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Purpose:Dickkopf-1 (DKK1) modulates Wnt signaling, promoting tumor growth, metastasis, and immunosuppression. High DKK1 expression has been detected in various tumor types—including biliary tract cancer (BTC)—and is associated with poor prognosis. DKN-01—a humanized mAb targeting DKK1—was evaluated in a phase I multicenter study in combination with gemcitabine and cisplatin in patients with unresectable or metastatic BTC with no prior systemic therapy for advanced disease.Patients and Methods:This study included a dose-escalation phase assessing DKN-01 at two dose levels (150 mg and 300 mg) combined with gemcitabine (1,000 mg/m2) and cisplatin (25 mg/m2) followed by dose expansion. Primary endpoints evaluated safety and tolerability; secondary endpoints evaluated efficacy, pharmacokinetics, and circulating biomarkers.Results:Fifty-one patients with intrahepatic cholangiocarcinoma (63%), extrahepatic cholangiocarcinoma (8%), and gallbladder cancer (29%) were enrolled. No dose-limiting toxicities were seen, and the expansion phase proceeded with DKN-01 300 mg (N = 47). The most frequent grade 3/4 treatment-emergent adverse events included neutropenia (60%), thrombocytopenia (34%), and anemia (23%). The objective response rate was 21.3% and median progression-free survival was 8.7 months (95% confidence interval, 5.4–10.3 months). Better outcomes were associated with biomarkers of angiogenesis inhibition (increased sVEGFR1 and lower VEGF-C) and reduced inflammation (lower IL6 and decreased TNFα).Conclusions:DKN-01 300 mg was well tolerated in this combination but did not appear to have additional activity beyond historically reported efficacy with gemcitabine/cisplatin alone. Exploratory pharmacokinetic and biomarker data indicate potential antiangiogenic and immunomodulatory activity of DKN-01/chemotherapy and the need for increased dose/intensity. A study with DKN-01 600 mg in combination with a PD-1 inhibitor in BTC is ongoing.

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Purpose: Dickkopf-1 (DKK1) modulates Wnt signaling, promoting tumor growth, metastasis, and immunosuppression. High DKK1 expression has been detected in various tumor types—including biliary tract cancer (BTC)—and is associated with poor prognosis. DKN-01—a humanized mAb targeting DKK1—was evaluated in a phase I multicenter study in combination with gemcitabine and cisplatin in patients with unresectable or metastatic BTC with no prior systemic therapy for advanced disease. Patients and Methods: This study included a dose-escalation phase assessing DKN-01 at two dose levels (150 mg and 300 mg) combined with gemcitabine (1,000 mg/m2) and cisplatin (25 mg/m2) followed by dose expansion. Primary endpoints evaluated safety and tolerability; secondary endpoints evaluated efficacy, pharmacokinetics, and circulating biomarkers. Results: Fifty-one patients with intrahepatic cholangiocarcinoma (63%), extrahepatic cholangiocarcinoma (8%), and gallbladder cancer (29%) were enrolled. No dose-limiting toxicities were seen, and the expansion phase proceeded with DKN-01 300 mg (N = 47). The most frequent grade 3/4 treatment-emergent adverse events included neutropenia (60%), thrombocytopenia (34%), and anemia (23%). The objective response rate was 21.3% and median progression-free survival was 8.7 months (95% confidence interval, 5.4–10.3 months). Better outcomes were associated with biomarkers of angiogenesis inhibition (increased sVEGFR1 and lower VEGF-C) and reduced inflammation (lower IL6 and decreased TNFα). Conclusions: DKN-01 300 mg was well tolerated in this combination but did not appear to have additional activity beyond historically reported efficacy with gemcitabine/cisplatin alone. Exploratory pharmacokinetic and biomarker data indicate potential antiangiogenic and immunomodulatory activity of DKN-01/chemotherapy and the need for increased dose/intensity. A study with DKN-01 600 mg in combination with a PD-1 inhibitor in BTC is ongoing.

The optimization of a series of thiazolopyridine S1P1 agonists with limited activity at the S1P3 receptor is reported. These efforts resulted in the discovery of 1-(3-fluoro-4-(5-(1-phenylcyclopropyl)thiazolo-[5,4-b]pyridin-2-yl)benzyl)azetidine-3-carboxylic acid (5d, AMG 369), a potent dual S1P1/S1P5 agonist with limited activity at S1P3 and no activity at S1P2/S1P4. Dosed orally at 0.1 mg/kg, 5d is shown to reduce blood lymphocyte counts 24 h postdose and delay the onset and reduce the severity of experimental autoimmune encephalomyelitis in rat.

