Quantitative systems pharmacology model-based investigation of adverse gastrointestinal events associated with prolonged treatment with PI3-kinase inhibitors.

Several PI3K inhibitors are in clinical development for the treatment of various forms of cancers, including pan-PI3K inhibitors targeting all four PI3K isoforms (α, ß, γ, and δ), and isoform-selective inhibitors. Diarrhea and immune-mediated colitis are among the adverse events observed with PI3K inhibition which limits the maximal tolerated dose. A quantitative systems pharmacology model was developed to investigate PI3K-inhibitor-induced colitis. The effects of individual PI3K isoforms on relevant cellular pathways were incorporated into a mechanistic representation of mucosal inflammation. A virtual clinical population captures the observed clinical variability in the onset timing and rates of diarrhea and colitis for seven clinically tested PI3K inhibitors. Model-based analysis suggests that colitis development is governed by both the inhibition of PI3Kδ, which drives T cell differentiation and proliferation, and PI3Kα, which regulates epithelial barrier integrity. Specifically, when PI3Kα is inhibited below a given threshold, epithelial barrier dysfunction precipitates an exaggerated T effector response due to PI3Kδ-inhibition, leading to risk of diarrhea and colitis. This synergy explains why the lowest diarrhea and colitis rates are seen with the weakest PI3Kδ inhibition (alpelisib), and higher rates are seen with strong PI3Kδ inhibition if PI3Kα is even mildly inhibited (e.g., idelalisib), whereas strong PI3Kδ inhibition in the absence of PI3Kα inhibition does not result in high colitis rates (umbralisib). Thus, the model-based analysis suggests that PI3Kα and δ inhibition play unique but synergistic roles in driving colitis. Finally, we explore if and how dose-regimen might influence colitis rates for molecules that inhibit both PI3Kα and PI3Kδ.

Intrinsic and Extrinsic Pharmacokinetic Variability of Small Molecule Targeted Cancer Therapy.

Pharmacokinetic (PK) variability in cancer clinical trials may be due to heterogeneous populations and identifying sources of variability is important. Use of healthy subjects in clinical pharmacology studies together with detailed knowledge of the characteristics of patients with cancer can allow for quick identification and quantification of factors affecting PK variability. PK data and sources of variability of 40 marketed molecularly targeted oncology therapeutics were compiled from regulatory approval documents covering an 18-year period (1999-2017). Variability in PK parameters was compared and contributors to variability were identified. The results show that PK variability was ~ 16% higher for peak plasma concentration (Cmax ) and area under the concentration time curve (AUC) in patients with cancer compared with healthy subjects. Several factors were identified as major contributors to variability including hepatic/renal impairment and cytochrome P450 inhibition/induction. Lower PK variability in healthy subjects may represent an opportunity to perform rapid and robust pharmacological and PK assessments to inform subsequent studies in the development of new cancer therapies.

In Vivo Stability Profiles of Anti-factor D Molecules Support Long-Acting Delivery Approaches.

The collection of aqueous humor (phase 1 b/2 Mahalo study) from patients dosed intravitreally with anti-factor D (AFD; FCFD4514S, lampalizumab), a humanized antibody fragment previously under investigation to treat geographic atrophy (GA) secondary to age-related macular degeneration, presented a unique opportunity to examine AFD properties in clinical samples. We investigated AFD stability and target-binding characteristics to set up strategies for engineering and evaluating optimized molecules that enable less frequent dosing. Two variants, AFD.v8 and AFD.v14, were evaluated as alternatives to AFD for longer-acting treatments. Mass spectrometry, surface plasmon resonance, and immunoassay were used to assess AFD stability and binding activity in aqueous humor samples from Mahalo patients. In vitro stability and binding activity of AFD, AFD.v8, and AFD.v14 were assessed in human vitreous humor versus buffer at 37 °C over 16 weeks and in vivo in rabbits over 28 days along with pharmacokinetic determinations. In human aqueous humor, AFD specific binding was >85% through 30 days, and deamidation was <3% through 60 days, consistent with the AFD stability and binding activity in vitreous humor from humans in vitro and rabbits in vivo. Target binding, stability, and rabbit pharmacokinetic parameters of AFD.v8 and AFD.v14 were similar to those of AFD. Physiological stability and activity of AFD translated across in vitro and in vivo studies in humans and rabbits. The two variants AFD.v8 and AFD.v14 demonstrated comparable potency and pharmacokinetics. These findings, along with previously demonstrated improved solubility of AFD.v8 and AFD.v14, provide proof-of-concept for developing other similar long-acting therapeutic variants.

