Development and validation of an ultra-performance liquid chromatography-tandem mass spectrometry method for the quantification of the antimalarial drug pyronaridine in human whole blood.

Malaria remains a major health concern, aggravated by emerging resistance of the parasite to existing treatments. The World Health Organization recently endorsed the use of artesunate-pyronaridine to treat uncomplicated malaria. However, there is a lack of clinical pharmacokinetic (PK) data of pyronaridine, particularly in special populations such as children and pregnant women. Existing methods for the quantification of pyronaridine in biological matrices to support PK studies exhibit several drawbacks. These include limited sensitivity, a large sample volume required, and extensive analysis time. To overcome these limitations, an ultra-performance reversed-phase liquid chromatography tandem-mass spectrometry method to determine pyronaridine was developed and validated according to international guidelines. The method enabled fast and accurate quantification of pyronaridine in whole blood across a clinically relevant concentration range of 0.500-500 ng/mL (r2 ≥ 0.9963), with a required sample volume of 50 µL. Pyronaridine was extracted from whole blood using liquid-liquid extraction, effectively eliminating the matrix effect and preventing ion enhancement or suppression. The method achieved a satisfactory reproducible sample preparation recovery of 77%, accuracy (as bias) and precision were within ±8.2% and ≤5.3%, respectively. Stability experiments demonstrated that pyronaridine was stable for up to 315 days when stored at -70°C. Adjustments to the chromatographic system substantially reduced carry-over and improved sensitivity compared to prior methods. The method was successfully applied to quantify pyronaridine in whole blood samples from a selection of pregnant malaria patients participating in the PYRAPREG clinical trial (PACTR202011812241529) in the Democratic Republic of the Congo, demonstrating its suitability to support future PK studies. Furthermore, the enhanced sensitivity allows for the determination of pyronaridine up to 42 days post-treatment initiation, enabling assessment of the terminal elimination half-life.

Intradermal naked DNA vaccination by DNA tattooing for mounting tumor-specific immunity in stage IV melanoma patients: a phase I clinical trial.

INTRODUCTION: Naked DNA vaccination could be a powerful and safe strategy to mount antigen-specific cellular immunity. We designed a phase I clinical trial to investigate the toxicity of naked DNA vaccines encoding CD8+ T-cell epitope from tumor-associated antigen MART-1 in patients with advanced melanoma. METHODS: This dose escalating phase Ia clinical trial investigates the toxicity and immunological response upon naked DNA vaccines encoding a CD8+ T-cell epitope from the tumor-associated antigen MART-1, genetically linked to the gene encoding domain 1 of subunit-tetanus toxin fragment C in patients with advanced melanoma (inoperable stage IIIC-IV, AJCC 7th edition). The vaccine was administrated via intradermal application using a permanent make-up or tattoo device. Safety was monitored according to CTCAE v.3.0 and skin biopsies and blood samples were obtained for immunologic monitoring. RESULTS: Nine pretreated, HLA-A*0201-positive patients with advanced melanoma expressing MART-1 and MHC class I, with a good performance status, and adequate organ function, were included. With a median follow-up of 5.9 months, DNA vaccination was safe, without treatment-related deaths. Common treatment-emergent adverse events of any grade were dermatologic reactions at the vaccination site (100%) and pain (56%). One patient experienced grade 4 toxicity, most likely related to tumor progression. One patient (11%) achieved stable disease, lasting 353 days. Immune analysis showed no increase in vaccine-induced T cell response in peripheral blood of five patients, but did show a MART-1 specific CD8+ T cell response at the tattoo administration site. The maximum dose administered was 2 mg due to lack of clinical activity. CONCLUSION: We showed that the developed DNA vaccine, applied using a novel intradermal application strategy, can be administered safely. Further research with improved vaccine formats is required to show possible clinical benefit of DNA vaccination.

The ABCB1 and ABCG2 efflux transporters limit brain disposition of the SYK inhibitors entospletinib and lanraplenib.

