Historically, thalidomide-induced congenital malformations have served as an important example of the enhanced susceptibility of developing embryos to chemical perturbation. The compound produced a wide variety of congenital malformations in humans, which were initially detected by an association with a relatively rare limb defect labeled phocomelia. Although true phocomelia in the most severe form is a transverse defect with intercalary absence of limb regions, it is proposed that thalidomide produces a longitudinal limb phenotype in humans under usual circumstances that can become transverse in severe cases with a preferential sensitivity of forelimb over hindlimb, preaxial over postaxial, and left more impacted than the corresponding non-autopod limb bones on the right. The thalidomide-induced limb phenotype in humans is described and followed by a hierarchical comparison with various laboratory animal species. Mechanistic studies have been hampered by the fact that only non-human primates and rabbits have malformations that are anatomically similar to humans. Included in this review are unpublished data on limb malformations produced by thalidomide in rhesus monkeys from experiments performed more than 50 years ago. The critical period in gestation for the induction of phocomelia may initiate prior to the development of the embryonic limb bud, which contrasts with other chemical and physical agents that are known to produce this phenotype. The importance of toxicokinetic parameters is reviewed including dose, enantiomers, absorption, distribution, and both non-enzymatic and enzymatic biotransformations. The limb embryopathy mechanism that provides a partial explanation of the limb phenotype is that cereblon binds to thalidomide creating a protein complex that ubiquitinates protein substrates (CRL4CRBN) that are not targets for the complex in the absence of the thalidomide. One of these neosubstrates is SALL4 which when mutated causes a syndrome that phenocopies aspects of thalidomide embryopathy. Other candidate neosubstrates for the complex that have been found in non-human species may contribute to an understanding of the limb defect including PLZF, p63, and various zinc finger transcription factors. It is proposed that it is important to consider the species-specificity of the compound when considering potential mechanistic pathways and that some of the more traditional mechanisms for explaining the embryopathy, such as anti-angiogenesis and redox perturbation, may contribute to a full understanding of this teratogen.

Supplementary Materials and Methods. Supplemental Table S1. Combination effects of BAY 80-6946 and lapatinib on caspase 9 activation in BT474 cells. Supplemental Table S2. Single dose i.v. pharmacokinetic parameter of BAY 80-6946 in female nude rats and ICR mice (n=3 per timepoint). Supplemental Table S3. Combination effects of BAY 80-6946 and paclitaxel in patient-derived NSCLC xenograft tumor model. Supplemental Figure S1: Chemical structure of reference compounds. Supplemental Figure S2. Nuclear translocation of FOXO1A in U2OS cells. Supplemental Figure S3. Caspase 9 activity in BT474 and BT20 cells measured 24 hours after exposure to BAY 80-6946 and reference drugs. Supplemental Figure S4. Single and multiple dose i.v. plasma concentration versus time profile of BAY 80-6946 in female nude rats (n=2 per timepoint).

Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada BC Children's Research Institute, Vancouver, British Columbia, Canada Division of Developmental Biology, University of Cincinnati, Cincinnati, Ohio Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Manitoba, Canada Department of Radiology, University of Manitoba, Winnipeg, Manitoba, Canada

Abstract Introduction: Several generations of PI3K inhibitors have been tested in clinic. However, thus far, clinical activity has been moderate. Different from other oral PI3K inhibitors dosed continuously, copanlisib (BAY 80-6946) is an intravenous PI3K inhibitor given intermittently to patients. Copanlisib dosed once weekly demonstrated clinical benefit with an improved safety profile, and therefore challenges the concept of default continuous dosing of PI3K inhibitors. However, it is still unclear if this concept can be generalized and whether ‘micropharmacokinetic parameters’ also contributed to the potent anti-tumor profile of copanlisib. Here, we report the characterization of binding kinetics for copanlisib, as well as the functional consequence in vivo. Methods: A set of PI3K inhibitors were characterized in 1) a kinetic probe competition assay (kPCA); 2) a cellular nanoBRET target engagement assay; 3) a cellular washout study with the assessment on pathway engagement; and 4) in vivo pharmacokinetics analysis. Results: Copanlisib showed nearly diffusion-controlled on- and relatively slow off-rates with kon = 3.45E+7 [M-1*s-1] and koff = 1.67E-3 [s-1] to PI3Kα. Consequently, it exhibited very high affinity to PI3Kα ( Ki ePCA = 9.31E-11[M] and KD kPCA = 4.77E-11 [M]). In a cellular nanoBRET target engagement assay, the apparent half-life (t1/2) of ca. 2 hours greatly surpassed the 6.9 min measured using kPCA. The high affinity to PI3Kα also translated into potent cellular pathway engagement demonstrated by inhibition of p-AKT and p-PRAS40 in the PIK3CAmut KPL4 cell line. In a cellular washout study, p-AKT and p-PRAS40 were assessed till 168 h after incubation with copanlisib for 1 h followed by a washout step. A dose- and time-dependent pathway engagement was observed even at 72 h post washout. This result indicated that in cells, copanlisib engages PI3Kα for an extremely long time, likely due to rebinding effects facilitated by the fast equilibration kinetics of the compound and its micropharmacokinetic properties. Interestingly, in vivo, BAY 80-6946 levels were approximately 100-fold higher in the tumor than in plasma at 48 hours and drug clearance from the tumor occurred more slowly than from plasma. This high and prolonged tumor exposure might be explained, at least in part, by the high expression of PI3Kα and long lasting target occupancy of copanlisib in tumors. Conclusion: Copanlisib demonstrated high affinity to PI3Kα with protracted target engagement at cellular and in vivo levels. This ‘micropharmacokinetic feature’ not only supports intermittent dosing but likely also explains the high exposure in tumors vs plasma, potent anti-tumor activity and good safety profiles. Citation Format: Amaury E. Fernández-Montalván, Victoria Georgi, James Vasta, Sarah Glaeske, Vera Puetter, Matthew B. Robers, Ursula Moenning, Andrea Sturz, Julien Lefranc, Karl Ziegelbauer, Michael Brands, Christian Stegmann, William J. Scott, Ningshu Liu. High target binding affinity with long lasting cellular target engagement and high dose intermittent schedule of PI3K inhibitor copanlisib contribute to the potent anti-tumor activity and good safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 160. doi:10.1158/1538-7445.AM2017-160

