Adibelivir (IM-250) is a novel helicase-primase inhibitor under development for the treatment of herpes simplex virus infections. A sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay was established for the quantification of adibelivir in human plasma and urine samples of a first-in-human clinical trial using a deuterated internal standard. This assay was validated based on the pertinent ICH M10 guideline for the linear range of 1-1000 ng/mL. The method fulfilled all criteria with a high inter-run accuracy of 100.0-101.4% and inter-run precision of 2.96-10.5%. The feasibility of the assay was shown for two healthy individuals by evaluating their pharmacokinetics of adibelivir. Additionally, the assay was adapted for the quantification of adibelivir in urine with a lowered calibration range of 0.1-100 ng/mL. The present work provides a fully validated assay for pharmacokinetic analyses of adibelivir in clinical development, e.g. in its first-in-human phase I trial (EudraCT 2022-003679-40).

The blood-brain barrier (BBB) paracellular permeability must be evaluated in various contexts in vitro and in vivo, including pharmacological evaluation of drug candidates, investigations of pathological changes in disease and model development. However, most available paracellular marker substances are either radioactive, lack sensitivity and thereby require large sample volumes, or they can influence the BBB themselves via osmotic pressure. Moreover, in drug permeability studies, an adequate paracellular marker should be detectable in the same sample as the compound being tested, ideally applying the same analytical technology. We rationally designed a novel permeability marker to be highly sensitively measurable with ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS); to be not a potential substrate of solute carriers or transporters; stable against enzymatic digestion and well tolerated. We evaluated this novel permeability marker in vitro in Transwell® models with four different cell types (MDCK II, hCMEC/D3, primary and from induced pluripotent stem cells derived brain capillary endothelial cells) and cross-validated these results with known fluorescence markers. We further analyzed its in vivo pharmacokinetics and brain-to-plasma ratio in healthy Swiss mice. Possible interactions with LAT-1 and PepT1 were evaluated with uptake and/or inhibition assays. Based on non-canonical and ᴅ-dipeptide structure, the developed marker ᴅ-methyl-tyrosinyl-ᴅ-ornithine (ᴅ-Tyr(Me)Orn) is hydrophilic and with low molecular weight (309 Da), it enables a low endogenous background and is readily available through peptide synthesis, also as isotopically labeled derivative. The developed UPLC-MS/MS quantification assay allows a highly sensitive measurement in different matrices (lower limit of quantification: 0.05 ng/mL cell medium, lysed cells, 0.1 ng/mL mouse plasma; 3 ng/g, mouse brain). In the well-known MDCK II Transwell® model, it revealed a suitable Papp of 5.13 ± 1.08 × 10− 7 cm/s. Using the marker ᴅ-Tyr(Me)Orn it was further possible to compare the tightness of three brain capillary endothelial cell models. The time dependent distribution and in vivo pharmacokinetics was determined in healthy Swiss mice and revealed a constantly low brain concentration and brain-to-plasma ratio. We developed a highly feasible new paracellular marker, easily adaptable in various experimental designs.

For the toxicokinetic evaluation of a lipid conjugate of the cell-penetrating peptide cyclic nonaarginine, we aimed to develop a highly sensitive plasma quantification. Due to the amphiphilic properties and high number of basic amino acids, plasma bioanalysis with LC-MS/MS instruments is difficult. Challenges in particular include chromatographic characteristics, minimal extraction recovery, and resistance against collision-induced dissociation. We developed a quantification of a nonextractable CPP-lipid conjugate by eliminating the lipid part by phospholipase D digestion, enabling the efficient extraction of a surrogate peptide for analysis. Chromatographic separation was feasible only with trifluoroacetic acid (TFA)-based mobile phases. Ion suppression caused by TFA was reversed by postcolumn addition of aqueous ammonia. Efficient extraction of the surrogate peptide fragment was achieved by protein precipitation with TFA. This enabled the highly sensitive quantification of the CPP-lipid conjugate in plasma in the low picomolar range (lower limit of quantification of 0.1 ng/mL; 34 pM). The assay was validated according to the pertinent recommendations of the ICH M10 guideline on bioanalytical method validation and applied to the determination of the intravenous pharmacokinetics of the CPP-lipid conjugate in Beagle dogs. Applicability was further demonstrated by validation of the assay for human plasma. The established strategy can be used as a general approach to the bioanalysis of amphiphilic lipid conjugates by using lipase digestion to generate extractable surrogate fragments. Additionally, TFA-based UPLC-MS/MS analysis with desolvation of TFA adducts by postcolumn addition of ammonia is feasible for the regulated, highly sensitive quantification of arginine-rich peptides.