The highly selective Spleen Tyrosine Kinase (SYK) inhibitors entospletinib and lanraplenib disrupt kinase activity and inhibit immune cell functions. They are developed for treatment of B-cell malignancies and autoimmunity diseases. The impact of P-gp/ABCB1 and BCRP/ABCG2 efflux transporters, OATP1a/1b uptake transporters and CYP3A drug-metabolizing enzymes on the oral pharmacokinetics of these drugs was assessed using mouse models. Entospletinib and lanraplenib were orally administered simultaneously at moderate dosages (10 mg/kg each) to female mice to assess the possibility of examining two structurally and mechanistically similar drugs at the same time, while reducing the number of experimental animals and sample-processing workload. The plasma pharmacokinetics of both drugs were not substantially restricted by Abcb1 or Abcg2. The brain-to-plasma ratios of entospletinib in Abcb1a/b-/-, Abcg2-/- and Abcb1a/b;Abcg2-/- mice were 1.7-, 1.8- and 2.9-fold higher, respectively, compared to those in wild-type mice. For lanraplenib these brain-to-plasma ratios were 3.0-, 1.3- and 10.4-fold higher, respectively. This transporter-mediated restriction of brain penetration for both drugs could be almost fully inhibited by coadministration of the dual ABCB1/ABCG2 inhibitor elacridar, without signs of acute toxicity. Oatp1a/b and human CYP3A4 did not seem to affect the pharmacokinetics of entospletinib and lanraplenib, but mouse Cyp3a may limit lanraplenib plasma exposure. Unexpectedly, entospletinib and lanraplenib increased each other's plasma exposure by 2.6- to 2.9-fold, indicating a significant drug-drug interaction. This interaction was, however, unlikely to be mediated through any of the studied transporters or CYP3A. The obtained insights may perhaps help to further improve the safety and efficacy of entospletinib and lanraplenib.

A multiplex UPLC-MS/MS method for the quantification of three PD-L1 checkpoint inhibitors, atezolizumab, avelumab, and durvalumab, in human serum.

BACKGROUND AND AIM: To support pharmacokinetic studies, a multiplex UPLC-MS/MS assay was developed and validated to quantify PD-L1 checkpoint inhibitors atezolizumab, avelumab, and durvalumab in serum. METHODS: A bottom-up sample pre-treatment procedure was developed to determine atezolizumab, avelumab, and durvalumab in serum. This procedure consisted of (1) precipitation of the monoclonal antibody with ammonium sulfate, (2) reduction with dithiothreitol, (3) denaturation with methanol, and (4) tryptic digestion of the protein. The unique signature peptides resulting after sample pre-treatment of the antibodies were measured using UPLC-MS/MS with a total run time of 11 minutes. The clinical application was evaluated by analyzing 114 atezolizumab patient samples. RESULTS: The developed method was found to be accurate and precise for all three analytes over a concentration range of 3.00-150 µg/mL. No endogenous interference was present in serum samples. Cross-interference experiments showed no cross-analyte interference and acceptable cross-internal standard interference. In addition, no substantial carry-over was observed. The stable isotopically labeled signature peptides were most effective in compensating for matrix effects. Recovery based on back-calculated concentrations of calibration standards and quality control samples was found to be high. The analytes were stable for at least three freeze-thaw cycles, for 42 hours at processing conditions, for at least two days at 2-8°C in the final extract, for five days before re-injection analysis at 4°C, and long-term for at least 11 months at -70°C. The assay was tested for its applicability in clinical practice. For this purpose, 114 atezolizumab patient samples were measured. CONCLUSION: A multiplex UPLC-MS/MS assay was developed and validated to quantify atezolizumab, avelumab, and durvalumab in human serum. The applicability of this method was demonstrated by the analysis of clinical atezolizumab samples. The method is suitable to support clinical pharmacokinetic studies involving atezolizumab, avelumab, or durvalumab.

Impact of loperamide on the pharmacokinetics and tissue disposition of ritonavir-boosted oral docetaxel therapy; a preclinical assessment.

PURPOSE: An oral docetaxel formulation boosted by the Cytochrome P450 (CYP) 3 A inhibitor ritonavir, ModraDoc006/r, is currently under clinical investigation. Based on clinical data, the incidence of grade 1-2 diarrhea is increased with this oral docetaxel formulation compared to the conventional intravenous administration. Loperamide, a frequently used diarrhea inhibitor, could be added to the regimen as symptomatic treatment. However, loperamide is also a substrate of the CYP3A enzyme, which could result in competition between ritonavir and loperamide for this protein. Therefore, we were interested in the impact of coadministered loperamide on the pharmacokinetics of ritonavir-boosted oral docetaxel. METHODS: We administered loperamide simultaneously or with an 8-hour delay to humanized CYP3A4 mice (with expression in liver and intestine) receiving oral ritonavir and docetaxel. Concentrations of docetaxel, ritonavir, loperamide and two of its active metabolites were measured. RESULTS: The plasma exposure (AUC and Cmax) of docetaxel was not altered during loperamide treatment, nor were the ritonavir plasma pharmacokinetics. However, the hepatic and intestinal dispositions of ritonavir were somewhat changed in the simultaneous, but not 8-hour loperamide treatment groups, possibly due to loperamide-induced delayed drug absorption. The pharmacokinetics of loperamide itself did not seem to be influenced by ritonavir. CONCLUSION: These results suggest that delayed loperamide administration can be added to ritonavir-boosted oral docetaxel treatment, without affecting the overall systemic exposure of docetaxel.

