Validation of a New Methodology to Create Oral Drugs beyond the Rule of 5 for Intracellular Tough Targets.

Establishing a technological platform for creating clinical compounds inhibiting intracellular protein-protein interactions (PPIs) can open the door to many valuable drugs. Although small molecules and antibodies are mainstream modalities, they are not suitable for a target protein that lacks a deep cavity for a small molecule to bind or a protein found in intracellular space out of an antibody's reach. One possible approach to access these targets is to utilize so-called middle-size cyclic peptides (defined here as those with a molecular weight of 1000-2000 g/mol). In this study, we validated a new methodology to create oral drugs beyond the rule of 5 for intracellular tough targets by elucidating structural features and physicochemical properties for drug-like cyclic peptides and developing library technologies to afford highly N-alkylated cyclic peptide hits. We discovered a KRAS inhibitory clinical compound (LUNA18) as the first example of our platform technology.

Development of Orally Bioavailable Peptides Targeting an Intracellular Protein: From a Hit to a Clinical KRAS Inhibitor.

Cyclic peptides as a therapeutic modality are attracting a lot of attention due to their potential for oral absorption and accessibility to intracellular tough targets. Here, starting with a drug-like hit discovered using an mRNA display library, we describe a chemical optimization that led to the orally available clinical compound known as LUNA18, an 11-mer cyclic peptide inhibitor for the intracellular tough target RAS. The key findings are as follows: (i) two peptide side chains were identified that each increase RAS affinity over 10-fold; (ii) physico-chemical properties (PCP) including Clog P can be adjusted by side-chain modification to increase membrane permeability; (iii) restriction of cyclic peptide conformation works effectively to adjust PCP and improve bio-activity; (iv) cellular efficacy was observed in peptides with a permeability of around 0.4 × 10-6 cm/s or more in a Caco-2 permeability assay; and (v) while keeping the cyclic peptide's main-chain conformation, we found one example where the RAS protein structure was changed dramatically through induced-fit to our peptide side chain. This study demonstrates how the chemical optimization of bio-active peptides can be achieved without scaffold hopping, much like the processes for small molecule drug discovery that are guided by Lipinski's rule of five. Our approach provides a versatile new strategy for generating peptide drugs starting from drug-like hits.

Discovery of CH7057288 as an Orally Bioavailable, Selective, and Potent pan-TRK Inhibitor.

Kinase fusions involving tropomyosin receptor kinases (TRKs) have been proven to act as strong oncogenic drivers and are therefore recognized as attractive therapeutic targets. We screened an in-house kinase-focused library and identified a promising hit compound with a unique tetracyclic scaffold. Compound 1 showed high TRK selectivity but moderate cell growth inhibitory activity as well as a potential risk of inducing CYP3A4. In this report, chemical modification intended to improve TRK inhibition and avoid CYP3A4 induction enabled us to identify an orally bioavailable, selective, and potent TRK inhibitor 7.

Evaluation of Methods to Assess CYP3A Induction Risk in Clinical Practice Using in Vitro Induction Parameters.

Established guidelines have recommended a number of methods based on in vitro data to assess the CYP3A induction risk of new chemical entities in clinical practice. In this study, we evaluated the predictability of various assessment methods. We collected in vitro parameters from a variety of literature that includes data on 19 batches of hepatocytes. Clinical CYP3A induction was predicted using 3 direct approaches-the fold-change, basic model, and mechanistic static models-as well as 5 correlation approaches, including the relative induction score (RIS) and the relative factor (RF) method. These predictions were then compared with data from 30 clinical inductions. Collected in vitro parameters varied greatly between hepatocyte batches. Direct assessment methods using fixed cut-off values provided a lot of false predictions due to hepatocyte variability, which can overlook induction risk or lead to needless clinical drug-drug interaction (DDI) studies. On the other hand, correlation methods with the cut-off values set for each batch of hepatocytes accurately predicted the induction risk. Among these, the AUCu/inducer concentrations for half the maximum induction (EC50) and the RF methods which use the area under the curve (AUC) of the unbound inducers for calculating induction potential showed an especially good correlation with clinical induction. Correlation methods were better at predicting clinical induction risk than the other methods, regardless of hepatocyte variability. The AUCu/EC50 and the RF methods in particular had a small number of false predictions, and can therefore be used to assess induction risk along with the other correlation methods recommended in guidelines.

Quantitative evaluation of hepatic and intestinal induction of CYP3A in clinical practice.

