Evaluation of Clinical Pharmacy Services for Phase 1 Clinical Trials.

BACKGROUND: Phase 1 clinical trials have challenges relative to later-phase clinical trials. As of April 2020, there were 71 active phase 1 cancer clinical trials at the Johns Hopkins Medicine Sidney Kimmel Comprehensive Cancer Center (SKCCC), and limited clinical pharmacy services are dedicated to the unique needs of phase 1 clinical trials. OBJECTIVES: To characterize the current phase 1 cancer-specific clinical pharmacy services at National Cancer Institute (NCI)-designated institutions, and to develop a framework for the implementation of these services at Johns Hopkins Medicine SKCCC. METHODS: We queried the current pharmacy practices for phase 1 cancer clinical trials at NCI-designated institutions through an e-mailed 20-question national online survey to 208 pharmacists. The recipients were asked to rate how often specific pharmacy services were performed, using a 4-point Likert scale of rarely/never (<10%), sometimes (10%-49%), often (50%-80%), or almost always (>80%). The services were grouped into pretrial implementation support, phase 1 trial implementation support, medication profile review, medication therapy management, and miscellaneous support. Using the survey results, a framework for phase 1 trial clinical pharmacy services was developed concurrently to prioritize protocol complexity, monitoring requirements, and clinical pharmacy interventions. RESULTS: Of the 208 surveys e-mailed, 45 recipients responded, for an overall survey response rate of 22%. The responses were divided into 2 subgroups for the institutions that currently conduct phase 1 cancer clinical trials, including institutions with >40 active phase 1 cancer clinical trials and institutions with ≤40 active phase 1 cancer clinical trials. The institutions with >40 active phase 1 cancer clinical trials were more likely to have pharmacists involved with direct participant care (47% vs 18.8%, respectively) and document medication lists for phase 1 trial participants (41% vs 18.8%, respectively) than institutions with ≤40 active phase 1 cancer clinical trials. The survey results assisted in developing a framework to classify drug regimens as platinum level (ie, higher complexity) or standard level (ie, lower or average complexity) to prioritize clinical pharmacy services based on their complexity level. CONCLUSION: Our analysis of current phase 1 clinical trial pharmacy practices at NCI institutions enabled the development of a framework for increased collaboration with research teams and phase 1 clinical trial-specific clinical pharmacy services within Johns Hopkins Medicine SKCCC.

Evaluation and clinical analyses of downstream targets of the Akt inhibitor GDC-0068.

PURPOSE: The oncogenic PI3K/Akt/mTOR pathway is an attractive therapeutic target in cancer. However, it is unknown whether the pathway blockade required for tumor growth inhibition is clinically achievable. Therefore, we conducted pharmacodynamic studies with GDC-0068, an ATP competitive, selective Akt1/2/3 inhibitor, in preclinical models and in patients treated with this compound. EXPERIMENTAL DESIGN: We used a reverse phase protein array (RPPA) platform to identify a biomarker set indicative of Akt inhibition in cell lines and human-tumor xenografts, and correlated the degree of pathway inhibition with antitumor activity. Akt pathway activity was measured using this biomarker set in pre- and post-dose tumor biopsies from patients treated with GDC-0068 in the dose escalation clinical trial. RESULTS: The set of biomarkers of Akt inhibition is composed of 10 phosphoproteins, including Akt and PRAS40, and is modulated in a dose-dependent fashion, both in vitro and in vivo. In human-tumor xenografts, this dose dependency significantly correlated with tumor growth inhibition. Tumor biopsies from patients treated with GDC-0068 at clinically achievable doses attained a degree of biomarker inhibition that correlated with tumor growth inhibition in preclinical models. In these clinical samples, compensatory feedback activation of ERK and HER3 was observed, consistent with preclinical observations. CONCLUSION: This study identified a set of biomarkers of Akt inhibition that can be used in the clinical setting to assess target engagement. Here, it was used to show that robust Akt inhibition in tumors from patients treated with GDC-0068 is achievable, supporting the clinical development of this compound in defined patient populations.

Potent non-benzoquinone ansamycin heat shock protein 90 inhibitors from genetic engineering of Streptomyces hygroscopicus.

Inhibition of the protein chaperone Hsp90 is a promising new approach to cancer therapy. We describe the preparation of potent non-benzoquinone ansamycins. One of these analogues, generated by feeding 3-amino-5-chlorobenzoic acid to a genetically engineered strain of Streptomyces hygroscopicus, shows high accumulation and long residence time in tumor tissue, is well-tolerated upon intravenous dosing, and is highly efficacious in the COLO205 mouse tumor xenograft model.

Identification of nuclear export inhibitors with potent anticancer activity in vivo.