Interplay of Ritonavir-Boosted Oral Cabazitaxel with the Organic Anion-Transporting Polypeptide (OATP) Uptake Transporters and Carboxylesterase 1 in Mice.

Intravenously administered chemotherapeutic cabazitaxel is used for palliative treatment of prostate cancer. An oral formulation would be more patient-friendly and reduce the need for hospitalization. We therefore study determinants of the oral pharmacokinetics of cabazitaxel in a ritonavir-boosted setting, which reduces the CYP3A-mediated first-pass metabolism of cabazitaxel. We here assessed the role of organic anion-transporting polypeptides (OATPs) in the disposition of orally boosted cabazitaxel and its active metabolites, using the Oatp1a/b-knockout and the OATP1B1/1B3-transgenic mice. These transporters may substantially affect plasma clearance and hepatic and intestinal drug disposition. The pharmacokinetics of cabazitaxel and DM2 were not significantly affected by Oatp1a/b and OATP1B1/1B3 activity. In contrast, the plasma AUC0-120 min of DM1 in Oatp1a/b-/- was 1.9-fold (p < 0.05) higher than that in wild-type mice, and that of docetaxel was 2.4-fold (p < 0.05) higher. We further observed impaired hepatic uptake and intestinal disposition for DM1 and docetaxel in the Oatp-ablated strains. None of these parameters showed rescue by the OATP1B1 or -1B3 transporters in the humanized mouse strains, suggesting a minimal role of OATP1B1/1B3. Ritonavir itself was also a potent substrate for mOatp1a/b, showing a 2.9-fold (p < 0.0001) increased plasma AUC0-120 min and 3.5-fold (p < 0.0001) decreased liver-to-plasma ratio in Oatp1a/b-/- compared to those in wild-type mice. Furthermore, we observed the tight binding of cabazitaxel and its active metabolites, including docetaxel, to plasma carboxylesterase (Ces1c) in mice, which may complicate the interpretation of pharmacokinetic and pharmacodynamic mouse studies. Collectively, these results will help to further optimize (pre)clinical research into the safety and efficacy of orally applied cabazitaxel.

Analytical Validation of a Volumetric Absorptive Microsampling Method for Therapeutic Drug Monitoring of the Oral Targeted Anticancer Agents, Abiraterone, Alectinib, Cabozantinib, Imatinib, Olaparib, and Sunitinib, and Metabolites.

BACKGROUND: Volumetric Absorptive Microsampling (VAMS) is a useful tool for therapeutic drug monitoring (TDM) of oral targeted anticancer agents. VAMS aims to improve safety and efficacy by enabling at-home blood sample collection by patients. This study aimed to develop and validate an ultra-high performance liquid chromatography-tandem mass spectrometry method for the quantitative determination of abiraterone, alectinib, cabozantinib, imatinib, olaparib, sunitinib, and the metabolites, Δ(4)-abiraterone (D4A), alectinib-M4, imatinib-M1, and N-desethyl sunitinib, in dried whole blood samples using VAMS to support TDM. METHODS: After the collection of 10 µL of whole blood sample using the VAMS device, the analytes were extracted from the tip using methanol with shaking, evaporated, and reconstituted in acetonitrile:0.1 mol/L ammonium hydroxide in water (1:1, vol/vol). The extracts were then analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry. Validation experiments based on the ICH M10 guideline were carried out, and stability was evaluated under shipping and storage conditions. VAMS specimens were collected in the outpatient clinic to demonstrate the applicability of the assay. RESULTS: The validated range of the method was considered accurate and precise for all analytes. Accordingly, the validation experiments met the relevant requirements, except for cross-analyte interference. Based on the stability data, shipment can be performed at room temperature within 14 days after sample collection and the VAMS specimen can be stored up to 9 months at -20 and -70°C. Samples from 59 patients were collected at the hospital. CONCLUSIONS: The developed method could be used to successfully quantify the concentrations of abiraterone, D4A, alectinib, alectinib-M4, cabozantinib, imatinib, imatinib-M1, olaparib, sunitinib, and N-desethyl sunitinib within the validated range using VAMS. Therefore, the method can be used to estimate the dried whole blood-to-plasma ratios for TDM in the clinic.