Background/Objectives: Bempedoic acid (BA) is a novel cholesterol-lowering agent with proven positive effects on cardiovascular endpoints. Because it is an inhibitor of the hepatic transporters OATP1B1 and OATP1B3, two uptake transporters regulating the intrahepatic availability of statins, it increases the systemic exposure of co-administered statins. This interaction could raise the risk of myopathy. We hypothesized that the drug interaction between BA and statins could be mitigated by staggered administration. Methods: This was a single-centre, open-label, randomized, two-arm, cross-over, phase I drug interaction trial in healthy volunteers (EudraCT-No: 2022-001096-13). The primary objective was to evaluate the OATP1B1 inhibitory effect of BA on exposure to pravastatin after simultaneous administration versus different schedules of staggered administration. A secondary objective was to evaluate the impact of SLCO1B1 genotypes (*1, *5, *15, *37) on pravastatin exposure. Pravastatin was administered in single oral doses of 40 mg at six visits. After a baseline visit with pravastatin alone, BA was dosed to steady state at the approved oral dose of 180 mg. Outcome measures were the area under the plasma concentration–time curve, extrapolated to infinity (AUC∞) and Cmax of pravastatin, 3α-hydroxy-pravastatin (pravastatin 3-iso), and pravastatin lactone, and their geometric mean ratios (GMRs) of different schedules of administration. Log-transformed AUC∞ and Cmax were compared with one-way ANOVA with a 90% confidence interval (CI). Results: Fourteen participants completed all visits. At BA steady state, the GMRs of pravastatin AUC∞ and Cmax were 1.80 (90% CI 1.31–2.46) and 1.95 (90% CI 1.40–2.72), respectively, compared to baseline. There was no significant difference in pravastatin exposure between simultaneous vs. staggered administration. There was no statistically significant difference in pravastatin 3-iso or pravastatin lactone between different administration modes. For the AUC∞ of pravastatin and pravastatin 3-iso, haplotype was a significant source of variation (63% and 20%, respectively), while the type of administration (simultaneous vs. staggered) had no significant impact. Conclusions: The increase in pravastatin exposure with concomitant intake of BA was larger than expected. There was no significant difference between simultaneous vs. staggered administration of pravastatin and BA, possibly due to a population that was heterogenous in SLCO1B1 haplotypes.

Lipid-based drug delivery systems can be surface-modified by lipid conjugates of the substance in question. The most important modifications include arginine-rich cell-penetrating peptides (CPP). A toxicokinetic evaluation of these lipid conjugates is im-portant during preclinical and clinical development of drug delivery formulations. Due to their amphiphilic properties and high number of basic amino acid residues, lipid conjugates of CPP exhibit difficult characteristics in regard to their plasma bioanalysis with LC-MS/MS instruments. These especially include challenging chromatography with extensive carry-over and minimal extrac-tion recovery, and, due to large numbers of basic amino acids and the resulting immobility of protons, resistance against collision-induced dissociation. We developed a surrogate quantification of a CPP-lipid conjugate relying on elimination of the lipid part by phospholipase D digestion. Chromatographic separation was only feasible with trifluoro acetic acid (TFA)-based mobile phases. Ion suppression caused by TFA was reversed by post-column addition of aqueous ammonia. Efficient extraction of the surrogate peptide fragment was achieved by protein precipitation with TFA. This enabled the highly sensitive quantification of the CPP-lipid conjugate in plasma in the low picomolar range (lower limit of quantification of 0.1 ng/mL; 34 pM). The assay was validated ac-cording to the pertinent recommendations of the ICH M10 guideline on bioanalytical method validation and applied to the deter-mination of the intravenous pharmacokinetics of the CPP-lipid conjugate in Beagle dogs. The established strategy can be used as a general approach to the bioanalysis of amphiphilic lipid conjugates and especially the TFA-based UPLC-MS/MS analysis with post-column desolvation of TFA adducts by ammonia is a feasible approach for the highly sensitive quantification of arginine-rich peptides and other related substances with challenging chromatographic characteristics.

