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Statistical analysis of in vivo tumor growth delay for dose reduction studies in SH-SY5Y xenografts and Felix-PDX.

Neuroblastoma cell line characteristics and Combination Index values in validation panel.

1.Western blot analysis of the cleaved caspase-3 and p53 pathways in Ceritinib and Ribociclib treated cells. 2.Combination ALK and CDK 4/6 inhibition leads to enhanced G0/G1 arrest.

Complete dataset for synergy screen.

Impact of ALK and CDK4 knockdown in NB-1691.

Overview of compound activity by cell line.

Purpose: Anaplastic lymphoma kinase (ALK) is the most frequently mutated oncogene in the pediatric cancer neuroblastoma. We performed an in vitro screen for synergistic drug combinations that target neuroblastomas with mutations in ALK to determine whether drug combinations could enhance antitumor efficacy.Experimental Design: We screened combinations of eight molecularly targeted agents against 17 comprehensively characterized human neuroblastoma-derived cell lines. We investigated the combination of ceritinib and ribociclib on in vitro proliferation, cell cycle, viability, caspase activation, and the cyclin D/CDK4/CDK6/RB and pALK signaling networks in cell lines with representative ALK status. We performed in vivo trials in CB17 SCID mice bearing conventional and patient-derived xenograft models comparing ceritinib alone, ribociclib alone, and the combination, with plasma pharmacokinetics to evaluate for drug–drug interactions.Results: The combination of ribociclib, a dual inhibitor of cyclin-dependent kinase (CDK) 4 and 6, and the ALK inhibitor ceritinib demonstrated higher cytotoxicity (P = 0.008) and synergy scores (P = 0.006) in cell lines with ALK mutations as compared with cell lines lacking mutations or alterations in ALK. Compared with either drug alone, combination therapy enhanced growth inhibition, cell-cycle arrest, and caspase-independent cell death. Combination therapy achieved complete regressions in neuroblastoma xenografts with ALK-F1174L and F1245C de novo resistance mutations and prevented the emergence of resistance. Murine ribociclib and ceritinib plasma concentrations were unaltered by combination therapy.Conclusions: This preclinical combination drug screen with in vivo validation has provided the rationale for a first-in-children trial of combination ceritinib and ribociclib in a molecularly selected pediatric population. Clin Cancer Res; 23(11); 2856–68. ©2016 AACR.

Supplementary Figure Legend

Statistical analysis of in vivo tumor growth delay and event-free survival in SH-SY5Y and NB-1691 xenografts.

Visceral leishmaniasis is responsible for up to 30,000 deaths every year. Current treatments have shortcomings that include toxicity and variable efficacy across endemic regions. Previously, we reported the discovery of GNF6702, a selective inhibitor of the kinetoplastid proteasome, which cleared parasites in murine models of leishmaniasis, Chagas disease, and human African trypanosomiasis. Here, we describe the discovery and characterization of LXE408, a structurally related kinetoplastid-selective proteasome inhibitor currently in Phase 1 human clinical trials. Furthermore, we present high-resolution cryo-EM structures of the Leishmania tarentolae proteasome in complex with LXE408, which provides a compelling explanation for the noncompetitive mode of binding of this novel class of inhibitors of the kinetoplastid proteasome.

NOD2 (nucleotide-binding oligomerization domain-containing protein 2) is an internal pattern recognition receptor that recognizes bacterial peptidoglycan and stimulates host immune responses. Dysfunction of NOD2 pathway has been associated with a number of autoinflammatory disorders. To date, direct inhibitors of NOD2 have not been described due to technical challenges of targeting the oligomeric protein complex. Receptor interacting protein kinase 2 (RIPK2) is an intracellular serine/threonine/tyrosine kinase, a key signaling partner, and an obligate kinase for NOD2. As such, RIPK2 represents an attractive target to probe the pathological roles of NOD2 pathway. To search for selective RIPK2 inhibitors, we employed virtual library screening (VLS) and structure based design that eventually led to a potent and selective RIPK2 inhibitor 8 with excellent oral bioavailability, which was used to evaluate the effects of inhibition of RIPK2 in various in vitro assays and ex vivo and in vivo pharmacodynamic models.