The export protein CRM1 is required for the nuclear export of a wide variety of cancer-related "cargo" proteins including p53, c-Abl, and FOXO-3A. Leptomycin B (LMB) is a highly specific inhibitor of CRM1 with significant in vitro potency but limited in vivo efficacy due to toxicity. We now report a series of semisynthetic LMB derivatives showing substantially improved therapeutic windows. Exposure of cancer cells to these compounds leads to a rapid and prolonged block of nuclear export and apoptosis. In contrast to what is observed in cancer cells, these agents induce cell cycle arrest, but not apoptosis, in normal lung fibroblasts. These new nuclear export inhibitors (NEI) maintain the high potency of LMB, are up to 16-fold better tolerated than LMB in vivo, and show significant efficacy in multiple mouse xenograft models. These NEIs show the potential of CRM1 inhibitors as novel and potent anticancer agents.

Targeted covalent inactivation of protein kinases by resorcylic acid lactone polyketides.

Resorcylic acid lactones containing a cis-enone are susceptible to Michael addition reactions and are potent inhibitors of several protein kinases. A structural-bioinformatics analysis identified a conserved Cys residue in the ATP-binding site of the kinases reported to be inhibited by cis-enone resorcylic acid lactones but absent in those that are not. Mining of the kinome database revealed that a subset of some 46 kinases contained this Cys residue. Screening a panel of 124 kinases with the resorcylic acid lactone hypothemycin showed that 18 of 19 targets containing the conserved Cys were inhibited. Kinetic analyses showed time-dependent inhibition, a hallmark of covalent inactivation, and biochemical studies of the interaction of extracellular signal-regulated kinase (ERK)2 with hypothemycin confirmed covalent adduct formation. Resorcylic acid lactones are unique among kinase inhibitors in that they target mitogen-activated protein (MAP) kinase pathways at four levels: mitogen receptors, MAP kinase kinase (MEK)1/2 and ERK1/2, and certain downstream ERK substrates. Cell lines dependent on the activation of Tyr kinase mitogen receptor targets of the resorcylic acid lactones were unusually sensitive toward hypothemycin and showed the expected inhibition of kinase phosphorylation due to inhibition of the mitogen receptors and/or MEK1/2 and ERK1/2. Among cells without mitogen receptor targets, those harboring an ERK pathway-activating B-RAF V600E mutation were selectively and potently inhibited by hypothemycin. Hypothemycin also prevented stimulated activation of the p38 cascade through inhibition of the Cys-containing targets MEK3/6 and TGF-beta-activated kinase 1 and of the JNK/SAPK (c-Jun N-terminal kinase/stress-activated protein kinase) cascade through inhibition of MEK4/7.

Chemobiosynthesis of novel 6-deoxyerythronolide B analogues by mutation of the loading module of 6-deoxyerythronolide B synthase 1.

Chemobiosynthesis (J. R. Jacobsen, C. R. Hutchinson, D. E. Cane, and C. Khosla, Science 277:367-369, 1997) is an important route for the production of polyketide analogues and has been used extensively for the production of analogues of 6-deoxyerythronolide B (6-dEB). Here we describe a new route for chemobiosynthesis using a version of 6-deoxyerythronolide B synthase (DEBS) that lacks the loading module. When the engineered DEBS was expressed in both Escherichia coli and Streptomyces coelicolor and fed a variety of acyl-thioesters, several novel 15-R-6-dEB analogues were produced. The simpler "monoketide" acyl-thioester substrates required for this route of 15-R-6-dEB chemobiosynthesis allow greater flexibility and provide a cost-effective alternative to diketide-thioester feeding to DEBS KS1(o) for the production of 15-R-6-dEB analogues. Moreover, the facile synthesis of the monoketide acyl-thioesters allowed investigation of alternative thioester carriers. Several alternatives to N-acetyl cysteamine were found to work efficiently, and one of these, methyl thioglycolate, was verified as a productive thioester carrier for mono- and diketide feeding in both E. coli and S. coelicolor.

6-Deoxyerythronolide B analogue production in Escherichia coli through metabolic pathway engineering.

The erythromycin precursor polyketide 6-deoxyerythronolide B (6-dEB) is produced from one propionyl-CoA starter unit and six (2S)-methylmalonyl-CoA extender units. In vitro studies have previously demonstrated that the loading module of 6-deoxyerythronolide B synthase (DEBS) exhibits relaxed substrate specificity and is able to accept butyryl-CoA, leading to the production of polyketides with butyrate starter units. We have shown that we can produce butyryl-CoA at levels of up to 50% of the total CoA pool in Escherichia coli cells that overexpress the acetoacetyl-CoA:acetyl-CoA transferase, AtoAD (EC 2.8.3.8), in media supplemented with butyrate. The DEBS polyketide synthase (PKS) used butyryl-CoA and methylmalonyl-CoA supplied in vivo by the AtoAD and methylmalonyl-CoA mutase pathways, respectively, to produce 15-methyl-6-dEB. Priming DEBS with endogenous butyryl-CoA affords an alternative and more direct route to 15-Me-6-dEB than that provided by the chemobiosynthesis method [Jacobsen, J. R., et al. (1997) Science 277, 367-369], which relies on priming a mutant DEBS with an exogenously fed diketide thioester. The approach described here demonstrates the utility of metabolic engineering in E. coli to introduce precursor pathways for the production of novel polyketides.