Evaluating drug-drug interaction risk associated with peptide analogues using advanced in vitro systems.

Drug-drug interaction (DDI) assessment of therapeutic peptides is an evolving area. The industry generally follows DDI guidelines for small molecules, but the translation of data generated with commonly used in vitro systems to in vivo is sparse. In the current study, we investigated the ability of advanced human hepatocyte in vitro systems namely HepatoPac, spheroids, and Liver-on-a-chip to assess potential changes in regulation of CYP1A2, CYP2B6, CYP3A4, SLCO1B1 and ABCC2 in the presence of selected therapeutic peptides, proteins, and small molecules. The peptide NN1177, a glucagon and GLP-1 receptor co-agonist, did not suppress mRNA expression or activity of CYP1A2, CYP2B6, and CYP3A4 in HepatoPac, spheroids, or Liver-on-a-chip; these findings were in contrast to the data obtained in sandwich cultured hepatocytes. No effect of NN1177 on SLCO1B1 and ABCC2 mRNA was observed in any of the complex systems. The induction magnitude differed across the systems (e.g., rifampicin induction of CYP3A4 mRNA ranged from 2.8-fold in spheroids to 81.2-fold in Liver-on-a-chip). Small molecules, obeticholic acid and abemaciclib, showed varying responses in HepatoPac, spheroids and Liver-on-a-chip, indicating a need for EC50 determinations to fully assess translatability data. HepatoPac, the most extensively investigated in this study (3 donors), showed high potential to investigate DDIs associated with CYP regulation by therapeutic peptides. Spheroids and Liver-on-a-chip were only assessed in one hepatocyte donor and further evaluations are required to confirm their potential. This study establishes an excellent foundation towards the establishment of more clinically-relevant in vitro tools for evaluation of potential DDIs with therapeutic peptides. Significance Statement At present, there are no guidelines for drug-drug interaction (DDI) assessment of therapeutic peptides. Existing in vitro methods recommended for assessing small molecule DDIs do not appear to translate well for peptide drugs, complicating drug development for these moieties. Here, we establish evidence that complex cellular systems have potential to be used as more clinically-relevant tools for the in vitro DDI evaluation of therapeutic peptides.

In vitro CYP450 enzyme down-regulation by GLP-1/glucagon co-agonist does not translate to observed drug-drug interactions in the clinic.

NN1177 is a glucagon/glucagon-like peptide 1 receptor co-agonist investigated for chronic weight management and treatment of non-alcoholic steatohepatitis. Here, we show concentration-dependent down-regulation of cytochrome P450 enzymes using freshly isolated human hepatocytes treated with this linear 29-amino acid peptide. Notably, reductions in CYP3A4 mRNA expression (57.2-71.7%) and activity (18.5-51.5%) were observed with a clinically-relevant concentration of 100 nM NN1177. CYP1A2 and CYP2B6 were also affected, but to a lesser extent. Physiological-based pharmacokinetic modelling simulated effects on CYP3A4 and CYP1A2 probe substrates (midazolam and caffeine, respectively) and revealed potential safety concerns related to drug-drug interactions (DDIs). To investigate the clinical relevance of observed in vitro CYP down-regulation, a phase 1 clinical cocktail study was initiated to assess the DDI potential. The study enrolled 45 study participants (BMI 23.0-29.9 kg/m2) to receive a Cooperstown 5+1 cocktail (midazolam, caffeine, omeprazole, dextromethorphan, and S-warfarin/vitamin K) alone and following steady state NN1177 exposure. The analysis of pharmacokinetic profiles for the cocktail drugs showed no significant effect from the co-administration of NN1177 on AUC0-inf for midazolam or S-warfarin. Omeprazole, caffeine, and dextromethorphan generally displayed decreases in AUC0-inf and Cmax following NN1177 co-administration. Thus, the in vitro observations were not reflected in the clinic. These findings highlight remaining challenges associated with standard in vitro systems used to predict DDIs for peptide-based drugs as well as the complexity of DDI trial design for these modalities. Overall, there is an urgent need for better pre-clinical models to assess potential drug-drug interaction risks associated with therapeutic peptides during drug development. Significance Statement This study highlights significant challenges associated with assessing drug-drug interaction risks for therapeutic peptides using in vitro systems, since potential concerns identified by standard assays did not translate to the clinical setting. Further research is required to guide investigators involved in peptide-based drug development towards better non-clinical models in order to more accurately evaluate potential drug-drug interactions.