Purpose: Anaplastic lymphoma kinase (ALK) is the most frequently mutated oncogene in the pediatric cancer neuroblastoma. We performed an in vitro screen for synergistic drug combinations that target neuroblastomas with mutations in ALK to determine whether drug combinations could enhance antitumor efficacy. Experimental Design: We screened combinations of eight molecularly targeted agents against 17 comprehensively characterized human neuroblastoma-derived cell lines. We investigated the combination of ceritinib and ribociclib on in vitro proliferation, cell cycle, viability, caspase activation, and the cyclin D/CDK4/CDK6/RB and pALK signaling networks in cell lines with representative ALK status. We performed in vivo trials in CB17 SCID mice bearing conventional and patient-derived xenograft models comparing ceritinib alone, ribociclib alone, and the combination, with plasma pharmacokinetics to evaluate for drug–drug interactions. Results: The combination of ribociclib, a dual inhibitor of cyclin-dependent kinase (CDK) 4 and 6, and the ALK inhibitor ceritinib demonstrated higher cytotoxicity (P = 0.008) and synergy scores (P = 0.006) in cell lines with ALK mutations as compared with cell lines lacking mutations or alterations in ALK. Compared with either drug alone, combination therapy enhanced growth inhibition, cell-cycle arrest, and caspase-independent cell death. Combination therapy achieved complete regressions in neuroblastoma xenografts with ALK-F1174L and F1245C de novo resistance mutations and prevented the emergence of resistance. Murine ribociclib and ceritinib plasma concentrations were unaltered by combination therapy. Conclusions: This preclinical combination drug screen with in vivo validation has provided the rationale for a first-in-children trial of combination ceritinib and ribociclib in a molecularly selected pediatric population. Clin Cancer Res; 23(11); 2856–68. ©2016 AACR.

Current anti-trypanosomal therapies suffer from problems of longer treatment duration, toxicity and inadequate efficacy, hence there is a need for safer, more efficacious and ‘easy to use’ oral drugs. Previously, we reported the discovery of the triazolopyrimidine (TP) class as selective kinetoplastid proteasome inhibitors with in vivo efficacy in mouse models of leishmaniasis, Chagas Disease and African trypanosomiasis (HAT). For the treatment of HAT, development compounds need to have excellent penetration to the brain to cure the meningoencephalic stage of the disease. Here we describe detailed biological and pharmacological characterization of triazolopyrimidine compounds in HAT specific assays. The TP class of compounds showed single digit nanomolar potency against Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense strains. These compounds are trypanocidal with concentration-time dependent kill and achieved relapse-free cure in vitro. Two compounds, GNF6702 and a new analog NITD689, showed favorable in vivo pharmacokinetics and significant brain penetration, which enabled oral dosing. They also achieved complete cure in both hemolymphatic (blood) and meningoencephalic (brain) infection of human African trypanosomiasis mouse models. Mode of action studies on this series confirmed the 20S proteasome as the target in T. brucei. These proteasome inhibitors have the potential for further development into promising new treatment for human African trypanosomiasis.

Chagas disease, leishmaniasis and sleeping sickness affect 20 million people worldwide and lead to more than 50,000 deaths annually1. The diseases are caused by infection with the kinetoplastid parasites Trypanosoma cruzi, Leishmania spp. and Trypanosoma brucei spp., respectively. These parasites have similar biology and genomic sequence, suggesting that all three diseases could be cured with drugs that modulate the activity of a conserved parasite target2. However, no such molecular targets or broad spectrum drugs have been identified to date. Here we describe a selective inhibitor of the kinetoplastid proteasome (GNF6702) with unprecedented in vivo efficacy, which cleared parasites from mice in all three models of infection. GNF6702 inhibits the kinetoplastid proteasome through a non-competitive mechanism, does not inhibit the mammalian proteasome or growth of mammalian cells, and is well-tolerated in mice. Our data provide genetic and chemical validation of the parasite proteasome as a promising therapeutic target for treatment of kinetoplastid infections, and underscore the possibility of developing a single class of drugs for these neglected diseases.

Deregulated kinase activities of tropomyosin receptor kinase (TRK) family members have been shown to be associated with tumorigenesis and poor prognosis in a variety of cancer types. In particular, several chromosomal rearrangements involving TRKA have been reported in colorectal, papillary thyroid, glioblastoma, melanoma, and lung tissue that are believed to be the key oncogenic driver in these tumors. By screening the Novartis compound collection, a novel imidazopyridazine TRK inhibitor was identified that served as a launching point for drug optimization. Structure guided drug design led to the identification of (R)-2-phenylpyrrolidine substituted imidazopyridazines as a series of potent, selective, orally bioavailable pan-TRK inhibitors achieving tumor regression in rats bearing KM12 xenografts. From this work the (R)-2-phenylpyrrolidine has emerged as an ideal moiety to incorporate in bicyclic TRK inhibitors by virtue of its shape complementarity to the hydrophobic pocket of TRKs.