Harnessing the Potential of Emerging Digital Health and Biological Sampling Technologies for Clinical Drug Development: Promise to Reality.

Advances in emerging innovative technologies have led to optimistic outlooks on their transformative potential for healthcare and clinical trials.1 Given the increased attention, this white paper by the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) presents perspectives on pharmaceutical and biotechnology industry trends for innovative digital health, adherence, and outpatient sampling technologies. As stimulus for cross-company scientific dialog points to consider for adoption, implementation, and recommendations to broaden uptake are proposed.

Racial and Ethnic Composition of Cancer Clinical Drug Trials: How Diverse Are We?

Many approved drugs demonstrate different pharmacokinetics, pharmacodynamics, and/or safety across racial and ethnic groups. The primary objective of the current study was to summarize the racial and ethnic makeup of cancer clinical drug trials using cancer drugs approved by the U.S. Food and Drug Administration (FDA) between January 1, 2010, and July 31, 2016. In clinical studies used for FDA approvals, 82.3% of participants identified as white, 10.2% as Asian, 2.3% as black, and 4.7% as Hispanic. Black participants made up 7.7% of U.S. and Canadian cancer clinical drug trials and 2.6% of global cancer clinical drug trials while Asian participants made up 13.5% of global cancer clinical drug trials but only 1.8% of U.S. and Canadian cancer clinical drug trials. The current study indicates that although cancer clinical drug trials have become more inclusive of Asian participants, other racial and ethnic minority groups remain under-represented. This may result in an inadequate understanding of drug safety and efficacy in many racial and ethnic populations.

Protein engineering to increase the potential of a therapeutic antibody Fab for long-acting delivery to the eye.

To date, ocular antibody therapies for the treatment of retinal diseases rely on injection of the drug into the vitreous chamber of the eye. Given the burden for patients undergoing this procedure, less frequent dosing through the use of long-acting delivery (LAD) technologies is highly desirable. These technologies usually require a highly concentrated formulation and the antibody must be stable against extended exposure to physiological conditions. Here we have increased the potential of a therapeutic antibody antigen-binding fragment (Fab) for LAD by using protein engineering to enhance the chemical and physical stability of the molecule. Structure-guided amino acid substitutions in a negatively charged complementarity determining region (CDR-L1) of an anti-factor D (AFD) Fab resulted in increased chemical stability and solubility. A variant of AFD (AFD.v8), which combines light chain substitutions (VL-D28S:D30E:D31S) with a substitution (VH-D61E) to stabilize a heavy chain isomerization site, retained complement factor D binding and inhibition potency and has properties suitable for LAD. This variant was amenable to high protein concentration (>250 mg/mL), low ionic strength formulation suitable for intravitreal injection. AFD.v8 had acceptable pharmacokinetic (PK) properties upon intravitreal injection in rabbits, and improved stability under both formulation and physiological conditions. Simulations of expected human PK behavior indicated greater exposure with a 25-mg dose enabled by the increased solubility of AFD.v8.

Pharmacogenomics in the age of personalized medicine.

The aim of personalized medicine is to offer the right treatment to the right person at the right dose, thus maximizing efficacy and minimizing toxicity for each individual patient. Pharmacogenomic approaches attempt to refine the aim of personalized medicine by utilizing an individual's germline and somatic DNA signatures to guide treatment. In this review, we highlight the current use of pharmacogenomic based biomarker information in drug labeling. We also present several case studies on the implementation of pharmacogenomic strategies in drug discovery and development. Lastly, we comment on current challenges to implementing pharmacogenomic based testing in the clinic.

Contribution of Antibody Hydrodynamic Size to Vitreal Clearance Revealed through Rabbit Studies Using a Species-Matched Fab.