In vitro drug-drug interactions of decitabine and tetrahydrouridine involving drug transporters and drug metabolising enzymes.

1. NDec is a novel, oral, fixed-dose formulation of decitabine and tetrahydrouridine that is currently being developed for the treatment of patients with sickle cell disease. Here, we examine the potential for both components of NDec to interact with key drug metabolising enzymes (tetrahydrouridine only) and drug transporters (decitabine and tetrahydrouridine).2. This study assessed the inhibition and induction of cytochrome P450 (CYP) enzymes by tetrahydrouridine, as well as the involvement of specific drug metabolising enzymes in tetrahydrouridine metabolism. Inhibition of efflux and uptake transporters by both decitabine and tetrahydrouridine was also studied.3. Tetrahydrouridine did not inhibit or induce relevant CYP enzymes at concentrations ranging from 0.1 to 100 µM. Metabolism of tetrahydrouridine did not occur in the presence of the human drug metabolising enzymes tested. Tetrahydrouridine showed weak inhibition towards the MATE2-K transporter (∼30% inhibition at 5 and 50 µM), which was not deemed clinically relevant. Tetrahydrouridine did not inhibit any of the remaining uptake or efflux transporters. Decitabine (0.5 and 5 µM) did not inhibit any of the evaluated uptake or efflux drug transporters.4. Data presented confirm that tetrahydrouridine and decitabine are unlikely to be involved in metabolism- or transporter-based drug-drug interactions.

Evaluation of 309 molecules as inducers of CYP3A4, CYP2B6, CYP1A2, OATP1B1, OCT1, MDR1, MRP2, MRP3 and BCRP in cryopreserved human hepatocytes in sandwich culture.

1. Regulation of hepatic metabolism or transport may lead to increase in drug clearance and compromise efficacy or safety. In this study, cryopreserved human hepatocytes were used to assess the effect of 309 compounds on the activity and mRNA expression (using qPCR techniques) of CYP1A2, CYP2B6 and CYP3A4, as well as mRNA expression of six hepatic transport proteins: OATP1B1 (SCLO1B1), OCT1 (SLC22A1), MDR1 (ABCB1), MRP2 (ABCC2), MRP3 (ABCC3) and BCRP (ABCG2). 2. The results showed that 6% of compounds induced CYP1A2 activity (1.5-fold increase); 30% induced CYP2B6 while 23% induced CYP3A4. qPCR data identified 16, 33 or 32% inducers of CYP1A2, CYP2B6 or CYP3A4, respectively. MRP2 was induced by 27 compounds followed by MDR1 (16)>BCRP (9)>OCT1 (8)>OATP1B1 (5)>MRP3 (2). 3. CYP3A4 appeared to be down-regulated (≥2-fold decrease in mRNA expression) by 53 compounds, 10 for CYP2B6, 6 for OCT1, 4 for BCRP, 2 for CYP1A2 and OATP1B1 and 1 for MDR1 and MRP2. 4. Structure-activity relationship analysis showed that CYP2B6 and CYP3A4 inducers are bulky lipophilic molecules with a higher number of heavy atoms and a lower number of hydrogen bond donors. Finally, a strategy for testing CYP inducers in drug discovery is proposed.

Reduced physiologically-based pharmacokinetic model of repaglinide: impact of OATP1B1 and CYP2C8 genotype and source of in vitro data on the prediction of drug-drug interaction risk.

PURPOSE: To investigate the effect of OATP1B1 genotype as a covariate on repaglinide pharmacokinetics and drug-drug interaction (DDIs) risk using a reduced physiologically-based pharmacokinetic (PBPK) model. METHODS: Twenty nine mean plasma concentration-time profiles for SLCO1B1 c.521T>C were used to estimate hepatic uptake clearance (CLuptake) in different genotype groups applying a population approach in NONMEM v.7.2. RESULTS: Estimated repaglinide CLuptake corresponded to 217 and 113 µL/min/10(6) cells for SLCO1B1 c.521TT/TC and CC, respectively. A significant effect of OATP1B1 genotype was seen on CLuptake (48% reduction for CC relative to wild type). Sensitivity analysis highlighted the impact of CLmet and CLdiff uncertainty on the CLuptake optimization using plasma data. Propagation of this uncertainty had a marginal effect on the prediction of repaglinide OATP1B1-mediated DDI with cyclosporine; however, sensitivity of the predicted magnitude of repaglinide metabolic DDI was high. In addition, the reduced PBPK model was used to assess the effect of both CYP2C8*3 and SLCO1B1 c.521T>C on repaglinide exposure by simulations; power calculations were performed to guide prospective DDI and pharmacogenetic studies. CONCLUSIONS: The application of reduced PBPK model for parameter optimization and limitations of this process associated with the use of plasma rather than tissue profiles are illustrated.