Ritonavir reverses resistance to docetaxel and cabazitaxel in prostate cancer cells with acquired resistance to docetaxel.

Aim: Docetaxel is a microtubule-stabilizing drug used for the treatment of several cancers, including prostate cancer. Resistance to docetaxel can either occur through intrinsic resistance or develop under therapeutic pressure, i.e., acquired resistance. A possible explanation for the occurrence of acquired resistance to docetaxel is increased drug efflux via P-glycoprotein (P-gp) drug transporters. Methods: We have generated docetaxel-resistant cell lines DU-145DOC10 and 22Rv1DOC8 by exposing parental cell lines DU-145DOC and 22Rv1 to increasing levels of docetaxel. Gene expression levels between DU-145DOC10 and 22Rv1DOC8 were compared with those of their respective originator cell lines. Both parental and resistant cell lines were treated with the taxane drugs docetaxel and cabazitaxel in combination with the P-gp/CYP3A4 inhibitor ritonavir and the P-gp inhibitor elacridar. Results: In the docetaxel-resistant cell lines DU-145DOC10 and 22Rv1DOC8, the ABCB1 (P-gp) gene was highly up-regulated. Expression of the P-gp protein was also significantly increased in the docetaxel-resistant cell lines in a Western blotting assay. The addition of ritonavir to docetaxel resulted in a return of the sensitivity to docetaxel in the DU-145DOC10 and 22Rv1DOC8 to a level similar to the sensitivity in the originator cells. We found that these docetaxel-resistant cell lines could also be re-sensitized to cabazitaxel in a similar manner. In a Caco-2 P-gp transporter assay, functional inhibition of P-gp-mediated transport of docetaxel with ritonavir was demonstrated. Conclusion: Our results demonstrate that ritonavir restores sensitivity to both docetaxel and cabazitaxel in docetaxel-resistant cell lines, most likely by inhibiting P-gp-mediated drug efflux.

Are novel oral oncolytics underdosed in obese patients?

PURPOSE: Data on the effects of obesity on drug exposure of oral targeted oncolytics is scarce. Therefore, the aim of this study was to investigate the influence of body weight and body mass index (BMI) on trough levels of oral oncolytics with an exposure-response relationship. The oral oncolytics of interest were abiraterone, alectinib, cabozantinib, crizotinib, imatinib, pazopanib, sunitinib and trametinib. METHODS: This retrospective cohort study included patients treated with the selected oral oncolytics at the standard dose, with a measured trough level at steady state and with available body weight. The Spearman's correlation test was used to determine the correlation between body weight and trough levels. The Fisher's exact text was used to compare the frequency of inadequate trough levels between BMI categories. RESULTS: 1265 patients were included across the different oral oncolytics. A negative correlation coefficient was observed between weight and trough levels for crizotinib (n = 75), imatinib (n = 201) and trametinib (n = 310), respectively, ρ = - 0.41, ρ = - 0.24 and ρ = - 0.23, all with a p-value < 0.001. For crizotinib, a higher percentage of patients with a body weight > 100 kg had inadequate trough levels. No statistically significant differences were observed in the frequency of inadequate trough levels between BMI categories. CONCLUSION: Higher body weight was only correlated with lower plasma trough levels for crizotinib, imatinib, and trametinib. Therefore, patients with a high body weight may require dose escalation to obtain adequate target levels when treated with these oral oncolytics.

Predictability of human exposure by human-CYP3A4-transgenic mouse models: A meta-analysis.