Neuroblastoma (NB) is one of the most common and deadly pediatric solid tumors. NB is characterized by clinical heterogeneity, from spontaneous regression to relentless progression despite intensive multimodality therapy. There is compelling evidence that members of the tropomyosin receptor kinase (Trk) family play important roles in these disparate clinical behaviors. Indeed, TrkB and its ligand, brain-derived neurotrophic factor (BDNF), are expressed in 50-60 % of high-risk NBs. The BDNF/TrkB autocrine pathway enhances survival, invasion, metastasis, angiogenesis and drug resistance.We tested a novel pan-Trk inhibitor, GNF-4256 (Genomics Institute of the Novartis Research Foundation), in vitro and in vivo in a nu/nu athymic xenograft mouse model to determine its efficacy in inhibiting the growth of TrkB-expressing human NB cells (SY5Y-TrkB). Additionally, we assessed the ability of GNF-4256 to enhance NB cell growth inhibition in vitro and in vivo, when combined with conventional chemotherapeutic agents, irinotecan and temozolomide (Irino-TMZ).GNF-4256 inhibits TrkB phosphorylation and the in vitro growth of TrkB-expressing NBs in a dose-dependent manner, with an IC₅₀ around 7 and 50 nM, respectively. Furthermore, GNF-4256 inhibits the growth of NB xenografts as a single agent (p0.0001 for mice treated at 40 or 100 mg/kg BID, compared to controls), and it significantly enhances the antitumor efficacy of irinotecan plus temozolomide (Irino-TMZ, p0.0071 compared to Irino-TMZ alone).Our data suggest that GNF-4256 is a potent and specific Trk inhibitor capable of significantly slowing SY5Y-TrkB growth, both in vitro and in vivo. More importantly, the addition of GNF-4256 significantly enhanced the antitumor efficacy of Irino-TMZ, as measured by in vitro and in vivo growth inhibition and increased event-free survival in a mouse xenograft model, without additional toxicity. These data strongly suggest that inhibition of TrkB with GNF-4256 can enhance the efficacy of current chemotherapeutic treatment for recurrent/refractory high-risk NBs with minimal or no additional toxicity.

Imidazopyridine 1 was identified from a phenotypic screen against P. falciparum (Pf) blood stages and subsequently optimized for activity on liver-stage schizonts of the rodent parasite P. yoelii (Py) as well as hypnozoites of the simian parasite P. cynomolgi (Pc). We applied these various assays to the cell-based lead optimization of the imidazopyrazines, exemplified by 3 (KAI407), and show that optimized compounds within the series with improved pharmacokinetic properties achieve causal prophylactic activity in vivo and may have the potential to target the dormant stages of P. vivax malaria.

The synthesis, preclinical profile, and in vivo efficacy in rat xenograft models of the novel and selective anaplastic lymphoma kinase inhibitor 15b (LDK378) are described. In this initial report, preliminary structure–activity relationships (SARs) are described as well as the rational design strategy employed to overcome the development deficiencies of the first generation ALK inhibitor 4 (TAE684). Compound 15b is currently in phase 1 and phase 2 clinical trials with substantial antitumor activity being observed in ALK-positive cancer patients.

Accumulation of β-amyloid (Aβ) in the brain is believed to contribute to the pathology of Alzheimer's Disease (AD). Aβ levels are controlled by the production of Aβ from amyloid precursor protein, degradation by proteases, and peripheral clearance. In this study we sought to determine whether enhancing clearance of plasma Aβ with a peripherally administered Aβ-degrading protease would reduce brain Aβ levels through a peripheral sink. Neprilysin (NEP) is a zinc-dependent metalloprotease that is one of the key Aβ-degrading enzymes in the brain. We developed a NEP fusion protein within vitrodegradation of Aβ and a 10 day plasma half-life in mouse. Intravenous administration of NEP to wild-type and APP23 transgenic mice resulted in dose-dependent clearance of plasma Aβ. However, this did not correspond to reduced levels of soluble brain Aβ with treatment up to 5 weeks in WT mice or formic acid-extractable brain Aβ with 3 month treatment in aged APP23. In contrast, intracranial injection of NEP resulted in an acute decrease in soluble brain Aβ. We found no change in amyloid precursor protein gene expression in mice treated with intravenous NEP, suggesting that the lack of effects in the brain following this route of administration was not caused by compensatory upregulation of Aβ production. Taken together, these results suggest a lack of a robust peripheral Aβ efflux sink through which brain amyloid burdens can be therapeutically reduced.

Successful drug discovery relies on the selection of drug candidates with good in vivo pharmacokinetic (PK) properties as well as appropriate preclinical efficacy and safety profiles. In vivo PK profiling is often a bottleneck in the discovery process. In this review, we focus on the tiered in vivo PK approaches implemented at the Genomics Institute of the Novartis Research Foundation (GNF), which includes snapshot PK, rapid PK and full PK studies. These in vivo PK approaches are well integrated within discovery research, allow tremendous flexibility and are highly efficient in supporting the diverse needs and increasing demand for in vivo profiling. The tiered in vivo PK studies expedite compound profiling and help guide the selection of more desirable compounds into efficacy models and for progression into development.

