parameters calculated in real-time. Noncompartmental and modeling approaches were used for analysis.ResultsEighteen infants were enrolled; mean gestational age at delivery was 26.7 weeks, and mean age and weight at enrollment were 42 days and 1519.0 grams, respectively. Seventeen completed pharmacokinetic assessments. Geometric mean dose and resulting 12 hour area-under-the-curve (AUC12) were 5.19 mg/kg and 52.7 mgxh/L, respectively. A total of 85 ganciclovir concentration-time data points were available for modeling. A one-compartment power covariate model with weight and serum creatinine on clearance and weight on distribution volume was used. Noncompartmental and modeled pharmacokinetic parameters were similar.ConclusionsThese are the first intravenous ganciclovir population pharmacokinetic data with covariate assessments in premature infants being treated for CMV disease. Results suggest an intravenous dose of 5 mg/kg/dose every 12 hours may be an appropriate starting regimen for treatment of premature infants with congenital CMV. Additional data are needed in this and other populations to better define optimal ganciclovir exposure targets.

In a randomized trial of maribavir or valganciclovir for treatment of first-episode cytomegalovirus infection after stem cell transplantation, maribavir re

Whether ganciclovir-resistant cytomegalovirus (ganR-CMV) can establish latency and reactivate absent any selective drug pressure is unknown and has implications for selecting empiric antiviral therapy...

IntroductionCytomegalovirus (CMV) is a leading infection-related cause of morbidity in immunocompromised patients, particularly solid organ and haematopoietic cell transplant recipients [1]. With advances in transplantation science, including novel immunosuppressive regimens and older age recipients, the risk and burden of CMV are increasing worldwide [2]. Ganciclovir (administered intravenously [i.v.]) or valganciclovir (administered orally) is the reference standard antiviral therapy for treatment of CMV. However, use of valganciclovir and ganciclovir is challenging due to the necessity of renal dose adjustment, confusion surrounding weight-based (ganciclovir) versus fixed (valganciclovir) dosing, myelotoxicity, and potential refractory infection or emergence of drug resistance [3]. These challenges underscore the critical importance of dose optimization as a fundamental antiviral stewardship intervention, as inadequate dosing may contribute to drug toxicity, resistance, and/or suboptimal clinical outcomes. This manuscript represents a critical reappraisal of historical pharmacokinetic/pharmacodynamic (PK/PD) targets and current dosing schemes for (val)ganciclovir and serves as a call to action for future research to optimize dosing and CMV treatment outcomes.Drug development and early pharmacokineticsGanciclovir (9-[(1,3-dihydroxy-2-propoxy)methyl]guanine), synthesized at Syntex Research, was first reported in 1983 [4]. In 1984, 26 patients with life-threatening CMV infection (22 of whom had AIDS, now referred to as advanced HIV disease) received i.v. ganciclovir via compassionate use at a dose of 5 mg/kg 2–3 times daily for a median of 14 days, based on murine model exposure extrapolations [5]. Early studies suggested doses ≥7.5 mg/kg/d i.v. for at least 10 days improved clinical response compared with lower doses, although relapses were common after treatment cessation of any dose [6]. Dosing consensus in the 1980s was anywhere from 7.5 mg/kg/d to 15 mg/kg/d i.v. in 2–3 divided doses for induction followed by ‘continued therapy’ (to attempt to prevent post-treatment recurrence) at 5–6 mg/kg/d administered 5–7 days per week [7]. Some experts noted “a dose of 2.5 mg/kg every 8 hours is known to result in average peak levels of 5 mg/L and trough levels of about 0.5 mg/L in adults, whereas CMV replication has been shown to be 50% inhibited at concentrations ranging from 0.1 to 2.75 mg/L. We usually begin all patients on a dose of 5 mg/kg every 12 hours” [8]. Accordingly, the first prospective randomized trial in haematopoietic cell transplant (HCT) recipients utilized an induction dose of 5 mg/kg i.v. ganciclovir twice daily for 7 days, followed by 5 mg/kg i.v. daily, extrapolating from regimens utilized in patients with advanced HIV disease [6]. This became standard of care (despite limited PK/PD justification for the dosing scheme and no comparative outcome data) after disease incidence decreased in the trial from 43% to 3% and CMV-related deaths from 17% to 0% [6].Subsequent trials investigating universal prophylaxis in HCT recipients failed due to increased neutropenia and treatment-resistant CMV in patients receiving ganciclovir compared with no prophylaxis [9,10]. Myelosuppression following ganciclovir administration is common, with up to 50% of HCT recipients suggested to suffer from bone marrow toxicity [11,12]. However, Ritchie et al. [13] found no significant association between trough or peak concentrations and haematologic toxicity endpoints. Similarly, a recent study by van der Wekken-Pas et al. [14] found no difference in clinical efficacy or toxicity between target trough levels