This is the first report quantitatively evaluating the clinical induction of CYP3A in the liver and the intestine.To evaluate hepatic induction, we collected literature data on endogenous biomarkers of hepatic CYP3A induction which we then used to calculate the fold-induction (inducer-mediated change in biomarker level). Literature data on decreases in the area under the curve (AUC) of alfentanil, a CYP3A substrate, caused by CYP3A inducers were also collected. We used the hepatic intrinsic clearance of alfentanil to calculate the hepatic induction ratio (inducer-mediated change in intrinsic clearance). For intestinal induction, the intestinal bioavailability (Fg) of alfentanil was used to calculate the intestinal induction ratio. We determined in vivo maximum induction (Emax) and the average unbound plasma concentration (Cav,u) required for half the maximum induction (EC50) for inducers using an Emax model analysis.In our results, fold-induction was comparable to the induction ratio at several inducer concentrations, and almost the maximum induction was achieved by a therapeutic dose. Induction ratios in the intestine were similar to the liver.Our findings suggest that, by knowing only hepatic induction ratios for common inducers, we can quantitatively predict the decreases in the AUC of substrates by CYP3A induction.

Risk of CYP2C9 induction analyzed by a relative factor approach with human hepatocytes.

By using the Relative Factor (RF) method-a method that can simply assess cytochrome P450 (CYP) induction risk based on a maximum induction effect model-we evaluated the risk of CYP2C9 induction and examined its relationship with risk of CYP3A4 induction. In cryopreserved human hepatocytes, the magnitude of CYP2C9 induction by eight drugs known to induce CYP3A4 was lower than the magnitude of CYP3A4 induction, but the magnitudes of induction of both were correlated. The RF values determined for CYP2C9 had a one-to-one linear relationship with values determined for CYP3A4, supporting reports that the induction mechanism of both enzymes is the same. Furthermore, clinical CYP2C9 induction data of compounds reported to induce CYP2C9 clinically were shown to be lower than those of CYP3A4. The thresholds for CYP2C9 induction risk assessment by the RF approach were determined to be at higher steady-state plasma concentrations than those for CYP3A4. Based on these results, induction of CYP2C9 was correlated with that of CYP3A4, and induction risk could be evaluated by the RF method using hepatocytes. The CYP2C9 induction risk of a compound was confirmed to be lower than its CYP3A4 induction risk.

Simple Evaluation Method for CYP3A4 Induction from Human Hepatocytes: The Relative Factor Approach with an Induction Detection Limit Concentration Based on the Emax Model.

We investigated the robustness and utility of the relative factor (RF) approach based on the maximum induction effect (Emax) model, using the mRNA induction data of 10 typical CYP3A4 inducers in cryopreserved human hepatocytes. The RF value is designated as the ratio of the induction detection limit concentration (IDLC) for a standard inducer, such as rifampicin (RIF) or phenobarbital (PB), to that for the compound (e.g., RFRIF is IDLCRIF/IDLCcpd; RFPB is IDLCPB/IDLCcpd). An important feature of the RF approach is that the profiles of the induction response curves on the logarithmic scale remain unchanged irrespective of inducers but are shifted parallel depending on the EC50 values. A key step in the RF approach is to convert the induction response curve by finding the IDLC of a standard inducer. The relative induction score was estimated not only from Emax and EC50 values but also from those calculated by the RF approach. These values showed good correlation, with a correlation coefficient of more than 0.974, which revealed the RF approach to be a robust analysis irrespective of its simplicity. Furthermore, the relationship between RFRIF or RFPB multiplied by the steady-state unbound plasma concentration and the in vivo induction ratio plotted using 10 typical inducers gives adequate thresholds for CYP3A4 drug-drug interaction risk assessment. In light of these findings, the simple RF approach using the IDLC value could be a useful method to adequately assess the risk of CYP3A4 induction in humans during drug discovery and development without evaluation of Emax and EC50.

Estimating the clinical risk of hypertension from VEGF signal inhibitors by a non-clinical approach using telemetered rats.

Anti-angiogenic drugs that target Vascular Endothelial Growth Factor (VEGF) signaling pathways caused hypertension as an adverse effect in clinical studies. Since the hypertension may limit the benefit provided for patients, the demand for non-clinical research that predicts the clinical risk of the hypertension has risen greatly. To clarify whether non-clinical research using rats can appropriately estimate the clinical risk of hypertension caused by VEGF signal inhibitors, we investigated the hemodynamic effects and pharmacokinetics (PK) of the VEGF signal inhibitors cediranib (0.1, 3, and 10 mg/kg), sunitinib (5, 10, and 40 mg/kg), and sorafenib (0.1, 1, and 5 mg/kg) in telemetered rats and examined the correlation between the non-clinical and the clinical hypertensive effect. The VEGF signal inhibitors significantly elevated blood pressure (BP) in rats within a few days of the initiation of dosing, and levels recovered after dosing ended. The trend of the hypertension was similar to that in clinical studies. We found that the AUC at which BP significantly increased by approximately 10 mmHg in rats was comparable to the clinical AUC at which moderate to severe hypertension occurred. These results represent correlations between the non-clinical and the clinical hypertensive effect of VEGF signal inhibitors, suggesting that non-clinical research using telemetered rats would be an effective approach to predict the clinical risk of hypertension caused by VEGF signal inhibitors.