Abstract The PI3K/AKT/mTOR pathway is frequently activated in human cancer. AKT, a central element in the pathway, is essential for tumor growth, proliferation, survival, invasion and metastasis. Activation of AKT is a key mechanism in resistance to chemo-, radio- and targeted therapies. Thus, AKT is considered an attractive drug target. Herein, we report on the preclinical profile and combinability of BAY 1125976, a potent, highly selective, allosteric AKT1/2 inhibitor, which is particularly effective in models with PI3K-AKT pathway aberrations. In biochemical assays, BAY 1125976 demonstrates equal potency against AKT1 and AKT2 in the low nanomolar range (IC50 ∼ 10 nM) while it displays weaker activity against AKT3 (IC50 ∼ 500 nM) and is inactive against ∼230 other protein/ lipid kinases (IC50 > 1 μM). Mechanistically, BAY 1125976 blocks AKT signalling by inhibiting the phosphorylation of AKT at both Thr308 and Ser473 (IC50 < 1 nM), as well as downstream phosphorylation of 4E-BP1 (IC50 < 50 nM). The strong inhibition of cellular p-AKT and downstream signalling translates to a broad inhibition of tumor cell proliferation in vitro. In particular, tumor cell lines carrying defects in the tumor suppressor PTEN, or oncogenic mutations in PIK3CA are most sensitive to BAY 1125976 treatment. Daily oral dosing of BAY 1125976 in human xenograft tumor models induces strong pharmacodynamic inhibition of AKT phosphorylation that correlates with drug exposure. In vivo, BAY 1125976 demonstrates dose-dependent anti-tumor efficacy in multiple xenograft tumor models of different histological types with PIK3CA mutations or PTEN deletions while being well tolerated. BAY 1125976 can be effectively combined with various anti-cancer therapies. In vitro combination profiling shows synergistic anti-proliferative effects with anti-hormonal therapeutics in breast and prostate cancer cell lines, which translates to enhanced anti-tumor efficacy with durable tumor regressions in vivo. Furthermore, in vivo combination of BAY 1125976 with external beam radiation results in strong additive to synergistic efficacy and significant tumor growth delay. Moreover, the combination of BAY 1125976 with the bone-targeting agent Radium 223 in a breast cancer bone metastasis model results in reduced tumor and metastases burden and increased necrotic and fibrotic bone area. In conclusion, BAY 1125976 is a highly selective, potent allosteric AKT1/2 inhibitor with strong in vitro and in vivo activity in tumor models with activated AKT signalling and strong synergistic activity in combination. Targeting AKT might also provide a promising strategy for overcoming chemo/radio-resistance and increasing radio-sensitization and radio-potentiation. Citation Format: Oliver Politz, Lars Baerfacker, Stuart Ince, William J. Scott, Roland Neuhaus, Ulf Boemer, Martin Michels, Dominik Mumberg, Franz von Nussbaum, Karl Ziegelbauer, Andrea Haegebarth. BAY 1125976, a highly selective and potent allosteric AKT1/2 inhibitor, for the treatment of cancers with aberrations in the PI3K-AKT-mTOR pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2050. doi:10.1158/1538-7445.AM2013-2050