On the basis of the initial success of optimization of a novel series of imidazolopiperazines, a second generation of compounds involving changes in the core piperazine ring was synthesized to improve antimalarial properties. These changes were carried out to further improve the potency and metabolic stability of the compounds by leveraging the outcome of a set of in vitro metabolic identification studies. The optimized 8,8-dimethyl imidazolopiperazine analogues exhibited improved potency, in vitro metabolic stability profile and, as a result, enhanced oral exposure in vivo in mice. The optimized compounds were found to be more efficacious than the current antimalarials in a malaria mouse model. They exhibit moderate oral exposure in rat pharmacokinetic studies to achieve sufficient multiples of the oral exposure at the efficacious dose in toxicology studies.

Adult neurogenesis occurs in mammals and provides a mechanism for continuous neural plasticity in the brain. However, little is known about the molecular mechanisms regulating hippocampal neural progenitor cells (NPCs) and whether their fate can be pharmacologically modulated to improve neural plasticity and regeneration. Here, we report the characterization of a small molecule (KHS101) that selectively induces a neuronal differentiation phenotype. Mechanism of action studies revealed a link of KHS101 to cell cycle exit and specific binding to the TACC3 protein, whose knockdown in NPCs recapitulates the KHS101-induced phenotype. Upon systemic administration, KHS101 distributed to the brain and resulted in a significant increase in neuronal differentiation in vivo. Our findings indicate that KHS101 accelerates neuronal differentiation by interaction with TACC3 and may provide a basis for pharmacological intervention directed at endogenous NPCs.

In an effort to find new pharmacological modalities to overcome resistance to ATP-binding-site inhibitors of Bcr-Abl, we recently reported the discovery of GNF-2, a selective allosteric Bcr-Abl inhibitor. Here, using solution NMR, X-ray crystallography, mutagenesis and hydrogen exchange mass spectrometry, we show that GNF-2 binds to the myristate-binding site of Abl, leading to changes in the structural dynamics of the ATP-binding site. GNF-5, an analogue of GNF-2 with improved pharmacokinetic properties, when used in combination with the ATP-competitive inhibitors imatinib or nilotinib, suppressed the emergence of resistance mutations in vitro, displayed additive inhibitory activity in biochemical and cellular assays against T315I mutant human Bcr-Abl and displayed in vivo efficacy against this recalcitrant mutant in a murine bone-marrow transplantation model. These results show that therapeutically relevant inhibition of Bcr-Abl activity can be achieved with inhibitors that bind to the myristate-binding site and that combining allosteric and ATP-competitive inhibitors can overcome resistance to either agent alone.

The discovery, synthesis, and optimization of compound 1 from a high-throughput screening hit to highly potent and selective peroxisome proliferator-activated receptor delta (PPARdelta) agonists are reported. The synthesis and structure-activity relationship in this series are described in detail. On the basis of a general schematic PPAR pharmacophore model, scaffold 1 was divided into headgroup, linker, and tailgroup and successively optimized for PPAR activation using in vitro PPAR transactivation assays. A (2-methylphenoxy)acetic acid headgroup, a flexible linker, and a five-membered heteroaromatic center ring with two hydrophobic aryl substituents were required for efficient and selective PPARdelta activation. The fine-tuning of these aryl substituents led to an array of highly potent and selective compounds such as compound 38c, displaying an excellent pharmacokinetic profile in mouse. In an in vivo acute dosing model, selected members of this array were shown to induce the expression of pyruvate dehydrogenase kinase-4 (PDK4) and uncoupling protein-3 (UCP3), genes that are known to be involved in energy homeostasis and regulated by PPARdelta in skeletal muscle.

Described in this article are strategies implemented to increase the throughput of in vivo rodent pharmacokinetic (PK) studies using the snapshot PK study design and automated methods for compound submission, sample processing, data analysis and reporting. Applying snapshot PK studies to categorize the oral exposure of >1300 discovery compounds as low, moderate or high resulted in an attrition rate of 86%. The follow up full PK studies on the remaining compounds found that 98% of the compounds were predicted in the correct (69%) or adjacent (29%) oral exposure category by the snapshot PK studies. These results demonstrate that the snapshot PK screen in rodents can serve as an effective and efficient in vivo tool in the compound selection process in drug discovery.