We have developed a tool Fab fragment of a rabbit monoclonal antibody that is useful for early evaluation in rabbit models of technologies for long acting delivery (LAD) of proteins to the eye. Using this Fab we show that vitreal clearance can be slowed through increased hydrodynamic size. Fab (G10rabFab) and Fab' (G10rabFab') fragments of a rabbit monoclonal antibody (G10rabIgG) were expressed in Chinese hamster ovary (CHO) cells and purified using antigen-based affinity chromatography. G10rabFab retains antigen-binding upon thermal stress (37 °C) for 8 weeks in phosphate-buffered saline (PBS) and can be detected in rabbit tissues using an antigen-based ELISA. Hydrodynamic radius, measured using quasi-elastic light scattering (QELS), was increased through site-specific modification of the G10rabFab' free cysteine with linear methoxy-polyethylene glycol(PEG)-maleimide of 20000 or 40000 molecular weight. Pharmacokinetic studies upon intravitreal dosing in New Zealand white rabbits were conducted on the G10rabFab and PEGylated G10rabFab'. Results of single and multidose pharmacokinetic experiments yield reproducible results and a vitreal half-life for G10rabFab of 3.2 days. Clearance from the eye is slowed through increased hydrodynamic size, with vitreal half-life showing a linear dependence on hydrodynamic radius (RH). A linear dependence of vitreal half-life on RH suggests that molecule diffusivity makes an important contribution to vitreal clearance. A method for prediction of vitreal half-life from RH measurements is proposed.

Identification of Tazarotenic Acid as the First Xenobiotic Substrate of Human Retinoic Acid Hydroxylase CYP26A1 and CYP26B1.

Cytochrome P450 (CYP) 26A1 and 26B1 are heme-containing enzymes responsible for metabolizing all-trans retinoic acid (at-RA). No crystal structures have been solved, and therefore homology models that provide structural information are extremely valuable for the development of inhibitors of cytochrome P450 family 26 (CYP26). The objectives of this study were to use homology models of CYP26A1 and CYP26B1 to characterize substrate binding characteristics, to compare structural aspects of their active sites, and to support the role of CYP26 in the metabolism of xenobiotics. Each model was verified by dockingat-RA in the active site and comparing the results to known metabolic profiles ofat-RA. The models were then used to predict the metabolic sites of tazarotenic acid with results verified by in vitro metabolite identification experiments. The CYP26A1 and CYP26B1 homology models predicted that the benzothiopyranyl moiety of tazarotenic acid would be oriented toward the heme of each enzyme and suggested that tazarotenic acid would be a substrate of CYP26A1 and CYP26B1. Metabolite identification experiments indicated that CYP26A1 and CYP26B1 oxidatively metabolized tazarotenic acid on the predicted moiety, with in vitro rates of metabolite formation by CYP26A1 and CYP26B1 being the highest across a panel of enzymes. Molecular analysis of the active sites estimated the active-site volumes of CYP26A1 and CYP26B1 to be 918 Å(3)and 977 Å(3), respectively. Overall, the homology models presented herein describe the enzyme characteristics leading to the metabolism of tazarotenic acid by CYP26A1 and CYP26B1 and support a potential role for the CYP26 enzymes in the metabolism of xenobiotics.

Assessment of Disease-Related Therapeutic Protein Drug-Drug Interaction for Etrolizumab in Patients With Moderately to Severely Active Ulcerative Colitis.

The efficacy and safety of etrolizumab, a humanized IgG1 mAb, were evaluated in patients with ulcerative colitis (UC) in a phase 2 study (EUCALYPTUS). The current study assessed the risk of therapeutic protein drug-drug interaction (TP-DDI) of etrolizumab on CYP3A activity in patients with UC. Literature review was performed to compare serum proinflammatory cytokine levels and pharmacokinetic (PK) parameters of CYP3A substrate drugs between patients with inflammatory bowel disease (IBD) and healthy subjects. Treatment effect of etrolizumab on CYP3A activity was evaluated by measuring colonic CYP3A4 mRNA expression and serum C-reactive protein (CRP) in EUCALYPTUS patients. Literature data suggested similar levels between IBD patients and healthy subjects for serum proinflammatory cytokines and PK parameters of CYP3A substrate drugs. Additionally, treatment with etrolizumab did not change colonic CYP3A4 mRNA expression or serum CRP levels in UC patients. In conclusion, our results indicate a low TP-DDI risk for etrolizumab in UC patients, particularly on medications metabolized by CYP3A.