We have discovered novel benzofuran-based S1P1 agonists with excellent in vitro potency and selectivity. 1-((4-(5-Benzylbenzofuran-2-yl)-3-fluorophenyl)methyl) azetidine-3-carboxylic acid (18) is a potent S1P1 agonist with >1000× selectivity over S1P3. It demonstrated a good in vitro ADME profile and excellent oral bioavailability across species. Dosed orally at 0.3 mg/kg, 18 significantly reduced blood lymphocyte counts 24 h postdose and demonstrated efficacy in a mouse EAE model of relapsing MS.

We have previously shown N-arylnaphthamides can be potent inhibitors of vascular endothelial growth factor receptors (VEGFRs). N-Alkyl and N-unsubstituted naphthamides were prepared and found to yield nanomolar inhibitors of VEGFR-2 (KDR) with an improved selectivity profile against a panel of tyrosine and serine/threonine kinases. The inhibitory activity of this series was retained at the cellular level. Naphthamides 3, 20, and 22 exhibited good pharmacokinetics following oral dosing and showed potent inhibition of VEGF-induced angiogenesis in the rat corneal model. Once-daily oral administration of 22 for 14 days led to 85% inhibition of established HT29 colon cancer and Calu-6 lung cancer xenografts at doses of 10 and 20 mg/kg, respectively.

The metabolism of pyrene to hydroxypyrene by CYP3A4 was investigated to determine the effect of cytochrome b5 (b5) on turnover kinetics. In the absence of b5, formation of hydroxypyrene in in vitro incubations showed a biphasic substrate-velocity curve where K(m1) and V(max1) were 1.3 microM and 0.5 pmol/min/pmol P450, respectively. The addition of testosterone to the incubation mixture completely abolished the second phase to yield a typical, hyperbolic curve, presumably through the disruption in the formation of a pi-pi stacked pyrene complex within the CYP3A4 active site. Finally, the addition of b5 yielded an increase hydroxypyrene formation that resulted in a sigmoidal substrate velocity curve. The V(max) was 15.7 pmol/min/pmol P450, the K(m) was 7.5 microM, and the Hill coefficient was greater than two. This demonstrated that b5 could directly induce positive cooperativity on CYP3A4 and that this biological factor needs to be carefully considered when included in in vitro P450 reactions.

Deregulation of the receptor tyrosine kinase c-Kit is associated with an increasing number of human diseases, including certain cancers and mast cell diseases. Interference of c-Kit signaling with multi-kinase inhibitors has been shown clinically to successfully treat gastrointestinal stromal tumors and mastocytosis. Targeted therapy of c-Kit activity may provide therapeutic advantages against off-target effects for non-oncology applications. A new structural class of c-Kit inhibitors is described, including in vitro c-Kit potency, kinase selectivity, and the observed binding mode.

The evaluation of a compound's pharmacokinetics (PK) and toxicokinetics (TK) is one critical component in understanding how a drug works in vivo. In the 1990s, one of the primary reasons for the failure of early clinical studies was poor or inadequate PK in humans. Since then, drug discovery and development has incorporated early assessments of preclinical PK into the lead optimization stage. As a consequence, the current primary reasons for failures during clinical development are lack of efficacy and/or toxicity. Scaling of preclinical data (both in vitro and in vivo) now facilitates estimates of human pharmacokinetics prior to the initiation of clinical studies. When those estimates point toward unfavorable PK or doses required for efficacy, this becomes part of the decision process on whether or not to take a particular compound into the clinic. Once a compound is selected for clinical development, a fundamental understanding of the PK and the TK of the compound is necessary to evaluate drug exposures obtained in the toxicology studies, which ensure a safe initial starting dose for first-in-human studies. Moreover, the same knowledge is instrumental in determining dose and dose frequency when testing efficacy. Thus, a basic understanding of the principles of PK is necessary to meet the current clinical challenges of toxicity and efficacy. This chapter includes an introduction to commonly used PK and TK parameters, a comparison of the PK characteristics, and challenges associated with small versus large molecules, as well as some practical considerations for the interpretation of PK/TK data.

An SAR campaign designed to increase polarity in the 'tail' region of benzothiazole 1 resulted in two series of structurally novel 5-and 6-substituted S1P(1) agonists. Structural optimization for potency ultimately delivered carboxamide (+)-11f, which in addition to possessing improved physicochemical properties relative to starting benzothiazole 1, also displayed good S1P(3) selectivity and acceptable in vivo lymphocyte-depleting activity.