The aim of the present study was to determine whether Cremophor EL is a suitable surfactant that can be routinely applied to pharmacokinetic (PK) studies in early drug discovery without influencing the intrinsic PK characteristics of the new chemical entities (NCEs). Cremophor EL, a polyoxyl 35 castor oil, has been used as a solubilization aid for water-insoluble compounds in pre-clinical drug discovery. The effect of Cremophor EL on the PK properties of NCEs was examined in seven structurally diverse discovery compounds after intravenous administration. Significant effects of Cremophor EL on plasma volume of distribution (Vss) and plasma clearance (CL) were observed in compounds with moderate to high Vss or CL. The plasma Vss decreased more than 2-fold and the Vss binning category decreased by one unit (e.g. from moderate to low Vss) in 6 of 7 test compounds. Two to five-fold reduction of CL was observed with these 6 compounds. Effect on the terminal half-life (T1/2) was minimal. Using one of these 7 NCEs, concentration dependent effect of Cremophor EL in the vehicle was also determined. Higher percentage of Cremophor EL in vehicle resulted in progressively increased alterations on the plasma CL and Vss. Taken together, these findings indicated that Cremophor EL altered the intrinsic PK properties of these discovery compounds in a concentration dependent manner.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase belonging to the insulin receptor superfamily. Expression of ALK in normal human tissues is only found in a subset of neural cells, however it is involved in the genesis of several cancers through genetic aberrations involving translocation of the kinase domain with multiple fusion partners (e.g., NPM-ALK in anaplastic large cell lymphoma ALCL or EML4-ALK in non-small cell lung cancer) or activating mutations in the full-length receptor resulting in ligand-independent constitutive activation (e.g., neuroblastoma). Here we are reporting the discovery of novel and selective anaplastic lymphoma kinase inhibitors from specific modifications of the 2,4-diaminopyridine core present in TAE684 and LDK378. Synthesis, structure activity relationships (SAR), absorption, distribution, metabolism, and excretion (ADME) profile, and in vivo efficacy in a mouse xenograft model of anaplastic large cell lymphoma are described.

Two CYP51 inhibitors, posaconazole and the ravuconazole prodrug E1224, were recently tested in clinical trials for efficacy in indeterminate Chagas disease. The results from these studies show that both drugs cleared parasites from the blood of infected patients at the end of the treatment but that parasitemia rebounded over the following months. In the current study, we sought to identify a dosing regimen of posaconazole that could permanently clear Trypanosoma cruzi from mice with experimental Chagas disease. Infected mice were treated with posaconazole or benznidazole, an established Chagas disease drug, and parasitological cure was defined as an absence of parasitemia recrudescence after immunosuppression. Twenty-day therapy with benznidazole (10 to 100 mg/kg of body weight/day) resulted in a dose-dependent increase in antiparasitic activity, and the 100-mg/kg regimen effected parasitological cure in all treated mice. In contrast, all mice remained infected after a 25-day treatment with posaconazole at all tested doses (10 to 100 mg/kg/day). Further extension of posaconazole therapy to 40 days resulted in only a marginal improvement of treatment outcome. We also observed similar differences in antiparasitic activity between benznidazole and posaconazole in acute T. cruzi heart infections. While benznidazole induced rapid, dose-dependent reductions in heart parasite burdens, the antiparasitic activity of posaconazole plateaued at low doses (3 to 10 mg/kg/day) despite increasing drug exposure in plasma. These observations are in good agreement with the outcomes of recent phase 2 trials with posaconazole and suggest that the efficacy models combined with the pharmacokinetic analysis employed here will be useful in predicting clinical outcomes of new drug candidates.

A series of PPARdelta-selective agonists was investigated and optimized for a favorable in vivo pharmacokinetic profile. Isoxazole LCI765 (17d) was found to be a potent and selective PPARdelta agonist with good in vivo PK properties in mouse (C(max)=5.1 microM, t(1/2)=3.1 h). LCI765 regulated expression of genes involved in energy homeostasis in relevant tissues when dosed orally in C57BL6 mice. A co-crystal structure of compound LCI765 and the LBD of PPARdelta is discussed.

The synthesis and structure-activity relationship of a series of arylaminoethyl amide cathepsin S inhibitors are reported. Optimization of P3 and P2 groups to improve overall physicochemical properties resulted in significant improvements in oral bioavailability over early lead compounds. An X-ray structure of compound 37 bound to the active site of cathepsin S is also reported.

A series of N α -2-benzoxazolyl-α-amino acid-(arylaminoethyl)amides were identified as potent, selective, and noncovalent inhibitors of cathepsin S. Structure–activity relationships including strategies for modulating the selectivities among cathepsins S, K, and L, and in vivo pharmacokinetics are discussed. A X-ray structure of compound 3 bound to the active site of cathepsin S is also reported.

The structure-based design, chemical synthesis, and biological evaluation of various 2-pyridone-containing human rhinovirus (HRV) 3C protease (3CP) inhibitors are described. These compounds are comprised of a peptidomimetic binding determinant and a Michael acceptor moiety, which forms an irreversible covalent adduct with the active site cysteine residue of the 3C enzyme. The 2-pyridone-containing inhibitors typically display improved 3CP inhibition properties relative to related peptide-derived molecules along with more favorable antiviral properties. The cocrystal structure of one pyridone-derived 3CP inhibitor complexed with HRV-2 3CP is also described along with certain ab initio conformation analyses. Optimization of the 2-pyridone-containing compounds is shown to provide several highly active 3CP inhibitors (k(obs)/[I]500,00 M(-1) s(-1)) that function as potent antirhinoviral agents (EC(50) =0.05 microM) against multiple virus serotypes in cell culture. One 2-pyridone-containing 3CP inhibitor is shown to be bioavailable in the dog after oral dosing (F = 48%).