Drug efflux transporters of the ATP-binding-cassette superfamily play a major role in the availability and concentration of drugs at their site of action. ABCC2 (MRP2) and ABCG2 (BCRP) are among the most important drug transporters that determine the pharmacokinetics of many drugs and whose overexpression is associated with cancer chemoresistance. ABCC2 and ABCG2 expression is frequently altered during treatment, thus influencing efficacy and toxicity. Currently, there are no routine approaches available to closely monitor transporter expression. Here, we developed and validated a UPLC-MS/MS method to quantify ABCC2 and ABCG2 in extracellular vesicles (EVs) from cell culture and plasma. In this way, an association between ABCC2 protein levels and transporter activity in HepG2 cells treated with rifampicin and hypericin and their derived EVs was observed. Although ABCG2 was detected in MCF7 cell-derived EVs, the transporter levels in the vesicles did not reflect the expression in the cells. An analysis of plasma EVs from healthy volunteers confirmed, for the first time at the protein level, the presence of both transporters in more than half of the samples. Our findings support the potential of analyzing ABC transporters, and especially ABCC2, in EVs to estimate the transporter expression in HepG2 cells.

Tacrolimus is metabolized by cytochrome P450 3A (CYP3A) and is susceptible to interactions with the CYP3A and P-glycoprotein inducer St. John's Wort (SJW). CYP3A isozymes are predominantly expressed in the small intestine and liver. Prolonged-release tacrolimus (PR-Tac) is largely absorbed in distal intestinal segments and is less susceptible to CYP3A inhibition. The effect of induction by SJW is unknown. In this randomized, crossover trial, 18 healthy volunteers received single oral tacrolimus doses (immediate-release [IR]-Tac or PR-Tac, 5 mg each) alone and during induction by SJW. Concentrations were quantified using ultra-high performance liquid chromatography coupled with tandem mass spectrometry and non-compartmental pharmacokinetics were evaluated. SJW decreased IR-Tac exposure (area under the concentration-time curve) to 73% (95% confidence interval 60%-88%) and maximum concentration (C-max) to 61% (52%-73%), and PR-Tac exposure to 67% (55%-81%) and C-max to 69% (58%-82%), with no statistical difference between the 2 formulations. The extent of interaction appeared to be less pronounced in volunteers with higher baseline CYP3A4 activity and in CYP3A5 expressors. In contrast to CYP3A inhibition, CYP3A induction by SJW showed a similar extent of interaction with both tacrolimus formulations. A higher metabolic baseline capacity appeared to attenuate the extent of induction by SJW.

Metformin is the gold standard substrate for evaluating potential inhibitors of the organic cation transporters (OCTs). Here, we established a UPLC-MS/MS assay to quantify metformin in cell pellets with a range of 0.05–50 ng/mL using 6-deuterated metformin as an internal standard. We used an ion-pairing chromatographic approach with heptafluorobutyric acid, making use of a reverse-phase column, and overcame the associated ion-suppression via previously established post-column injection of aqueous ammonia. The assay was validated according to the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) recommendations for bioanalytical methods. The established extraction procedure was simple, very fast and ensured almost 100% recovery of the analyte. The exceptionally sharp peak form and retention of the ion-pairing chromatography are superior to other methods and allow us to measure as sensitively as 0.05 ng/mL. We used the herein established and validated method to develop a cellular OCT inhibition assay by using metformin as a substrate and human embryonic kidney cells (HEK) overexpressing the OCTs 1-3. The method presented may be useful for identifying new OCT inhibitors, but also for drug–drug interactions and other pharmacokinetic studies, where accurate quantification of low metformin amounts in relevant tissues is mandatory.