of 1–2 mg/L versus 2–4 mg/L. On the contrary, the study by Märtson et al. [15] did show correlation between white blood cell to the highest trough concentration and the area under the concentration-time curve (AUC24h), suggesting an exposure–response relationship for myelosuppression does exist, although a specific target or threshold for toxicity has not been identified and exposure–toxicity relationships are inconsistent.Valganciclovir, the oral prodrug of ganciclovir, received U.S. Food and Drug Administration approval in April 2004. According to pharmacokinetic (PK) data of patients with HIV summarized in the prescribing information [16], the AUC of valganciclovir 900 mg orally once daily (29.1 ± 9.7 mg*h/L, derived from three studies of 57 patients) was comparable to the AUC of ganciclovir 5 mg/kg i.v. once daily (26.5 ± 5.9 mg*h/L derived from four studies of 68 patients). Despite this ‘comparable exposure,’ decades of clinical experience have revealed substantial interpatient variability in (val)ganciclovir exposure. Notably, trough concentrations correlate poorly with AUC (r ∼ 0.6), peak concentrations are approximately 40% lower with oral valganciclovir than with intravenous ganciclovir, and fixed oral dosing generally results in higher overall exposure compared with weight-based intravenous administration [15,17].In a study of 96 liver transplant recipients receiving valganciclovir prophylaxis at standard (900 mg/d) or reduced (450 mg/d) doses, rates of CMV disease were similar (23% vs. 26%, p=0.71), but neutropenia occurred more frequently with standard dosing (60% vs. 10%, p <0.001) [18]. No ganciclovir-resistant CMV was detected in the standard-dose group (0/35), whereas two cases occurred in the low-dose group (2/61, 3.3%). Most data exploring an exposure–efficacy relationship or a threshold for resistance development remain limited by retrospective design, heterogeneous patient populations, concomitant medications, variation in ganciclovir formulations and dosing, poor understanding of the included patient population’s burden of immunosuppression, CMV testing methods (e.g. viral culture versus prestandardized PCR technology versus modern-day) and limited PK/PD sampling.The PK/PD target for efficacy and the toxicodynamic threshold of ganciclovir is unknown across all patient populations and indications. Based on a single pharmacodynamic (PD) study in solid organ transplant recipients, an AUC24 target of 50 mg*h/L for prophylaxis and 80–120 mg*h/L for treatment has been proposed [19,20]. This commentary explores this study and addresses the critical gap in our understanding of PK/PD targets for ganciclovir. Detailed reviews of ganciclovir therapeutic drug monitoring, PK, population modelling approaches, and bioassay techniques are available elsewhere [[20], [21], [22], [23]].The Wiltshire et al. [19] studyThe Wiltshire et al. [19] study is a foundational reference, which has been used to define the only described quantitative PK/PD target for ganciclovir prophylaxis against CMV. Here, we will critically investigate the origin and applicability of this target.A total of 372 solid organ transplant recipients (D+/R−) were randomized 2:1 to receive either valganciclovir 900 mg once daily (n = 245) or oral ganciclovir 100 mg three times daily (n = 127) for CMV prophylaxis for 100 days. The study included liver, kidney, heart and kidney-pancreas recipients. This is a heterogeneous patient population with different immunosuppressive regimens, CMV risk, and graft function. The PK profiles were available from 240 patients. The systemic ganciclovir exposure was demonstrated using AUC. The mean AUC24h with valganciclovir was 46.3 ± 15.2 mg*h/L compared with 28.0 ± 10.9 mg*h/L with oral ganciclovir. This represented a 1.65-fold increase in AUC24h for valganciclovir. Thereafter, logistic regression analyses were done to calculate the risk for CMV viremia during prophylaxis. On day 100, an AUC24h threshold of 50 mg*h/L predicted the risk for viremia in 1.3% of patients; the risk was eight times higher for an AUC24h threshold of 25 mg*h/L. A similar trend was observed for 4 months after transplantation, where an AUC24h of 33 mg*h/L was associated with a 20% risk of viremia, whereas an AUC24h of 50 mg*h/L corresponded to a 10% risk.For patients with PK data, there was no correlation between higher ganciclovir exposure and anaemia and a weak tendency with higher ganciclovir exposure and increased neutropenia. At 4 months post-transplant, median predicted incidences of neutropenia of 15% and 20% were associated with AUCs of 39 and 61 mg*h/L, respectively.The AUC24h target of 50 mg*h/L for prophylaxis from Wiltshire et al. [19] has since been used as a benchmark in several subsequent clinical PK studies and dosing algorithms. However, it is important to note that although this was a valuable study in understanding the PD of ganciclovir in the context of CMV prophylaxis, the study was not designed to investigate the PK/PD target for treatment of CMV, nor is it robustly validated for CMV prophylaxis in either the study population or other immunocompromised hosts.The way forwardPK/PD target findingIn