Evaluation of Fluorophotometry to Assess the Vitreal Pharmacokinetics of Protein Therapeutics.

PURPOSE: In this work, we assessed the ability of fluorophotometry to measure the vitreal pharmacokinetics (PK) of fluorescently-labeled ranibizumab in the rabbit after intravitreal injection. We compared these values to those obtained using enzyme-linked immunosorbent assays (ELISA). Data obtained in this study were also compared to historical ranibizumab ocular PK data, either measured in-house or previously published. METHODS: Three individual in vivo studies were performed in New Zealand White rabbits to assess the feasibility of using fluorophotometry to measure rabbit ocular PK of ranibizumab; explore the dynamic range of dosing fluorescently-labeled ranibizumab; and directly compare ranibizumab concentrations and calculated PK parameters measured by vitreal fluorophotometry to those measured using ELISA. RESULTS: In direct comparisons between fluorophotometry and ELISA, the calculated clearance (CL) values were 0.26 and 0.21 mL/day, the volumes of distribution at steady state (Vss) were 0.80 and 0.94 mL, the half-lives (t1/2) were 3.1 and 2.9 days and the dose normalized areas under the curve (AUC/D) were 4.7 and 3.9 µg·day/mL/µg, respectively. These values fell within the ranges of 0.13 to 0.44 mL/day for CL, 0.5 to 1.8 mL for Vss, 2.8 to 3.5 days for t1/2, and 2.3 to 7.9 µg·day/mL/µg for AUC/D that have been either measured previously in-house or published elsewhere. CONCLUSIONS: Although not suitable for measuring retinal concentrations, fluorophotometry is a valuable, noninvasive method to measure vitreous concentrations of protein therapeutics after intravitreal injection.

Pharmacokinetics, metabolism and distribution of PEGs and PEGylated proteins: quo vadis?

The pharmacokinetics (PK), metabolism and biodistribution of polyethylene glycol (PEG) in PEGylated proteins are important to understand the increased cellular vacuolation reported in various tissues in animals. The tissue distribution profile of PEGylated proteins and 'metabolic' PEG is guided largely by absolute PEG load, PEG molecular weight and, where applicable, receptor-mediated uptake via the protein moiety. High molecular weight PEGs show slow renal clearance, and consequently have a greater potential to accumulate within cells. The intracellular nonbiodegradable PEG can accumulate within the lysosome ultimately causing distension and vacuolation observed by standard histological examinations. Improved bioanalytical methodologies will contribute to the identification of specific PK parameters including distribution behavior to support development of PEGylated proteins as therapeutics.

Human UGT1A4 and UGT1A3 conjugate 25-hydroxyvitamin D3: metabolite structure, kinetics, inducibility, and interindividual variability.

25-Hydroxyvitamin D3 (25OHD3) is used as a clinical biomarker for assessment of vitamin D status. Blood levels of 25OHD3 represent a balance between its formation rate and clearance by several oxidative and conjugative processes. In the present study, the identity of human uridine 5'-diphosphoglucuronyltransferases (UGTs) capable of catalyzing the 25OHD3 glucuronidation reaction was investigated. Two isozymes, UGT1A4 and UGT1A3, were identified as the principal catalysts of 25OHD3 glucuronidation in human liver. Three 25OHD3 monoglucuronides (25OHD3-25-glucuronide, 25OHD3-3-glucuronide, and 5,6-trans-25OHD3-25-glucuronide) were generated by recombinant UGT1A4/UGT1A3, human liver microsomes, and human hepatocytes. The kinetics of 25OHD3 glucuronide formation in all systems tested conformed to the Michaelis-Menten model. An association between the UGT1A4*3 (Leu48Val) gene polymorphism with the rates of glucuronide formation was also investigated using human liver microsomes isolated from 80 genotyped livers. A variant allele dose effect was observed: the homozygous UGT1A4*3 livers (GG) had the highest glucuronidation activity, whereas the wild type (TT) had the lowest activity. Induction of UGT1A4 and UGT1A3 gene expression was also determined in human hepatocytes treated with pregnane X receptor/constitutive androstane receptor agonists, such as rifampin, carbamazepine, and phenobarbital. Although UGT mRNA levels were increased significantly by all of the known pregnane X receptor/constitutive androstane receptor agonists tested, rifampin, the most potent of the inducers, significantly induced total 25OHD3 glucuronide formation activity in human hepatocytes measured after 2, but not 4 and 24 hours, of incubation. Finally, the presence of 25OHD3-3-glucuronide in both human plasma and bile was confirmed, suggesting that the glucuronidation pathway might be physiologically relevant and contribute to vitamin D homeostasis in humans.