Angiogenesis inhibitors identified by cell-based high-throughput screening: synthesis, structure-activity relationships and biological evaluation of 3-[(E)-styryl]benzamides that specifically inhibit endothelial cell proliferation.

Proliferation of endothelial cells is critical for angiogenesis. We report orally available, in vivo active antiangiogenic agents which specifically inhibit endothelial cell proliferation. After identifying human umbilical vein endothelial cell (HUVEC) proliferation inhibitors from a cell-based high-throughput screening (HTS), we eliminated those compounds which showed cytotoxicity against HCT116 and vascular endothelial growth factor receptor 2 (VEGFR-2) inhibitory activity. Evaluations in human Calu-6 xenograft model delivered lead compound 1. Following extensive lead optimization and alteration of the scaffold we discovered 32f and 32g, which both inhibited the proliferation and tube formation of HUVEC without showing inhibitory activity against any of 25 kinases or cytotoxicity against either normal fibroblasts or 40 cancer cell lines. Upon oral administration, 32f and 32g had good pharmacokinetic profiles and potent antitumor activity and decreased microvessel density (MVD) in Calu-6 xenograft model. Combination therapy with a VEGFR inhibitor enhanced the in vivo efficacy. These results suggest that 32f and 32g may have potential for use in cancer treatment.

The selective class I PI3K inhibitor CH5132799 targets human cancers harboring oncogenic PIK3CA mutations.

PURPOSE: The phosphatidylinositol 3-kinase (PI3K) pathway plays a central role in cell proliferation and survival in human cancer. PIK3CA mutations, which are found in many cancer patients, activate the PI3K pathway, resulting in cancer development and progression. We previously identified CH5132799 as a novel PI3K inhibitor. Thus, this study aimed to clarify the biochemical and antitumor activity of CH5132799 and elucidate the correlation between CH5132799 response and genetic alterations in the PI3K pathway. EXPERIMENTAL DESIGN: Kinase inhibitory activity was profiled in cell-free assays. A large panel of human breast, ovarian, prostate, and endometrial cancer cell lines, as well as xenograft models, were used to evaluate the antitumor activity of CH5132799, followed by analysis for genetic alterations. Effects on Akt phosphorylation induced by mTORC1 inhibition were tested with CH5132799 and compared with mTORC1 and PI3K/mTOR inhibitors. RESULTS: CH5132799 selectively inhibited class I PI3Ks and PI3Kα mutants in in vitro kinase assays. Tumors harboring PIK3CA mutations were significantly sensitive to CH5132799 in vitro and were remarkably regressed by CH5132799 in in vivo mouse xenograft models. In combination with trastuzumab, tumors disappeared in the trastuzumab-insensitive breast cancer model with the PIK3CA mutation. Moreover, CH5132799 did not reverse a negative feedback loop of PI3K/Akt/mTOR signaling and induced regression against tumors regrown after long-term mTORC1 inhibitor treatment. CONCLUSIONS: CH5132799 is a selective class I PI3K inhibitor with potent antitumor activity against tumors harboring the PIK3CA mutations. Prediction of CH5132799 response on the basis of PIK3CA mutations could enable patient stratification in clinical settings.

Discovery and biological activity of a novel class I PI3K inhibitor, CH5132799.

Phosphatidylinositol 3-kinase (PI3K) is a lipid kinase and a promising therapeutic target for cancer. Using structure-based drug design (SBDD), we have identified novel PI3K inhibitors with a dihydropyrrolopyrimidine skeleton. Metabolic stability of the first lead series was drastically improved by replacing phenol with aminopyrimidine moiety. CH5132799, a novel class I PI3K inhibitor, exhibited a strong inhibitory activity especially against PI3Kα (IC(50)=0.014 µM). In human tumor cell lines with PI3K pathway activation, CH5132799 showed potent antiproliferative activity. CH5132799 is orally available and showed significant antitumor activity in PI3K pathway-activated human cancer xenograft models in mice.

Prediction of drug-induced QT interval prolongation in telemetered common marmosets.