Abstract The PI3K pathway plays critical roles in cancer cell growth and survival, as well as in intrinsic and acquired resistance to both chemotherapy and targeted agents. These essential roles have led to the clinical development of PI3K pathway inhibitors. Due to the complexity derived from the existence of various PI3K isoforms (≥,α,α,α), and their differential roles in signal transduction, as well as cancer pathology, development of PI3K inhibitors with differential pharmacological profiles would allow exploration in different indications, combinations and dosing regimens. Having identified BAY 80-6946, an intravenously dosed, highly potent and selective PI3K inhibitor which is particularly effective in PIK3CAmut and/or Her2+ tumors, we sought to develop a novel oral PI3K inhibitor particularly effective in PTEN-loss tumors with coexisting mutation or amplification of PIK3CA and/or activation of PI3Kα (e.g., through RTKs). Herein we report the pharmacological profile of a highly selective PI3Kα/α-balanced inhibitor, BAY 1082439. BAY 1082439 has an IC50 ratio of 1:3 in biochemical assays of PI3Kα (4.9 nM) vs. PI3Kα (15.0 nM), and >1000-fold selectivity against mTOR kinase. The balanced PI3Kα and PI3Kα activity of BAY 1082439 is also reflected in cellular mechanistic (p-AKT473) and proliferation assays in PI3Kα- (KPL4, BT474) vs. PI3Kα-driven (PC3, LNCaP) tumor cells. In vivo, BAY 1082439 showed clear advantages over the strong PI3Kα inhibitor BAY 80-6946 in PTEN/PI3Kα-driven tumor models (e.g., PC3 and HEC-1B), when the two compounds were compared at their MTDs. Furthermore, BAY 1082439 has unique pharmacokinetic (PK) properties with very high plasma free fractions across all species tested (33-50%), large Vss, high clearance and intermediate T1/2. The relationship of PK vs. PD and the efficacy vs. dosing regimens were investigated. BAY 1082439 showed strong p-AKT inhibition at 2 and 5 hours post-treatment while p-AKT returned to levels comparable to the vehicle group at 24 hours in all 4 tumor models tested. Interestingly, with once daily dosing, BAY 1082439 could induce tumor regression in KPL4 (PIK3CAmut and HER2+), and tumor stasis in HEC-1B (PTENdel) and in HEC-1A (PIK3CAmut) tumor models, suggesting that continuous inhibition of p-AKT may not be required for anti-tumor efficacy. In addition, comparison of different dosing regimens (QD, Q2D, D1-2/W, D1-3/W, D1-4/W to QW) at MTD indicated that QD and QW dosing produced optimal anti-tumor efficacy. These results support the hypothesis that strong pathway inhibition for a certain time period, rather than maintaining constant inhibition might lead to optimal anti-tumor efficacy along with a maximal therapeutic window. In conclusion, BAY 1082439 represents a new type of PI3K inhibitor with unique pharmacological and pharmacodynamic properties to be further explored in clinical development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2799. doi:1538-7445.AM2012-2799

BACKGROUND:The 49-year history of the Teratology Society is reviewed. An abbreviated history is outlined in table form, with listings of the Warkany Lectures, the Continuing Education Courses, and officers of the society. The original article was updated to include the years 2000 to 2010.METHODS:A year-by-year description of the events is given, including the scientific and social content of the annual meetings and changes in the business of the society, in many cases using comments from the past presidents. The valuable and unique diversity of the members is discussed and illustrated, presenting the disciplines and main research areas of the presidents. The number of submitted abstracts and the various categories are tabulated, averaging the number and type over successive periods. A significant increase in the number of abstracts dealing with epidemiology and developmental biology is evident. The society's development is compared to that of a human, and the question was asked by Shephard et al. (2000): Have we reached the maturational stage of old age or senescence, or is the society still maturing gracefully? This question needs further discussion by all the members. By 2010, many positive changes are happening to revitalize the society.RESULTS:During the past 50 years, we have developed the scientific basis to prevent birth defects caused by rubella, alcoholism, and folate deficiency, as well as other prenatal exposures. We are now taking advantage of advances in many fields to begin shaping the Teratology Society of the 21st century.CONCLUSIONS:We must now engage in political battles to obtain the resources needed to conduct further research and to implement prevention programs, as well as to provide care and rehabilitation for persons with birth defects.

BACKGROUND:A variety of drugs, environmental chemicals, and physical agents induce a common limb malformation in the offspring of pregnant mice exposed on day 9 of gestation. This malformation, postaxial, right-sided forelimb ectrodactyly, is thought to arise via an alteration of hedgehog signaling.METHODS:We have studied two of these teratogens, acetazolamide and cadmium, using the technique of microarray analysis of limb bud ectoderm and mesoderm to search for changes in gene expression that could indicate a common pathway to postaxial limb reduction.RESULTS:Results indicated a generalized up-regulation of gene expression after exposure to acetazolamide but a generalized down-regulation due to cadmium exposure. An intriguing observation was a cadmium-induced reduction of Mt1 and Mt2 expression in the limb bud mesoderm indicating a lowering of embryonic zinc.CONCLUSIONS:We propose that these two teratogens and others (valproic acid and ethanol) lower sonic hedgehog signaling by perturbation of zinc function in the sonic hedgehog protein.