IL-22 regulates iron availability in vivo through the induction of hepcidin.

Iron is a trace element important for the proper folding and function of various proteins. Physiological regulation of iron stores is of critical importance for RBC production and antimicrobial defense. Hepcidin is a key regulator of iron levels within the body. Under conditions of iron deficiency, hepcidin expression is reduced to promote increased iron uptake from the diet and release from cells, whereas during conditions of iron excess, induction of hepcidin restricts iron uptake and movement within the body. The cytokine IL-6 is well established as an important inducer of hepcidin. The presence of this cytokine during inflammatory states can induce hepcidin production, iron deficiency, and anemia. In this study, we show that IL-22 also influences hepcidin production in vivo. Injection of mice with exogenous mouse IgG1 Fc fused to the N terminus of mouse IL-22 (Fc-IL-22), an IL-22R agonist with prolonged and enhanced functional potency, induced hepcidin production, with a subsequent decrease in circulating serum iron and hemoglobin levels and a concomitant increase in iron accumulation within the spleen. This response was independent of IL-6 and was attenuated in the absence of the IL-22R-associated signaling kinase, Tyk2. Ab-mediated blockade of hepcidin partially reversed the effects on iron biology caused by IL-22R stimulation. Taken together, these data suggest that exogenous IL-22 regulates hepcidin production to physiologically influence iron usage.

Critical review of preclinical approaches to investigate cytochrome p450-mediated therapeutic protein drug-drug interactions and recommendations for best practices: a white paper.

Drug-drug interactions (DDIs) between therapeutic proteins (TPs) and small-molecule drugs have recently drawn the attention of regulatory agencies, the pharmaceutical industry, and academia. TP-DDIs are mainly caused by proinflammatory cytokine or cytokine modulator-mediated effects on the expression of cytochrome P450 enzymes. To build consensus among industry and regulatory agencies on expectations and challenges in this area, a working group was initiated to review the preclinical state of the art. This white paper represents the observations and recommendations of the working group on the value of in vitro human hepatocyte studies for the prediction of clinical TP-DDI. The white paper was developed following a "Workshop on Recent Advances in the Investigation of Therapeutic Protein Drug-Drug Interactions: Preclinical and Clinical Approaches" held at the Food and Drug Administration White Oak Conference Center on June 4 and 5, 2012. Results of a workshop poll, cross-laboratory data comparisons, and the overall recommendations of the in vitro working group are presented herein. The working group observed that evaluation of TP-DDI for anticytokine monoclonal antibodies is currently best accomplished with a clinical study in patients with inflammatory disease. Treatment-induced changes in appropriate biomarkers in phase 2 and 3 studies may indicate the potential for a clinically measurable treatment effect on cytochrome P450 enzymes. Cytokine-mediated DDIs observed with anti-inflammatory TPs cannot currently be predicted using in vitro data. Future success in predicting clinical TP-DDIs will require an understanding of disease biology, physiologically relevant in vitro systems, and more examples of well conducted clinical TP-DDI trials.

Evaluation of UGT protein interactions in human hepatocytes: effect of siRNA down regulation of UGT1A9 and UGT2B7 on propofol glucuronidation in human hepatocytes.

Previous experiments performed in recombinant systems have suggested that protein-protein interactions occur between the UGTs and may play a significant role in modulating enzyme activity. However, evidence of UGT protein-protein interactions either in vivo or in more physiologically relevant in vitro systems has yet to be demonstrated. In this study, we examined oligomerization and its ability to affect glucuronidation in plated human hepatocytes. siRNA down regulation experiments and activity studies were used to examine changes in metabolite formation of one UGT isoform due to down regulation of a second UGT isoform. Selective siRNA directed towards UGT1A9 or UGT2B7 resulted in significant and selective decreases in their respective mRNA levels. As expected, the metabolism of the UGT1A9 substrate propofol decreased with UGT1A9 down regulation. Interestingly, UGT1A9 activity, but not UGT1A9 mRNA expression, was also diminished when UGT2B7 expression was selectively inhibited, implying potential interactions between the two isoforms. Minor changes to UGT1A4, UGT2B4 and UGT2B7 activity were also observed when UGT1A9 expression was selectively down regulated. To our knowledge, this represents the first piece of evidence that UGT protein-protein interactions occur in human hepatocytes and suggests that expression levels of UGT2B7 may directly impact the glucuronidation activity of selective UGT1A9 substrates.