basic and clinical virology, IC50 and IC90 (half-maximal and 90% inhibitory concentrations) and EC50 and EC90 (half-maximal and 90% effective concentrations) are utilized to study drug effects. The IC50 and IC90 represent the drug concentrations required to inhibit a specific biochemical function by 50% or 90% in biochemical assays focusing on a single target. However, EC50 and EC90 measure the drug concentrations that induce 50% or 90% of the maximal antiviral response in cell-based assays such as reducing viral plaque formation or suppressing viral load in cell culture. Inhibitory concentration values reflect target-specific inhibition, and effective concentration values capture the overall biological effect on viral replication (e.g. drug uptake, intracellular metabolism). These parameters should be considered in PK/PD target finding, which is not widely investigated in antiviral therapy. For example, time above IC50 or IC90 (time > IC50/IC90) or EC50/EC90 (time > EC50/EC90) has been shown to be an effective parameter for protease inhibitors like atazanavir, where prolonged exposure above the effective concentration ensured sustained viral suppression [24]. It was shown in the hollow-fibre infection model that using a continuous infusion to maintain drug levels at 4 × EC50 completely suppressed replication, whereas an equivalent daily AUC delivered as peak-and-valley dosing failed to maintain control.The host immune status affected by immunosuppressive regimens can have an impact on antiviral efficacy and virus replication, which makes extrapolating data from varying immunosuppressed populations often difficult [25]. In addition to host variation, IC50 and EC50/EC90 values vary by virus strain due to large inter-lab and strain variability. Evaluation of time over IC50/IC90 (EC50/EC90) as an alternative or complementary PD index would be particularly valuable for resistant or rapidly replicating CMV strains, where sustained drug pressure above inhibitory concentrations may be more important than total daily exposure.Incorporating peak concentration into a potential PK/PD target is another likely scenario, as the purpose of antiviral treatment is to suppress the viral load below the limit of detection to avoid any replication. This approach would help achieve sufficiently high antiviral peak concentrations that can rapidly drive viral loads below the limit of detection early in the infection period. This strategy could halt viral replication before resistance or persistence can emerge. One could theorize that varying PK/PD targets could be utilized in early versus late infection.Another difficult challenge in deriving a PK/PD target for ganciclovir is its rate of phosphorylation. As with any nucleoside analogues, ganciclovir needs the host cell to phosphorylate into monophosphate, diphosphate, and thereafter triphosphate that then competes with the viral DNA polymerase [26]. This halts the viral replication in the cell. Currently, the PK/PD targets are based on the nonphosphorylated state of the drug in plasma. It is important to consider the potential saturable step in the phosphorylation of the drug, which can be driven by different PK than the parent drug [27]. Billat et al. [28] have demonstrated in renal transplant patients that the development of neutropenia was significantly associated with intracellular ganciclovir triphosphate (GSV-TP) accumulation rather than plasma ganciclovir exposure. This finding shows that intracellular phosphorylated metabolites can drive toxic effects. An efficacy effect has been observed for another nucleoside analogue, tenofovir from tenofovir disoproxil fumarate, where the active intracellular metabolite (tenofovir diphosphate) determines antiviral activity and predictor of efficacy in pre-exposure prophylaxis [29], [30]. Understanding intracellular phosphorylation kinetics and their clinical implications is essential, as the active triphosphate form determines antiviral efficacy but cannot be easily measured in routine clinical practice.Clinical considerations in PK/PD target findingResearchers must also consider the relevance of PK/PD target development for prophylaxis versus treatment indications and whether these targets vary in different types of immunocompromised hosts. Indeed, the viral threshold at which treatment is started may differ depending on the risk profile and immune status of the patient; identifying viral load thresholds for treatment initiation could significantly improve patient outcomes. The most important means of controlling viral replication and preventing CMV reactivation is the adaptive immune response in the form of T cell-mediated cellular immunity, particularly CD8 cytotoxic T lymphocytes. CD4 T cells also respond to the virus and are associated with protection from CMV disease. The complexity of the host immune system is nearly impossible to model alongside antiviral kinetics to determine optimal prevention and treatment exposures.Moving forward, different PK/PD targets should be investigated in translational PK/PD studies, where ideally both the parent drug and the phosphorylated form of the drug are analysed in a