Herein, we demonstrate that Lrp6-mediated R-spondin 2 signaling through the canonical Wnt pathway is required for normal morphogenesis of the respiratory tract and limbs. We show that the footless insertional mutation creates a severe hypomorphic R-spondin 2 allele (Rspo2Tg). The predicted protein encoded by Rspo2Tg neither bound the cell surface nor activated the canonical Wnt signaling reporter TOPFLASH. Rspo2 activation of TOPFLASH was dependent upon the second EGF-like repeat of Lrp6. Rspo2Tg/Tg mice had severe malformations of laryngeal-tracheal cartilages, limbs and palate, and lung hypoplasia consistent with sites of Rspo2 expression. Rspo2Tg/Tg lung defects were associated with reduced branching, a reduction in TOPGAL reporter activity, and reduced expression of the downstream Wnt target Irx3. Interbreeding the Rspo2Tg and Lrp6- alleles resulted in more severe defects consisting of marked lung hypoplasia and absence of tracheal-bronchial rings, laryngeal structures and all limb skeletal elements.

Novel anthranilamides were surprisingly found to exert additional activity on B-RAF. Corresponding thiophene, pyrazole, and thiazole core analogs were prepared as VEGFR-2 inhibitors with c-KIT, and B-RAF activity. Compounds in the phenyl, thiophene, and thiazole series are in vivo active.

A series of 4,5-disubstituted cis-pyrrolidinones was investigated as inhibitors of 17beta-HSD II for the treatment of osteoporosis. Biochemical data for several compounds are given. Compound 42 was selected as the lead candidate.

Proc Amer Assoc Cancer Res, Volume 47, 2006 3769 Therapies aimed at inhibiting tumor angiogenesis and lymphangiogenesis are expected to deprive malignant tissues of their oxygen and nutrient supplies, and to also prevent tumors from eliminating metabolic wastes. This, in turn, would inhibit the tumor growth and metastatic progression that accompanies most advanced cancers. High throughput screening followed by lead optimization led to the development of a diaryl urea containing a distal 2-cyanopyridine as the program lead. This lead compound possesses potent activity against both VEGFR3 and VEGFR2 but no activity against c-ErbB1/2, FLT3, FGFR1, LCK and many other kinases. Several in vivo studies were carried out to determine the effect of the lead compound on tumor growth and metastasis. In an MDA-MB-231 breast tumor model, the cyanopyridine inhibited tumor growth by 85% when dosed once a day for 9 days at 10 mg/kg (mpk). In a separate study using the MDA-MB-231 model, dosing was stopped after 9 days and tumor growth was monitored for 55 days. A growth delay of 36 days was observed, along with 4 of 10 tumors achieving complete regression and 5 of 10 achieving partial regression. Moreover, several intermittent dosing-schedules were tested in the MDA-MB-231 model. When dosed orally at 30 mpk every other day (Q2Dx5), every third day (Q3Dx4) or every fourth day (Q4Dx3), the cyanopyridine inhibited tumor growth by 96%, 82% and 73%, respectively. The lead compound proved active in a variety of models of tumor growth. When dosed at 30 mpk, the cyanopyridine inhibited tumor growth by 78% in a LOX melanoma model, by 86% in a 22RV-1 prostate model, and by 78% in a Colo205 colon model. In an HCT-15 colon model (MDR) and an H460 NSCLC model, the lead compound inhibited tumor growth by 67% and 42%, respectively, when dosed daily at 10 mpk. CD-31 and α-smooth muscle actin levels in tumors from the MDA-MB-231 model were analyzed by immunohistochemistry, showing a strong decrease in tumor angiogenesis. Furthermore, the effect of the compound on cancer cell metastasis was assessed in an MDA-MB-435 orthotopic model. Eight weeks after tumor implantation in the fat pad, the compound was dosed daily for 21 days. Lung examination by histology (H&E) revealed numerous foci in lungs of control groups, but no foci were observed in lungs of the compound-treated group. Similar results were obtained in a LOX melanoma model in which real-time PCR was used to detect lung and lymph node micro-metastases. Overall, these results show the ability of the lead compound to inhibit tumor growth and metastasis in multiple subcutaneous and orthotopic tumor models.

4,5-Disubstituted cis-pyrrolidinones were investigated as inhibitors of type II 17beta-hydroxysteroid dehydrogenase (17beta-HSD). Early structure-activity relationship patterns for this class of compounds are discussed.