First-in-human dose predictions are primarily based on no-observed-adverse-effect levels in animal studies. Predictions from these animal models are only as effective as their ability to predict human results. To narrow the gap between human and animals, researchers have, among other things, focused on the replacement of animal cytochrome P450 (CYP) enzymes with their human counterparts (called humanization), especially in mice. Whereas research in humanized mice is extensive, the emphasis has been particularly on qualitative rather than quantitative predictions. Because the CYP3A4 enzyme is most involved in the metabolism of clinically used drugs, most benefit was expected from CYP3A4 models. There are several applications of these mouse models regarding in vivo CYP3A4 functionality, one of which might be their capacity to help improve first-in-human (FIH) dose predictions for CYP3A4-metabolized drugs. To evaluate whether human-CYP3A4-transgenic mouse models are better predictors of human exposure compared to the wild-type mouse model, we performed a meta-analysis comparing both mouse models in their ability to accurately predict human exposure of small-molecule drugs metabolized by CYP3A4. Results showed that, in general, the human-CYP3A4-transgenic mouse model had similar accuracy in the prediction of human exposure compared to the wild-type mouse model, suggesting that there is limited added value in humanization of the mouse Cyp3a enzymes if the primary aim is to acquire more accurate FIH dose predictions. Despite the results of this meta-analysis, corrections for interspecies differences through extension of human-CYP3A4-transgenic mouse models with pharmacokinetic modeling approaches seems a promising contribution to more accurate quantitative predictions of human pharmacokinetics.

Is Higher Docetaxel Clearance in Prostate Cancer Patients Explained by Higher CYP3A? An In Vivo Phenotyping Study with Midazolam.

Patients with prostate cancer (PCa) have a lower docetaxel exposure for both intravenous (1.8-fold) and oral administration (2.4-fold) than patients with other solid cancers, which could influence efficacy and toxicity. An altered metabolism by cytochrome P450 3A (CYP3A) due to castration status might explain the observed difference in docetaxel pharmacokinetics. In this in vivo phenotyping, pharmacokinetic study, CYP3A activity defined by midazolam clearance (CL) was compared between patients with PCa and male patients with other solid tumors. All patients with solid tumors who did not use CYP3A-modulating drugs were eligible for participation. Patients received 2 mg midazolam orally and 1 mg midazolam intravenously on 2 consecutive days. Plasma concentrations were measured with a validated liquid chromatography-tandem mass spectrometry method. Genotyping was performed for CYP3A4 and CYP3A5. Nine patients were included in each group. Oral midazolam CL was 1.26-fold higher in patients with PCa compared to patients with other solid tumors (geometric mean [coefficient of variation], 94.1 [33.5%] L/h vs 74.4 [39.1%] L/h, respectively; P = .08). Intravenous midazolam CL did not significantly differ between the 2 groups (P = .93). Moreover, the metabolic ratio of midazolam to 1'-hydroxy midazolam did not differ between the 2 groups for both oral administration (P = .67) and intravenous administration (P = .26). CYP3A4 and CYP3A5 genotypes did not influence midazolam pharmacokinetics. The observed difference in docetaxel pharmacokinetics between both patient groups therefore appears to be explained neither by a difference in midazolam CL nor by a difference in metabolic conversion rate of midazolam.

Clinical Relevance of High Plasma Trough Levels of the Kinase Inhibitors Crizotinib, Alectinib, Osimertinib, Dabrafenib, and Trametinib in NSCLC Patients.

BACKGROUND: the study aims to evaluate whether high plasma trough levels of the kinase inhibitors (K.I.s) crizotinib, alectinib, osimertinib, dabrafenib, and trametinib were associated with a higher risk of toxicity in non-small-cell lung cancer patients. METHODS: In this retrospective cohort study, patients with non-small-cell lung cancer treated with the selected K.I.s were included if at least one plasma trough level at steady state (C min,ss ) was available. Data were extracted from electronic medical records and laboratory databases. The high group for each K.I. was defined as 10% of patients with the highest first C min,ss . The remaining patients were placed in the non-high group. The frequency of dose-limiting toxicities (DLTs), defined as adverse events leading to dose reduction, dose interruption, or permanent discontinuation, was compared between the 2 groups. RESULTS: A total of 542 patients were included in the different K.I. groups. A high C min,ss of crizotinib (n = 96), alectinib (n = 105), osimertinib (n = 227), dabrafenib (n = 52), and trametinib (n = 62) correlated with a C min,ss ≥490, ≥870, ≥405, ≥150, and ≥25 ng/mL, respectively. DLTs were more common in the alectinib high group than in the alectinib non-high group (64% vs. 29%, P = 0.036). Liver toxicity was observed in 4 (36%) patients in the high group and 5 (5%) patients in the non-high group ( P = 0.007). For other K.I.s, no significant differences were observed in the frequency of DLTs between the high and non-high groups. CONCLUSIONS: For alectinib, high C min,ss was correlated with a higher risk of DLT. No differences in the frequency of DLTs were observed between the high and non-high groups for crizotinib, osimertinib, dabrafenib, and trametinib.