Characterizing the relationship between the pharmacokinetics (PK, concentration vs. time) and pharmacodynamics (PD, effect vs. time) is an important tool in the discovery and development of new drugs in the pharmaceutical industry. The purpose of this publication is to serve as a guide for drug discovery scientists towards optimal design and conduct of PK/PD studies in the research phase. This review is a result of the collaborative efforts of DMPK scientists from various Metabolism and Pharmacokinetic (MAP) departments of the global organization Novartis Institute of Biomedical Research (NIBR). We recommend that PK/PD strategies be implemented in early research phases of drug discovery projects to enable successful transition to drug development. Effective PK/PD study design, analysis, and interpretation can help scientists elucidate the relationship between PK and PD, understand the mechanism of drug action and identify PK properties for further improvement and optimal compound design. Additionally, PK/PD modeling can help increase the translation of in vitro compound potency to the in vivo setting, reduce the number of in vivo animal studies, and improve translation of findings from preclinical species into the clinical setting. This review focuses on three important elements of successful PK/PD studies, namely partnership among key scientists involved in the study execution; parameters that influence study designs; and data analysis and interpretation. Specific examples and case studies are highlighted to help demonstrate key points for consideration. The intent is to provide a broad PK/PD foundation for colleagues in the pharmaceutical industry and serve as a tool to promote appropriate discussions on early research project teams with key scientists involved in PK/PD studies.

Systematic SAR optimization of the GPR119 agonist lead 1, derived from an internal HTS campaign, led to compound 29. Compound 29 displays significantly improved in vitro activity and oral exposure, leading to GLP1 elevation in acutely dosed mice and reduced glucose excursion in an OGTT study in rats at doses ⩾10 mg/kg.

Objective: We tested the hypothesis that the mechanism, rate, and extent of in vivo placental transfer of dideoxynucleoside drugs against human immunodeficiency virus can be predicted by the in vitro perfused human placenta and the drug octanol-water partition coefficient. Study Design: Near-term pregnant macaques ( Macaca nemestrina ) underwent long-term catheterization for the administration of 4 dideoxynucleosides against human immunodeficiency virus: zidovudine, didanosine, zalcitabine, and stavudine. Maternal plasma, fetal plasma, and amniotic fluid concentrations were determined frequently after intravenous bolus and/or infusion of the drugs administered into the maternal or fetal circulation on separate occasions. Antipyrine was included in all experiments as a marker of placental blood flow. The mechanism, rate, and extent of placental transfer of the 4 dideoxynucleosides in perfused human placenta were determined and compared with the findings obtained by others. Results: The mechanism and rate of the antipyrine-normalized placental transfer of the 4 dideoxynucleosides in perfused human placenta were highly correlated with those observed in vivo. The extent of placental transfer (fetal/maternal steady-state plasma concentration ratio) was also highly correlated with both the antipyrine-normalized placental transfer clearance (clearance index) determined in the in vitro perfused human placenta model ( r 2 = 0.95, in vitro clearance-index model) and the drug octanol-water partition coefficient ( r 2 = 0.99, in vitro partition-coefficient model). To determine the predictive capacity of these correlative models, we predicted the fetal/maternal steady-state plasma concentration ratio of each drug after excluding the data on that drug from the model fit. Both in vitro models to predict in vivo placental transfer of drug models resulted in good predictions of the observed fetal/maternal steady-state plasma concentration ratio (mean error: in vitro clearance-index model=−1.2%; in vitro partition-coefficient model=3.9%). Conclusions: We propose that our models will accurately predict the extent of placental transfer of dideoxynucleoside drugs against human immunodeficiency virus. The models may also be applicable to other classes of drugs, regardless of therapeutic category, provided that these drugs passively diffuse across the placenta. Such a result will expedite phase 1 clinical trials of drugs in pregnant women. (Am J Obstet Gynecol 1999;180:198−206.)

Screening hit 5 was identified in a biochemical screen for GPR119 agonists. Compound 5 was structurally novel, displayed modest biochemical activity and no oral exposure, but was structurally distinct from typical GPR119 agonist scaffolds. Systematic optimization led to compound 36 with significantly improved in vitro activity and oral exposure, to elevate GLP1 acutely in an in vivo mouse model at a dose of 10mg/kg.