The most important dose-limiting factor of the anthracycline idarubicin is the high risk of cardiotoxicity, in which the secondary alcohol metabolite idarubicinol plays an important role. It is not yet clear which enzymes are most important for the formation of idarubicinol and which inhibitors might be suitable to suppress this metabolic step and thus would be promising concomitant drugs to reduce idarubicin-associated cardiotoxicity. We, therefore, established and validated a mass spectrometry method for intracellular quantification of idarubicin and idarubicinol and investigated idarubicinol formation in different cell lines and its inhibition by known inhibitors of the aldo–keto reductases AKR1A1, AKR1B1, and AKR1C3 and the carbonyl reductases CBR1/3. The enzyme expression pattern differed among the cell lines with dominant expression of CBR1/3 in HEK293 and MCF-7 and very high expression of AKR1C3 in HepG2 cells. In HEK293 and MCF-7 cells, menadione was the most potent inhibitor (IC 50 = 1.6 and 9.8 µM), while in HepG2 cells, ranirestat was most potent (IC 50 = 0.4 µM), suggesting that ranirestat is not a selective AKR1B1 inhibitor, but also an AKR1C3 inhibitor. Over-expression of AKR1C3 verified the importance of AKR1C3 for idarubicinol formation and showed that ranirestat is also a potent inhibitor of this enzyme. Taken together, our study underlines the importance of AKR1C3 and CBR1 for the reduction of idarubicin and identifies potent inhibitors of metabolic formation of the cardiotoxic idarubicinol, which should now be tested in vivo to evaluate whether such combinations can increase the cardiac safety of idarubicin therapies while preserving its efficacy.

Background Pediatric low-grade gliomas (pLGG) are the most common pediatric central nervous system tumors, with driving alterations typically occurring in the MAPK pathway. The ERK1/2 inhibitor ulixertinib (BVD-523) has shown promising responses in adult patients with mitogen-activated protein kinase (MAPK)-driven solid tumors. Methods We investigated the antitumoral activity of ulixertinib monotherapy as well as in combination with MEK inhibitors (MEKi), BH3-mimetics, or chemotherapy in pLGG. Patient-derived pLGG models reflecting the two most common alterations in the disease, KIAA1549:BRAF-fusion and BRAFV600E mutation (DKFZ-BT66 and BT40, respectively) were used for in vitro and in vivo (zebrafish embryos and mice) efficacy testing. Results Ulixertinib inhibited MAPK pathway activity in both models, and reduced cell viability in BT40 with clinically achievable concentrations in the low nanomolar range. Combination treatment of ulixertinib with MEKi or BH3-mimetics showed strong evidence of antiproliferative synergy in vitro. Ulixertinib showed on-target activity in all tested combinations. In vivo, sufficient penetrance of the drug into brain tumor tissue in concentrations above the in vitro IC50 and reduction of MAPK pathway activity was achieved. In a preclinical mouse trial, ulixertinib mono- and combined therapies slowed tumor growth and increased survival. Conclusions These data indicate a high clinical potential of ulixertinib for the treatment of pLGG and strongly support its first clinical evaluation in pLGG as single agent and in combination therapy in a currently planned international phase I/II umbrella trial.

Supplementary Figure S1: Western blot analysis of protein lysates derived from HNO97 Cells; Supplementary Figure S2: ITGαVβ6 expression in vitro and in situ; Supplementary Figure S3: Binding properties of SFPF-10; Supplementary Figure S4: Evaluation of 125I-SFITGv6 binding to breast- and colon carcinoma cell lines and the internalization and efflux of 125I-SFITGv6 from HNO97 cells; Supplementary Figure S5: Histochemical peptide staining of brain metastasis; Supplementary Figure S6: Histochemical peptide staining of carcinoma-free lymph nodes.

S1: Peptide sequences of 16 phage-derived clones; S2: Tumor-to-Tissue Ratio of 177Lu-DOTA-SFITGv6 in HNO97 xenografts; S3: Maximum Standard Uptake values (SUVmax) for PET/CT with 18F-FDG and the 68Ga-DOTA-SFITGv6 for one HNSCC patient; S4: Maximum Standard Uptake values (SUVmax) for PET/CT with 18F-FDG and the 68Ga-DOTA-SFITGv6 for one NSCLC patient