Development and validation of an LC-MS/MS method for the quantification of KRASG12C inhibitor opnurasib in several mouse matrices and its application in a pharmacokinetic mouse study.

Opnurasib (JDQ-443) is a highly potent and promising KRASG12C inhibitor that is currently under clinical investigation. Results of the ongoing clinical research demonstrated the acceptable safety profile and clinical activity of this drug candidate as a single agent for patients with NSCLC harboring KRASG12C mutations. In this early stage of development, a deeper insight into pharmacokinetic properties in both preclinical and clinical investigations of this drug is very important. Thus, a reliable quantification method is required. To date, no quantitative bioanalytical assay of opnurasib was publicly available. In this study we present a validated assay to quantify opnurasib in mouse plasma and eight mouse tissue-related matrices utilizing liquid chromatography-tandem mass spectrometry. Erlotinib was used as internal standard and acetonitrile was utilized to treat 10 µl of the sample with protein precipitation in a 96-well plate format. Separation and detection were achieved using a BEH C18 column under basic chromatographic conditions and a triple quadrupole mass spectrometer, respectively. We have fully validated this assay for mouse plasma and partially for eight mouse tissue-related matrices over the range of 2-2000 ng/ml. The accuracy and precision of the assay fulfilled international guidelines (EMA & U.S. FDA) over the validated range. The method was proven selective and sensitive to quantify opnurasib down to 2 ng/ml in all investigated matrices. The recoveries of both analyte and internal standard in mouse plasma were ∼100 % with no significant matrix effect in any of the matrices. Opnurasib in mouse plasma was stable up to 12 h at room temperature, and up to 8 h at room temperature in tissue homogenates (except for kidney up to 4 h). This presented method has been successfully applied to quantify opnurasib in preclinical samples from a mouse study and demonstrated its usability to support preclinical pharmacokinetic studies.

Coadministration of ABCB1/P-glycoprotein inhibitor elacridar improves tissue distribution of ritonavir-boosted oral cabazitaxel in mice.

Developing an oral formulation for the chemotherapeutic cabazitaxel might improve its patient-friendliness, costs, and potentially exposure profile. Cabazitaxel oral availability is restricted by CYP3A-mediated first-pass metabolism, but can be substantially boosted with the CYP3A inhibitor ritonavir. We here tested whether adding the ABCB1/P-glycoprotein inhibitor elacridar to ritonavir-boosted oral cabazitaxel could further improve its tissue exposure using wild-type, CYP3A4-humanized and Abcb1a/b-/- mice. The plasma AUC0-2h of cabazitaxel was increased 2.3- and 1.9-fold in the ritonavir- and ritonavir-plus-elacridar groups of wild-type, and 10.5- and 8.8-fold in CYP3A4-humanized mice. Elacridar coadministration did not influence cabazitaxel plasma exposure. The brain-to-plasma ratio of cabazitaxel was not increased in the ritonavir group, 7.3-fold in the elacridar group and 13.4-fold in the combined booster group in wild-type mice. This was 0.4-, 4.6- and 3.6-fold in CYP3A4-humanized mice, illustrating that Abcb1 limited cabazitaxel brain exposure also during ritonavir boosting. Ritonavir itself was also a potent substrate for the Abcb1 efflux transporter, limiting its oral availability (3.3-fold) and brain penetration (10.6-fold). Both processes were fully reversed by elacridar. The tissue disposition of ritonavir-boosted oral cabazitaxel could thus be markedly enhanced by elacridar coadministration without affecting the plasma exposure. This approach should be verified in selected patient populations.

Severe Immune-Related Enteritis after In Utero Exposure to Pembrolizumab.

Immune checkpoint blockade has become standard treatment for many types of cancer. Such therapy is indicated most often in patients with advanced or metastatic disease but has been increasingly used as adjuvant therapy in those with early-stage disease. Adverse events include immune-related organ inflammation resembling autoimmune diseases. We describe a case of severe immune-related gastroenterocolitis in a 4-month-old infant who presented with intractable diarrhea and failure to thrive after in utero exposure to pembrolizumab. Known causes of the symptoms were ruled out, and the diagnosis of pembrolizumab-induced immune-related gastroenterocolitis was supported by the results of histopathological assays, immunophenotyping, and analysis of the level of antibodies against programmed cell death protein 1 (PD-1). The infant's condition was successfully treated with prednisolone and infliximab.