Enhancement of hepatic 4-hydroxylation of 25-hydroxyvitamin D3 through CYP3A4 induction in vitro and in vivo: implications for drug-induced osteomalacia.

Long-term therapy with certain drugs, especially cytochrome P450 (P450; CYP)-inducing agents, confers an increased risk of osteomalacia that is attributed to vitamin D deficiency. Human CYP24A1, CYP3A4, and CYP27B1 catalyze the inactivation and activation of vitamin D and have been implicated in the adverse drug response. In this study, the inducibility of these enzymes and monohydroxylation of 25-hydroxyvitamin D3 (25OHD3) were evaluated after exposure to P450-inducing drugs. With human hepatocytes, treatment with phenobarbital, hyperforin, carbamazepine, and rifampin significantly increased the levels of CYP3A4, but not CYP24A1 or CYP27B1 mRNA. In addition, rifampin pretreatment resulted in an 8-fold increase in formation of the major metabolite of 25OHD3, 4ß,25(OH)2D3. This inductive effect was blocked by the addition of 6',7'-dihydroxybergamottin, a selective CYP3A4 inhibitor. With human renal proximal tubular HK-2 cells, treatment with the same inducers did not alter CYP3A4, CYP24A1, or CYP27B1 expression. 24R,25(OH)2 D3 was the predominant monohydroxy metabolite produced from 25OHD3, but its formation was unaffected by the inducers. With healthy volunteers, the mean plasma concentration of 4ß,25(OH)2D3 was increased 60% (p < 0.01) after short-term rifampin administration. This was accompanied by a statistically significant reduction in plasma 1α,25(OH)2D3 (-10%; p = 0.03), and a nonsignificant change in 24R,25(OH)2D3 (-8%; p = 0.09) levels. Further analysis revealed a negative correlation between the increase in 4ß,25(OH)2D3 and decrease in 1α,25(OH)2D3 levels. Examination of the plasma monohydroxy metabolite/25OHD3 ratios indicated selective induction of the CYP3A4-dependent 4ß-hydroxylation pathway of 25OHD3 elimination. These results suggest that induction of hepatic CYP3A4 may be important in the etiology of drug-induced osteomalacia.

Quantitative prediction of CYP2B6 induction by estradiol during pregnancy: potential explanation for increased methadone clearance during pregnancy.

There is considerable evidence that pregnancy changes the disposition of drugs in an enzyme- and gestational stage-specific manner. On the basis of probe drug studies, the activity of CYP3A4 and CYP2D6 increases and CYP1A2 decreases during human pregnancy. However, no studies of CYP2B6 activity during human pregnancy have been conducted. In rodent models and in HepG2 cells, CYP2B enzymes have been shown to be regulated by estradiol. Because estradiol concentrations increase by ∼50-fold during human pregnancy, it was hypothesized that the increasing estradiol concentrations during human pregnancy would result in induction of CYP2B6 activity. Hepatocytes from three female donors were treated with estradiol, and the EC(50) and E(max) were measured for CYP2B6 mRNA and bupropion hydroxylation activity. The measured values were used to predict the magnitude of CYP2B6 induction during human pregnancy. At 100 nM total estradiol, a concentration achievable during the third trimester of pregnancy, CYP2B6 activity was predicted to increase by 1.5-3-fold, based on increased CYP2B6 activity and mRNA. When the E(max) and EC(50) values were compared with those for carbamazepine and rifampin, estradiol was found to be as potent an inducer of CYP2B6 as rifampin and carbamazepine. These data suggest that, during human pregnancy, the increasing estradiol concentrations will result in increased clearance of drugs that have CYP2B6-mediated clearance pathways. This could in part explain the observed increase in methadone clearance during pregnancy.