Description of methods, results, legends of supplementary figures

Compared to rifampicin (600 mg/day), standard doses of rifabutin (300 mg/day) have a lower risk of drug–drug interactions due to induction of cytochrome P450 3A4 (CYP3A4) or P-glycoprotein (Pgp/ABCB1) mediated by the pregnane X receptor (PXR). However, clinical comparisons with equal rifamycin doses or in vitro experiments respecting actual intracellular concentrations are lacking. Thus, the genuine pharmacological differences and the potential molecular mechanisms of the discordant perpetrator effects are unknown. Consequently, the cellular uptake kinetics (mass spectrometry), PXR activation (luciferase reporter gene assays), and impact on CYP3A4 and Pgp/ABCB1 expression and activity (polymerase chain reaction, enzymatic assays, flow cytometry) were evaluated in LS180 cells after treatment with different rifampicin or rifabutin concentrations for variable exposure times and eventually normalized to actual intracellular concentrations. In addition, inhibitory effects on CYP3A4 and Pgp activities were investigated. While rifampicin is poorly taken up by LS180 cells, it strongly activates PXR and leads to enhanced expression and activity of CYP3A4 and Pgp. In contrast, rifabutin is a significantly less potent and less efficient PXR activator and gene inducer, despite sixfold to eightfold higher intracellular accumulation. Finally, rifabutin is a potent inhibitor of Pgp (IC50 = 0.3 µM) compared to rifampicin (IC50 = 12.9 µM). Together, rifampicin and rifabutin significantly differ by their effects on the regulation and function of CYP3A4 and Pgp, even when controlled for intracellular concentrations. Rifabutin’s concurrent Pgp inhibitory action might partly compensate the inducing effects, explaining its weaker clinical perpetrator characteristics.

Purpose Early antiviral treatment with nirmatrelvir/ritonavir is recommended for SARS-CoV-2-infected patients at high risk for severe courses. Such patients are usually chronically ill and susceptible to adverse drug interactions caused by ritonavir. We investigated the interactions of short-term low-dose ritonavir therapy with atorvastatin and rosuvastatin, two statins commonly used in this population. Method We assessed exposure changes (area under the concentration–time curve (AUC ∞ ) and maximum concentration ( C max )) of a single dose of 10 mg atorvastatin and 10 mg rosuvastatin before and on the fifth day of ritonavir treatment (2 × 100 mg/day) in healthy volunteers and developed a semi-mechanistic pharmacokinetic model to estimate dose adjustment of atorvastatin during ritonavir treatment. Results By the fifth day of ritonavir treatment, the AUC ∞ of atorvastatin increased 4.76-fold and C max 3.78-fold, and concurrently, the concentration of atorvastatin metabolites decreased to values below the lower limit of quantification. Pharmacokinetic modelling indicated that a stepwise reduction in atorvastatin dose during ritonavir treatment with a stepwise increase up to 4 days after ritonavir discontinuation can keep atorvastatin exposure within safe and effective margins. Rosuvastatin pharmacokinetics were only mildly modified; ritonavir significantly increased the C max 1.94-fold, while AUC ∞ was unchanged. Conclusion Atorvastatin doses should likely be adjusted during nirmatrelvir/ritonavir treatment. For patients on a 20-mg dose, we recommend half of the original dose. In patients taking 40 mg or more, a quarter of the dose should be taken until 2 days after discontinuation of nirmatrelvir/ritonavir. Patients receiving rosuvastatin do not need to change their treatment regimen. Trial Registration EudraCT number: 2021–006634-39. DRKS00027838.

Oral delivery of peptides is severely limited by their instability and poor absorption in the gastrointestinal tract. In contrast to coadministration strategies using medium‐chain fatty acids, which have recently gained regulatory approval with low oral bioavailabilities ≤ 1% (Rybelsus and Mycapssa), efforts to clinically implement delivery systems based on nanocarriers have not been successful to date. The approved drug‐delivery formulations show fairly accurate correlation between clinical results and nonrodent mammal bioavailability, including Beagle dogs for Rybelsus, indicating that Beagle dogs represent a translationally relevant model. Here, a nanocarrier formulation for the oral administration of peptide therapeutics is reported with systemic targets consisting of liposomes decorated with cyclic cell‐penetrating peptides, which significantly increase oral bioavailability in translationally relevant Beagle dogs. This nanocarrier formulation is optimized using the glycopeptide vancomycin, and results in a considerable oral bioavailability of 3.9%. Further, this nanocarrier system increases the oral bioavailability of the large linear peptide therapeutic exenatide 20‐fold, and consistently achieves effective plasma concentrations in Beagle dogs.

The authors would like to make the following corrections to the publication [...].