In Vitro Interaction of Tetrahydrouridine with Key Human Nucleoside Transporters.

NDec is a novel combination of oral decitabine and tetrahydrouridine that is currently under clinical development for the treatment of sickle cell disease (SCD). Here, we investigate the potential for the tetrahydrouridine component of NDec to act as an inhibitor or substrate of key concentrative nucleoside transporters (CNT1-3) and equilibrative nucleoside transporters (ENT1-2). Nucleoside transporter inhibition and tetrahydrouridine accumulation assays were performed using Madin-Darby canine kidney strain II (MDCKII) cells overexpressing human CNT1, CNT2, CNT3, ENT1, and ENT2 transporters. Results showed that tetrahydrouridine did not influence CNT- or ENT-mediated uridine/adenosine accumulation in MDCKII cells at the concentrations tested (25 and 250 µM). Accumulation of tetrahydrouridine in MDCKII cells was initially shown to be mediated by CNT3 and ENT2. However, while time- and concentration-dependence experiments showed active accumulation of tetrahydrouridine in CNT3-expressing cells, allowing for estimation of Km (3,140 µM) and Vmax (1,600 pmol/mg protein/min), accumulation of tetrahydrouridine was not observed in ENT2-expressing cells. Potent CNT3 inhibitors are a class of drugs not generally prescribed to patients with SCD, except in certain specific circumstances. These data suggest that NDec can be administered safely with drugs that act as substrates and inhibitors of the nucleoside transporters included in this study.

Industry Perspective on Therapeutic Peptide Drug-Drug Interaction Assessments During Drug Development: A European Federation of Pharmaceutical Industries and Associations White Paper.

Drug-drug interaction (DDI) assessments are well defined in health authority guidelines for small molecule drugs, and US Food and Drug Administration (FDA) draft guidance is now available for therapeutic proteins. However, there are currently no regulatory guidelines outlining DDI assessments for therapeutic peptides, which poses significant uncertainty and challenges during drug development for this heterogenous class of molecules. A cross-industry peptide DDI working group consisting of experts from 10 leading companies was formed under the sponsorship of the European Federation of Pharmaceutical Industries and Associations. We aimed to capture the range of DDI studies undertaken for peptide drugs by (i) anonymously surveying relevant companies involved in peptide drug development to better understand DDI study type/timing currently performed and (ii) compiling a database containing in vitro / clinical DDI data from submission packages for recently approved peptide drugs. Our analyses highlight significant gaps and uncertainty in the field. For example, the reported timing of in vitro peptide DDI studies, if performed, vary substantially across responding companies from early research to phase III. Nearly all in vitro cytochrome P450 / transporter inhibition studies reported in the survey were negative. For the few positive hits reported, no clinical follow-up studies were performed, questioning the clinical relevance of these findings. Furthermore, available submission packages reveal DDI likelihood is low for peptides >2 kDa, making it reasonable to adopt a risk-based approach during drug development for larger peptides. By benchmarking the landscape of peptide DDI activities across the industry, we set the stage for future discussions with health authorities on harmonizing peptide DDI approaches.

Therapeutic Protein Drug Interactions: A White Paper From the International Consortium for Innovation and Quality in Pharmaceutical Development.