Physiologically-based pharmacokinetic model to predict doxorubicin and paclitaxel exposure in infants through breast milk.

Limited information is available concerning infant exposure and safety when breastfed by mothers receiving chemotherapy. Whereas defining distribution to breast milk is important to infer drug exposure, infant pharmacokinetics also determine to what extent the infant will be exposed to potential toxic effects. We aimed to assess the impact of chemotherapy containing breast milk on infants by predicting systemic and local (intestinal) exposure of paclitaxel and doxorubicin in infants through breast milk using a physiologically-based pharmacokinetic (PBPK) approach. Whole-body PBPK models of i.v. paclitaxel and doxorubicin were extended from the literature, with an oral absorption component to enable predictions in infants receiving paclitaxel or doxorubicin-containing breast milk. For safety considerations, worst-case scenarios were explored. Finally, paclitaxel and doxorubicin exposures in plasma and intestinal tissue of infants following feeding of breast milk from paclitaxel- or doxorubicin-treated mothers were simulated and breast milk discarding strategies were evaluated. The upper 95th percentile of the predicted peak concentrations in peripheral venous blood were 3.48 and 0.74 nM (0.4%-1.7% and 0.1%-1.8% of on-treatment) for paclitaxel and doxorubicin, respectively. Intestinal exposure reached peak concentrations of 1.0 and 140 µM for paclitaxel and doxorubicin, respectively. Discarding breast milk for the first 3 days after maternal chemotherapy administration reduced systemic and tissue exposures even further, to over 90% and 80% for paclitaxel and doxorubicin, respectively. PBPK simulations of chemotherapy exposure in infants after breastfeeding with chemotherapy containing breast milk suggest that particularly local gastrointestinal adverse events should be monitored, whereas systemic adverse events are not expected.

Cortisol as Biomarker for CYP17-Inhibition is Associated with Therapy Outcome of Abiraterone Acetate.

BACKGROUND: Abiraterone acetate is an irreversible 17α-hydroxylase/C17, 20-lyase (CYP17) inhibitor approved for the treatment of metastatic castration-resistant prostate cancer (mCRPC) patients. Inhibition of this enzyme leads to low testosterone and cortisol levels in blood. There is growing evidence that clinical efficacy of abiraterone is related to the rate of suppression of serum testosterone. However, quantification of very low levels of circulating testosterone is challenging. We therefore aimed to investigate whether circulating cortisol levels could be used as a surrogate biomarker for CYP17 inhibition in patients with mCRPC treated with abiraterone acetate. PATIENTS AND METHODS: mCRPC patients treated with abiraterone acetate were included. Abiraterone and cortisol levels were measured with a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS). On treatment cortisol and abiraterone concentrations were related to treatment response and progression free survival. RESULTS: In total 117 patients were included with a median cortisol concentration of 1.13 ng/ml (range: 0.03 - 82.2) and median abiraterone trough concentration (Cmin) of 10.2 ng/ml (range: 0.58 - 92.1). In the survival analyses, abiraterone Cmin ≥ 8.4 ng/mL and cortisol < 2.24 ng/mL were associated with a longer prostate-specific antigen (PSA) independent progression-free survival than patients with an abiraterone concentration ≥ 8.4 ng/mL and a cortisol concentration ≥ 2.24 ng/mL (13.8 months vs. 3.7 months). CONCLUSION: Our study shows that cortisol is not an independent predictor of abiraterone response in patients with mCRPC, but it is of added value in combination with abiraterone levels, to predict a response on abiraterone.

High accumulation of nivolumab in human breast milk: A case report.

Nivolumab is an immunotherapeutic monoclonal antibody (mAb) that is used for the treatment of several types of cancer. The evidence on its use during lactation is lacking. Here, we report on a 39-year-old woman with metastasized melanoma who was treated with 480 mg nivolumab every four weeks during lactation. Breast milk samples were collected over the course of 34 days, including two cycles of nivolumab. The highest measured concentration of nivolumab during the first cycle was 503 ng/mL at day 13. The cumulative relative infant dose (RID) over the first cycle (28 days) was 9.8 %. The highest overall measured nivolumab concentration was 519 ng/mL at day 33, five days after administration of the second nivolumab cycle. Nivolumab seems to accumulate in breast milk over two consecutive cycles, hence the RIDs of consecutive cycles are expected to be higher. To draw further conclusions regarding safety of breastfeeding during nivolumab therapy, more information about the oral bioavailability of nivolumab in newborns, the nivolumab steady-state concentrations in breast milk and its pharmacodynamic effects are needed.