variety of populations, including adult and paediatric patients. Thereafter, these concentrations should be related to viral load reduction and emergence of resistant mutations. In the clinical context, transplant population studies would also benefit from combining trough concentration monitoring with intracellular drug concentrations [for example, in peripheral blood mononuclear cells (PBMCs)] and incorporating patients’ immune status as this has a significant effect on treatment outcomes. Evaluation of time over IC50/IC90 (EC50/EC90) as an alternative or complementary index would be useful, especially in resistant or rapidly replicating CMV strains. Lastly, model-informed precision dosing tools should incorporate viral loads and neutrophil counts alongside drug PK to consider the individual treatment response in drug dosing. Furthermore, tissue-specific drug distribution for invasive CMV disease should be accounted for when establishing targets. Most historical dosing has been based on CMV retinitis in persons with HIV, where crossing the blood–eye barrier was critical and often required intravitreal ganciclovir supplementation. However, the PK exposure within tissues may differ significantly for patients with CMV colitis, pneumonitis, or DNAemia.Lastly, specific steps to further establish the PK/PD of ganciclovir against CMV should be taken. In addition to exploring intracellular ganciclovir triphosphate exposure versus plasma exposure, population PK modelling that incorporates transplanted organ, renal function, immunosuppression regimens, and viral kinetics should be investigated. In the longer term, quantitative systems pharmacology may be useful to investigate complex host-virus-drug interactions. Finally, high-risk patient populations such as lung transplant recipients (the highest CMV disease incidence), paediatric recipients, and D+/R− serostatus patients carrying the highest risk for CMV across all organ types should be prioritized.Optimal (val)ganciclovir dosing strategies for CMV are unknown but should be data-driven and informed by translational PK/PD studies across diverse patient populations. Prospective, population-specific PK/PD studies to define optimal dosing in various types of immunocompromised populations should be performed. Given the lack of defined efficacy or safety targets for (val)ganciclovir, therapeutic drug monitoring practices vary across institutions and remain limited globally. Further clinical studies would be valuable to establish such targets.Declaration of competing interestE.K.M. has participated on an advisory board for Merck related to cytomegalovirus prevention and treatment.Declaration of generative AI and AI-assisted technologies in the manuscript preparation processAI is not used in the preparation of this manuscript.CRediT authorship contribution statementErin K. McCreary: Writing – review & editing, Writing – original draft, Conceptualization. Navaneeth Narayanan: Writing – review & editing, Writing – original draft. Thomas Tängdén: Writing – review & editing, Writing – original draft. Anne-Grete Märtson: Writing – review & editing, Writing – original draft, Project administration, Conceptualization.Funding StatementNo specific funding was received for this article. A-G.M. is funded by the Dutch Research Council (NWO), project ‘A Fresh Look at Antiviral Treatment: A Translational Pharmacology Approach’ with file number 09150162310191 of the research programme VENI. T.T. acknowledges financial support from the Swedish Research Council (grant number 2020-02320) and Sweden’s Innovation Agency VINNOVA (grant number 2021-02699).AcknowledgementsThe SIDP Publications Committee provided thoughtful peer review and endorses the concepts and content of this manuscript in its final form.

ABSTRACT Valganciclovir (VGCV) is the first-line drug for preemptive therapy of cytomegalovirus (CMV) infection. However, even at standard doses, plasma concentrations of the active metabolite ganciclovir (GCV) show substantial inter-individual variability. To ensure therapeutic efficacy and minimize adverse effects, therapeutic drug monitoring (TDM) based on the area under the concentration–time curve (AUC) is essential. Yet, conventional TDM via venous blood sampling is invasive and unsuitable for frequent monitoring. In this study, we aimed to develop a simple and minimally invasive method for GCV quantification using dried blood spots (DBS) combined with liquid chromatography–tandem mass spectrometry (LC–MS/MS). The method demonstrated a good linearity over a concentration range of 0.25–16 μg/mL and satisfied the validation criteria for accuracy and precision. It showed acceptable stability for up to 7 days under refrigerated conditions. Methanol was identified as the optimal extraction solvent, allowing for a simplified sample pretreatment without the need for ultrasonic processing. While hematocrit levels affected spot size and quantification accuracy, reliable measurements were obtained within the 30%–50% hematocrit range. The established DBS-based LC–MS/MS method provides a promising, minimally invasive approach for TDM of GCV in the management of CMV infections.