Our objective was to determine the effect of pregnancy, mode of administration and neonatal age on the pharmacokinetics of the anti-HIV drug zalcitabine (2',3'-dideoxycytidine; ddC) in the pigtailed macaque (Macaca nemestrina). Zalcitabine was administered as an i.v. bolus dose to pregnant dams (n = 3) at term and at 6 weeks post-partum. No significant differences were found between the pre- and post-partum systemic plasma clearance, steady-state volume of distribution or terminal plasma half-life of zalcitabine, indicating that pregnancy does not affect the pharmacokinetics of the drug in the macaque. The observed maternal plasma, fetal plasma and amniotic fluid concentration-time profiles were compared with profiles that were simulated using pharmacokinetic parameter estimates obtained in an earlier constant i.v. infusion study in pregnant macaques. The fetal:maternal ratio of the area under the simulated zalcitabine plasma concentration-time profile after an i.v. bolus dose (0.58) was close to the earlier observed fetal:maternal steady-state plasma concentration ratio after i.v. infusion of the drug (0.58 +/- 0.05). The excellent agreement between observed and simulated fetal:maternal ratio of zalcitabine demonstrates that the steady-state infusion experimental design can be used to estimate the drug exposure to the fetus after a single dose. To determine the influence of age on the pharmacokinetics of zalcitabine, the drug was administered as a single i.v. bolus dose to four infant macaques serially at the ages of 1-2 weeks, 1 month and 4 months. The systemic plasma clearance of zalcitabine was significantly smaller and the terminal plasma half-life significantly longer at age 1-2 weeks than at 1 and 4 months of age. If replicated in humans, these substantial age-dependent changes in the pharmacokinetics of zalcitabine would warrant smaller and less frequent dosing with zalcitabine in HIV-infected neonates than in older children and adults.

The synthesis, preclinical profile, and in vivo efficacy in rat xenograft models of the novel and selective anaplastic lymphoma kinase inhibitor 15b (LDK378) are described. In this initial report, preliminary structure-activity relationships (SARs) are described as well as the rational design strategy employed to overcome the development deficiencies of the first generation ALK inhibitor 4 (TAE684). Compound 15b is currently in phase 1 and phase 2 clinical trials with substantial antitumor activity being observed in ALK-positive cancer patients.

OBJECTIVE: Our purpose was to determine whether the anti-human immunodeficiency virus drug zalcitabine (2',3'-dideoxycytidine) is actively transferred across the placenta in the near-term Macaca nemestrina. STUDY DESIGN: Constant rate infusions of zalcitabine (1.31 μg/min/kg) and antipyrine (66.7 μg/min/kg) were administered to the dam through the femoral vein ( n = 4) or to the fetus through the carotid artery ( n = 3) in a randomized cross-over design. Zalcitabine was also administered at a 10-fold higher infusion rate to the dam ( n = 3). The effect of zidovudine on the transplacental transfer of zalcitabine was studied by coinfusing the two drugs to the dam ( n = 2). Samples from maternal plasma, fetal plasma, and amniotic fluid were collected at regular intervals during the 30-hour infusions. RESULTS: The maternal-fetal transfer clearance of zalcitabine (0.41 ± 0.16 ml/min/kg) was not significantly different from the fetal-maternal transfer clearance of the drug (0.66 ± 0.30 ml/min/kg). Moreover, the steady-state fetal-maternal plasma concentration ratios of zalcitabine after the low (0.58 ± 0.06) and high (0.66 ± 0.10) infusion rates to the dam were similar. This ratio was not substantially changed (0.69) when zalcitabine was coadministered with zidovudine. The placental transfer rate of zalcitabine was 11% (±4%) that of antipyrine. CONCLUSION: The placental transfer of zalcitabine is passive and unaffected by simultaneous administration of zidovudine. In Macaca nemestrina the average fetal exposure to zalcitabine is approximately 60% of the maternal exposure. (AM J OBSTET GYNECOL 1996;174:856-63.)

A novel series of alkoxyimino derivatives as S1P1 agonists were discovered through de novo design using FTY720 as the chemical starting point. Extensive structure–activity relationship studies led to the discovery of (E)-1-(4-(1-(((4-cyclohexyl-3-(trifluoromethyl)benzyl)oxy)imino)ethyl)-2-ethylbenzyl)azetidine-3-carboxylic acid (32, BAF312, Siponimod), which has recently completed phase 2 clinical trials in patients with relapsing–remitting multiple sclerosis.

Through scaffold morphing of a known Smoothened antagonist Antag691, a series of novel phenyl imidazole derivatives were developed. Structure-activity-relationship studies and lead optimization led to the discovery of potent, selective and orally bioavailable Smoothened antagonist 19 that is suitable for in vivo studies.