Survival of Patients With Cancer With DPYD Variant Alleles and Dose-Individualized Fluoropyrimidine Therapy-A Matched-Pair Analysis.

PURPOSE: DPYD-guided fluoropyrimidine dosing improves patient safety in carriers of DPYD variant alleles. However, the impact on treatment outcome in these patients is largely unknown. Therefore, progression-free survival (PFS) and overall survival (OS) were compared between DPYD variant carriers treated with a reduced dose and DPYD wild-type controls receiving a full fluoropyrimidine dose in a retrospective matched-pair survival analysis. METHODS: Data from a prospective multicenter study (ClinicalTrials.gov identifier: NCT02324452) in which DPYD variant carriers received a 25% (c.1236G>A and c.2846A>T) or 50% (DPYD*2A and c.1679T>G) reduced dose and data from DPYD variant carriers treated with a similarly reduced dose of fluoropyrimidines identified during routine clinical care were obtained. Each DPYD variant carrier was matched to three DPYD wild-type controls treated with a standard dose. Survival analyses were performed using Kaplan-Meier estimates and Cox regression. RESULTS: In total, 156 DPYD variant carriers and 775 DPYD wild-type controls were available for analysis. Sixty-one c.1236G>A, 25 DPYD*2A, 13 c.2846A>T, and-when pooled-93 DPYD variant carriers could each be matched to three unique DPYD wild-type controls. For pooled DPYD variant carriers, PFS (hazard ratio [HR], 1.23; 95% CI, 1.00 to 1.51; P = .053) and OS (HR, 0.95; 95% CI, 0.75 to 1.51; P = .698) were not negatively affected by DPYD-guided dose individualization. In the subgroup analyses, a shorter PFS (HR, 1.43; 95% CI, 1.10 to 1.86; P = .007) was found in c.1236G>A variant carriers, whereas no differences were found for DPYD*2A and c.2846A>T carriers. CONCLUSION: In this exploratory analysis, DPYD-guided fluoropyrimidine dosing does not negatively affect PFS and OS in pooled DPYD variant carriers. Close monitoring with early dose modifications based on toxicity is recommended, especially for c.1236G>A carriers receiving a reduced starting dose.

Pharmacokinetics of the KRASG12C inhibitor adagrasib is limited by CYP3A and ABCB1, and influenced by binding to mouse plasma carboxylesterase 1c.

Adagrasib (Krazati™) is the second FDA-approved specific KRASG12C inhibitor for non-small cell lung cancer (NSCLC) patients harboring this mutation. The impact of the drug efflux transporters ABCB1 and ABCG2, and the drug-metabolizing enzymes CYP3A and carboxylesterase 1 (CES1) on the pharmacokinetics of oral adagrasib were studied using genetically modified mouse models. Adagrasib was potently transported by human ABCB1 and modestly by mouse Abcg2 in vitro. In Abcb1a/b-/- and Abcb1a/b;Abcg2-/- mice, the brain-to-plasma ratios were enhanced by 33- and 55-fold, respectively, compared to wild-type mice, whereas ratios in Abcg2-/- mice remained unchanged. The influence of ABC transporters was completely reversed by coadministration of the dual ABCB1/ABCG2 inhibitor elacridar, increasing the brain penetration in wild-type mice by 41-fold while no signs of acute CNS toxicity were observed. Tumor ABCB1 overexpression may thus confer adagrasib resistance. Whereas the ABC transporters did not affect adagrasib plasma exposure, CYP3A and Ces1 strongly impacted its apparent oral availability. The plasma AUC0-8 h was significantly enhanced by 2.3-fold in Cyp3a-/- compared to wild-type mice, and subsequently 4.3-fold reduced in transgenic CYP3A4 mice, indicating substantial CYP3A-mediated metabolism. Adagrasib plasma exposure was strongly reduced in Ces1-/- compared to wild-type mice, but tissue exposure was slightly increased, suggesting that adagrasib binds to plasma Ces1c in mice and is perhaps metabolized by Ces1. This binding could complicate interpretation of mouse studies, especially since humans lack circulating CES1 enzyme(s). Our results may be useful to further optimize the clinical safety and efficacy of adagrasib, and give more insight into potential drug-drug interactions risks.