The calcium-dependent serine endoprotease PACE4 is evaluated as a therapeutic target for prostate cancer. The peptide Ac-[d-Leu]LLLRVK-amba inhibits PACE4 with high affinity and has shown efficacy in preclinical mice xenograft models of prostate cancer. To support in vivo examinations of the potential therapeutic peptide Ac-[d-Leu]LLLRVK-amba, we established a highly sensitive assay for its quantification in mouse whole blood microsamples based on UPLC-MS/MS determination. Ac-[d-Leu]LLLRVK-amba was very labile during sample processing, which was particularly pronounced in plasma. High resolution mass spectrometric investigations of the metabolism/degradation in plasma revealed that no peptide bond hydrolysis generated products were formed, leaving the cause of the observed consumption of the peptide elusive. As a consequence, whole-blood quantification was developed relying on the immediate snap-freezing of blood samples after collection and immediate sample processing after serial thawing to ensure accurate and reliable quantification. The assay was validated according to the applicable recommendations of the FDA and EMA in a range of 10–10,000 ng/mL and applied to determine the pharmacokinetics of Ac-[d-Leu]LLLRVK-amba after intravenous and intraperitoneal administration to mice. Individual pharmacokinetic profiles were assessed using four microsamplings per animal. Intraperitoneal absorption was found to be efficient, demonstrating that this well-manageable route of administration is feasible for preclinical efficacy experiments with Ac-[d-Leu]LLLRVK-amba.

The solute carrier L-type amino acid transporter 1 (LAT-1/SLC7A5) is a viable target for drug delivery to the central nervous system (CNS) and tumors due to its high abundance at the blood–brain barrier and in tumor tissue. LAT-1 is only localized on the cell surface as a heterodimer with CD98, which is not required for transporter function. To support future CNS drug-delivery development based on LAT-1 targeting, we established an ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) assay for stable isotopically labeled leucine ([13C6, 15N]-L-leucine), with a dynamic range of 0.1–1000 ng/mL that can be applied for the functional testing of LAT-1 activity when combined with specific inhibitors and, consequently, the LAT-1 inhibition capacity of new compounds. The assay was established in a 96-well format, facilitating high-throughput experiments, and, hence, can support the screening for novel inhibitors. Applicable recommendations of the US Food and Drug Administration and European Medicines Agency for bioanalytical method validation were followed to validate the assay. The assay was applied to investigate the IC50 of two well-known LAT-1 inhibitors on hCMEC/D3 cells: the highly specific LAT-1 inhibitor JPH203, which was also used to demonstrate LAT-1 specific uptake, and the general system L inhibitor BCH. In addition, the [13C6, 15N]-L-leucine uptake was determined on two human brain capillary endothelial cell lines (NKIM-6 and hCMEC/D3), which were characterized for their expressional differences of LAT-1 at the protein and mRNA level and the surface amount of CD98. The IC50 values of the inhibitors were in concordance with previously reported values. Furthermore, the [13C6, 15N]-L-leucine uptake was significantly higher in hCMEC/D3 cells compared to NKIM-6 cells, which correlated with higher expression of LAT-1 and a higher surface amount of CD98. Therefore, the UPLC-MS/MS quantification of ([13C6, 15N]-L-leucine is a feasible strategy for the functional characterization of LAT-1 activity in cells or tissue.

The human peptide transporter hPepT1 (SLC15A1), physiologically transporting dipeptides and tripeptides generated during food digestion, also plays a role in the uptake of small bioactive peptides and peptide-like drugs. Moreover, it might be addressed in prodrug strategies of poorly absorbed drugs. We hypothesised that the cyclic drug peptides octreotide and pasireotide could be substrates of this transporter because their diameter can resemble the size of dipeptides or tripeptides due to their strong structural curvature and because they reach the systemic circulation in Beagle dogs. For investigating possible hPepT1 substrate characteristics, we generated and characterised a CHO-K1 cell line overexpressing SLC15A1 by transfection and selection via magnetic beads. Possible inhibition of the uptake of the prototypical substrate Gly-Sar by octreotide and pasireotide was screened, followed by quantifying the uptake of the cyclic peptides in cells overexpressing SLC15A1 compared with the parental cell line. Although inhibition of Gly-Sar uptake was observed, uptake of octreotide and pasireotide was not increased in SLC15A1 overexpressing cells, indicating a lack of transport by hPepT1. Our data clearly indicate that octreotide and pasireotide are nonsubstrate inhibitors of hPepT1 and that their oral bioavailability cannot be explained by absorption via hPepT1.