To determine if there is active efflux of zidovudine (ZDV) and 2′,3′-dideoxyinosine (ddl) out of the cerebrospinal fluid (CSF), and if this efflux is saturable, we investigated the steady-state CSF/plasma concentration ratio of the two drugs when administered alone or in combination. Constant-rate infusions of ZDV, ddl or both were administered to seven macaques (Macaca nemestrina) through a chronic venous catheter for a minimum of 28 hr. Antipyrine, a marker of passive diffusion, was coinfused in all experiments. Blood (5 mL) and CSF samples (0.5–1 mL) were collected by venous and lumbar/thoracic punctures, respectively, at 24 and 28 hr after beginning the infusion. When ZDV and ddl were administered alone, the steady-state CSF/plasma concentration ratios were significantly different from unity (ZDV, 0.20 ± 0.08; ddI, 0.09 ± 0.04) and were independent of the plasma concentration (P > 0.05). In contrast, the CSF/plasma concentration ratio of antipyrine (0.82 ± 0.19) was close but significantly smaller than unity (P > 0.05). The CSF/ plasma concentration ratios after simultaneous administration of ZDV and ddI were not significantly different (P > 0.05) from those obtained after administration of the drugs alone. These results suggest that ZDV and ddI are actively transported out of the CSF; however, within the concentration range studied, this efflux is neither saturable nor mutually competitive. Concomitant administration of ZDV and ddI did not produce a systemic interaction in the animals, indicating that the pharmacokinetics of either drug is unaffected by the presence of the other.

Most malaria drug development focuses on parasite stages detected in red blood cells, even though, to achieve eradication, next-generation drugs active against both erythrocytic and exo-erythrocytic forms would be preferable. We applied a multifactorial approach to a set of >4000 commercially available compounds with previously demonstrated blood-stage activity (median inhibitory concentration < 1 micromolar) and identified chemical scaffolds with potent activity against both forms. From this screen, we identified an imidazolopiperazine scaffold series that was highly enriched among compounds active against Plasmodium liver stages. The orally bioavailable lead imidazolopiperazine confers complete causal prophylactic protection (15 milligrams/kilogram) in rodent models of malaria and shows potent in vivo blood-stage therapeutic activity. The open-source chemical tools resulting from our effort provide starting points for future drug discovery programs, as well as opportunities for researchers to investigate the biology of exo-erythrocytic forms.

Neurotrophins and their receptors (TRKs) play key roles in the development of the nervous system and the maintenance of the neural network. Accumulating evidence points to their role in malignant transformations, chemotaxis, metastasis, and survival signaling and may contribute to the pathogenesis of a variety of tumors of both neural and non-neural origin. By screening the GNF kinase collection, a series of novel oxindole inhibitors of TRKs were identified. Optimization led to the identification of GNF-5837 (22), a potent, selective, and orally bioavailable pan-TRK inhibitor that inhibited tumor growth in a mouse xenograft model derived from RIE cells expressing both TRKA and NGF. The properties of 22 make it a good tool for the elucidation of TRK biology in cancer and other nononcology indications.

[Image: see text] Starting from a hit series from a GNF compound library collection and based on a cell-based proliferation assay of Plasmodium falciparum, a novel imidazolopiperazine scaffold was optimized. SAR for this series of compounds is discussed, focusing on optimization of cellular potency against wild-type and drug resistant parasites and improvement of physiochemical and pharmacokinetic properties. The lead compounds in this series showed good potencies in vitro and decent oral exposure levels in vivo. In a Plasmodium berghei mouse infection model, one lead compound lowered the parasitemia level by 99.4% after administration of 100 mg/kg single oral dose and prolonged mice survival by an average of 17.0 days. The lead compounds were also well-tolerated in the preliminary in vitro toxicity studies and represents an interesting lead for drug development.

The second generation of Bcr-Abl inhibitors nilotinib, dasatinib, and bosutinib developed to override imatinib resistance are not active against the T315I “gatekeeper” mutation. Here we describe a type-II T315I inhibitor 2 (GNF-7), based upon a 3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one scaffold which is capable of potently inhibiting wild-type and T315I Bcr-Abl as well as other clinically relevant Bcr-Abl mutants such as G250E, Q252H, Y253H, E255K, E255V, F317L, and M351T in biochemical and cellular assays. In addition, compound 2 displayed significant in vivo efficacy against T315I-Bcr-Abl without appreciable toxicity in a bioluminescent xenograft mouse model using a transformed T315I-Bcr-Abl-Ba/F3 cell line that has a stable luciferase expression. Compound 2 is among the first type-II inhibitors capable of inhibiting T315I to be described and will serve as a valuable lead to design the third generation Bcr-Abl kinase inhibitors.

A novel family of 1H-imidazol-2-yl-pyrimidine-4,6-diamines has been identified with potent activity against the erythrocyte-stage of Plasmodium falciparum (Pf), the most common causative agent of malaria. A systematic SAR study resulted in the identification of compound 40 which exhibits good potency against both wild-type and drug resistant parasites and exhibits good in vivo pharmacokinetic properties.