Phosphoinositide 3-kinase α (PI3Kα) is a lipid kinase that plays a key regulatory role in several cellular processes. The mutation or amplification of this kinase in humans has been implicated in the growth of multiple tumor types. Consequently, PI3Kα has become a target of intense research for drug discovery. Our studies began with the identification of benzothiazole compound 1 from a high throughput screen. Extensive SAR studies led to the discovery of sulfonamide 45 as an early lead, based on its in vitro cellular potency. Subsequent modifications of the central pyrimidine ring dramatically improved enzyme and cellular potency and led to the identification of chloropyridine 70. Further arylsulfonamide SAR studies optimized in vitro clearance and led to the identification of 82 as a potent dual inhibitor of PI3K and mTOR. This molecule exhibited potent enzyme and cell activity, low clearance, and high oral bioavailability. In addition, compound 82 demonstrated tumor growth inhibition in U-87 MG, A549, and HCT116 tumor xenograft models.

Optimization of a benzofuranyl S1P1 agonist lead compound (3) led to the discovery of 1-(3-fluoro-4-(5-(2-fluorobenzyl)benzo[d]thiazol-2-yl)benzyl)azetidine-3-carboxylic acid (14), a potent S1P1 agonist with minimal activity at S1P3. Dosed orally at 0.3 mg/kg, 14 significantly reduced blood lymphocyte counts 24 h postdose and attenuated a delayed type hypersensitivity (DTH) response to antigen challenge.

A potent and selective c-Kit inhibitor 20 was identified through a structure–activity relationship study. In an in vivo mouse model of mast cell activation, 20 blocked the SCF-induced histamine release with an EC50 of 26 nM.

A series of naphthyl-based compounds were synthesized as potential inhibitors of vascular endothelial growth factor (VEGF) receptors. Investigations of structure–activity relationships led to the identification of a series of naphthamides that are potent inhibitors of the VEGF receptor tyrosine kinase family. Numerous analogues demonstrated low nanomolar inhibition of VEGF-dependent human umbilical vein endothelial cell (HUVEC) proliferation, and of these several compounds possessed favorable pharmacokinetic (PK) profiles. In particular, compound 48 demonstrated significant antitumor efficacy against established HT29 human colon adenocarcinoma xenografts implanted in athymic mice. A full account of the preparation, structure–activity relationships, pharmacokinetic properties, and pharmacology of analogues within this series is presented.

A ligand-based model is reported that predicts the Ki values for cytochrome P450 2C9 (CYP2C9) inhibitors. This CoMFA model was used to predict the affinity of 14 structurally diverse compounds not in the training set and appears to be robust. The mean error of the predictions is 6 microM. The experimentally measured Ki values of the 14 compounds range from 0.1 to 48 microM. Leave-one-out cross-validated partial least-squares gives a q2 value of between 0.6 and 0.8 for the various models which indicates internal consistency. Random assignment of biological data to structure leads to negative q2 values. These models are useful in that they establish a pharmacophore for binding to CYP2C9 that can be tested with site-directed mutagenesis. These models can also be used to screen for potential drug interactions and to design compounds that will not bind to this enzyme with high affinity.

BACKGROUND: Changes either in the number or in the responsiveness of hematopoietic progenitors may be a major factor accounting for age-related changes in stimulus driven hematopoiesis. METHODS: To test these hypotheses, we compared the relative proportions and the responsiveness of CD34+ bone marrow cells from healthy young (20-30 yrs) and healthy elderly (70-80 yrs) volunteers to G-CSF, GM-CSF, and IL-3 in an in vitro marrow culture system. RESULTS: There was no age-related difference either in the proportion of CD34+ marrow cells or in the proportion of a more mature CD34+ subset, defined as CD34+, CD33+ cells. Maximal colony formation by CD34+ cells stimulated with a combination of G-CSF, GM-CSF, and IL-3 was similar in the two groups, but the dose-response studies with individual growth factors revealed a 2-fold decrease in sensitivity of the elderly subjects' cells to G-CSF (p < .01). CONCLUSIONS: Aging has little impact on the marrow content of early precursors of the neutrophil lineage. There is, however, a significant difference in the in vitro proliferative response of these cells to the lineage specific growth factor G-CSF. This alteration may account for the greater propensity in elderly populations for the development of neutropenia with severe infections and chemotherapy.