Typically, therapeutic proteins (TPs) have a low risk for eliciting meaningful drug interactions (DIs). However, there are select instances where TP drug interactions (TP-DIs) of clinical concern can occur. This white paper discusses the various types of TP-DIs involving mechanisms such as changes in disease state, target-mediated drug disposition, neonatal Fc receptor (FcRn), or antidrug antibodies formation. The nature of TP drug interaction being investigated should determine whether the examination is conducted as a standalone TP-DI study in healthy participants, in patients, or assessed via population pharmacokinetic analysis. DIs involving antibody-drug conjugates are discussed briefly, but the primary focus here will be DIs involving cytokine modulation. Cytokine modulation can occur directly by certain TPs, or indirectly due to moderate to severe inflammation, infection, or injury. Disease states that have been shown to result in indirect disease-DIs that are clinically meaningful have been listed (i.e., typically a twofold change in the systemic exposure of a coadministered sensitive cytochrome P450 substrate drug). Type of disease and severity of inflammation should be the primary drivers for risk assessment for disease-DIs. While more clinical inflammatory marker data needs to be collected, the use of two or more clinical inflammatory markers (such as C-reactive protein, albumin, or interleukin 6) may help broadly categorize whether the predicted magnitude of inflammatory disease-DI risk is negligible, weak, or moderate to strong. Based on current knowledge, clinical DI studies are not necessary for all TPs, and should no longer be conducted in certain disease patient populations such as psoriasis, which do not have sufficient systemic inflammation to cause a meaningful indirect disease-DI.

A comprehensive assessment of repaglinide metabolic pathways: impact of choice of in vitro system and relative enzyme contribution to in vitro clearance.

Repaglinide is presently recommended by the U.S. Food and Drug Administration as a clinical CYP2C8 probe, yet current in vitro and clinical data are inconsistent concerning the role of this enzyme in repaglinide elimination. The aim of the current study was to perform a comprehensive investigation of repaglinide metabolic pathways and assess their contribution to the overall clearance. Formation of four repaglinide metabolites was characterized using in vitro systems with differential complexity. Full kinetic profiles for the formation of M1, M2, M4, and repaglinide glucuronide were obtained in pooled cryopreserved human hepatocytes, human liver microsomes, human S9 fractions, and recombinant cytochrome P450 enzymes. Distinct differences in clearance ratios were observed between CYP3A4 and CYP2C8 for M1 and M4 formation, resulting in a 60-fold M1/M4 ratio in recombinant (r) CYP3A4, in contrast to 0.05 in rCYP2C8. Unbound K(m) values were within 2-fold for each metabolite across all in vitro systems investigated. A major system difference was seen in clearances for the formation of M2, which is suggested to be a main metabolite of repaglinide in vivo. An approximately 7-fold higher unbound intrinsic clearance was observed in hepatocytes and S9 fractions in comparison to microsomes; the involvement of aldehyde dehydrogenase in M2 formation was shown for the first time. This systematic analysis revealed a comparable in vitro contribution from CYP2C8 and CYP3A4 to the metabolism of repaglinide (<50%), whereas the contribution of glucuronidation ranged from 2 to 20%, depending on the in vitro system used. The repaglinide M4 metabolic pathway is proposed as a specific CYP2C8 probe for the assessment of drug-drug interactions.

Progress curve mechanistic modeling approach for assessing time-dependent inhibition of CYP3A4.

A progress curve method for assessing time-dependent inhibition of CYP3A4 is based on simultaneous quantification of probe substrate metabolite and inhibitor concentrations during the experiment. Therefore, it may overcome some of the issues associated with the traditional two-step method and estimation of inactivation rate (k(inact)) and irreversible inhibition (K(I)) constants. In the current study, seven time-dependent inhibitors were investigated using a progress curve method and recombinant CYP3A4. A novel mechanistic modeling approach was applied to determine inhibition parameters using both inhibitor and probe metabolite data. Progress curves generated for clarithromycin, erythromycin, diltiazem, and N-desmethyldiltiazem were described well by the mechanistic mechanism-based inhibition (MBI) model. In contrast, mibefradil, ritonavir, and verapamil required extension of the model and inclusion of competitive inhibition term for the metabolite. In addition, this analysis indicated that verapamil itself causes minimal MBI, and the formation of inhibitory metabolites was responsible for the irreversible loss of CYP3A4 activity. The k(inact) and K(I) estimates determined in the current study were compared with literature data generated using the conventional two-step method. In the current study, the inactivation efficiency (k(inact)/K(I)) for clarithromycin, ritonavir, and erythromycin were up to 7-fold higher, whereas k(inact)/K(I) for mibefradil, N-desmethyldiltiazem, and diltiazem were, on average, 2- to 4.8-fold lower than previously reported estimates. Use of human liver microsomes instead of recombinant CYP3A4 resulted in 5-fold lower k(inact)/K(I) for erythromycin. In conclusion, the progress curve method has shown a greater mechanistic insight when determining kinetic parameters for MBI in addition to providing a more comprehensive experimental protocol.