Drug-induced QT interval prolongation is a critical issue in development of new chemical entities, so the pharmaceutical industry needs to evaluate risk as early as possible. Common marmosets have been in the limelight in early-stage development due to their small size, which requires only a small amount of test drug. The purpose of this study was to determine the utility of telemetered common marmosets for predicting drug-induced QT interval prolongation. Telemetry transmitters were implanted in common marmosets (male and female), and QT and RR intervals were measured. The QT interval was corrected for the RR interval by applying Bazett's and Fridericia's correction formulas and individual rate correction. Individual correction showed the least slope for the linear regression of corrected QT (QTc) intervals against RR intervals, indicating that it dissociated changes in heart rate most effectively. With the individual correction method, the QT-prolonging drugs (astemizole, dl-sotalol) showed QTc interval prolongations and the non-QT-prolonging drugs (dl-propranolol, nifedipine) did not show QTc interval prolongations. The plasma concentrations of astemizole and dl-sotalol associated with QTc interval prolongations in common marmosets were similar to those in humans, suggesting that the sensitivity of common marmosets would be appropriate for evaluating risk of drug-induced QT interval prolongation. In conclusion, telemetry studies in common marmosets are useful for predicting clinical QT prolonging potential of drugs in early stage development and require only a small amount of test drug.

Cloning and functional expression of a novel marmoset cytochrome P450 2D enzyme, CYP2D30: comparison with the known marmoset CYP2D19.

Using a primer set designed on the cDNA encoding the known marmoset cytochrome P450 2D19 (CYP2D19), a cDNA encoding a novel CYP2D enzyme (CYP2D30) was cloned from the liver of a female marmoset bred at Kyoto University (KYU). In addition, a cDNA encoding CYP2D19 was cloned from the liver of a female marmoset bred at Kagoshima University (KAU). CYP2D30 and CYP2D19 showed homologies of 93.6 and 93.4% in their nucleotide and amino acid sequences, respectively. Reverse transcription polymerase chain reaction (RT-PCR) and digestion with NdeI demonstrated that the KYU-marmoset liver contained mainly mRNA for CYP2D30, while the KAU-marmoset liver contained mainly mRNA for CYP2D19. Marmoset CYP2D30, like human CYP2D6, exhibited high debrisoquine (DB) 4-hydroxylase activity and relatively low DB 5-, 6-, 7- and 8-hydroxylase activities, whereas CYP2D19 lacked DB 4-hydroxylase but exhibited marked 5-, 6-, 7- and 8-hydroxylase activities. The two marmoset recombinant enzymes showed enantioselective bufuralol (BF) 1"-hydroxylase activities, similar to CYP2D6. BF 1"-hydroxylation by CYP2D30 exhibited product-enantioselectivity of (1"R-OH-BF << 1"S-OH-BF), similar to that observed with human CYP2D6, whereas CYP2D19 showed a reversed selectivity of (1"R-OH-BF > or = 1"S-OH-BF). BF 1"-hydroxylation in marmoset liver microsomes from both sources was inhibited by antibodies raised against rat CYP2D1 in a concentration-dependent manner. A known inhibitor of CYP2D6, quinidine, effectively inhibited the BF 1"-hydroxylation activities in liver microsomal fractions prepared from KYU- and KAU-marmosets. These results suggest that CYP2D19 and CYP2D30 proteins can be expressed as functional enzymes in marmoset livers, although it is unresolved whether both enzymes coexist in the same marmoset liver.

Stereoselectivity in the oxidation of bufuralol, a chiral substrate, by human cytochrome P450s.

Bufuralol (BF), a nonselective beta-adrenoceptor blocking agent, has a chiral center in its molecule, yielding the enantiomers 1'R-BF and 1'S-BF. beta-Adrenoceptor blocking potency is much higher in 1'S-BF than in 1'R-BF. One of the metabolic pathways of BF is 1"-hydroxylation of an ethyl group attached at the aromatic 7-position forming a carbinol metabolite (1"-hydroxybufuralol, 1"-OH-BF), and further oxidation (or dehydrogenation) produces a ketone metabolite (1-oxobufuralol, 1"-Oxo-BF). Both 1"-OH-BF and 1"-Oxo-BF are known to have beta-adrenoceptor blocking activities comparable to or higher than those of the parent drug. The 1"-hydroxylation introduces another chiral center into the BF molecule and four 1"-OH-BF diastereomers are formed from BF racemate in mammals, including humans, making elucidation of the metabolic profiles complicated. HPLC methods employing derivatization, reversed phase, or chiral columns have been developed to efficiently separate the four 1"-OH-BF diastereomers formed from BF enantiomers or racemate. Accumulated in vitro experimental results revealed that 1'R-BF is a much more preferential substrate than 1'S-BR for BF 1"-hydroxylation in human liver microsomes. Kinetic studies using recombinant human cytochrome P450 (CYP) enzymes indicate that CYP2D6 serves as a major BF 1"-hydroxylase and that CYP1A2 and CYP2C19 also contribute to BF 1"-hydroxylation in human livers. This mini-review summarizes the knowledge reported so far on the pharmacology of BF and its metabolites and the profiles of BF metabolism, especially focusing on the stereoselectivity in the oxidation of BF mainly in human livers and recombinant CYP enzymes.