Amonafide, a topoisomerase II inhibitor, is unaffected by P-glycoprotein-mediated efflux.

Over-expression of P-glycoprotein (Pgp+) has been related to resistance to classical Topo II inhibitors used in the treatment of AML and is common in patients with poor-prognosis, such as those with secondary AML (sAML). Since clinical trials with amonafide, a unique ATP-independent Topo II inhibitor, in combination with cytarabine, have shown significant efficacy for remission induction in patients with sAML, we compared the cytotoxic effect of amonafide (amonafide l-malate, Xanafide) to the classical Topo II inhibitors (daunorubicin, doxorubicin, idarubicin, etoposide, and mitoxantrone) in K562 leukemia cells and in the MDR subline, K562/DOX. Pgp expression was found to be approximately 6.5-fold greater in K562/DOX and causes the rapid efflux of these drugs from the leukemia cell. As a consequence, the LC(50) values for the classical Topo II inhibitor drugs tested were each increased up to 3 log units. A similar result was also observed in murine P388 and P388/ADR leukemia cells. Addition of cyclosporin A reversed K562/DOX resistance for the classical Topo II inhibitors, decreasing their LC(50) values to the levels observed with wild type cells but had no effect on amonafide potency in Pgp+ or wild type cells. Further examination of amonafide in bidirectional Caco-2 and MDR1-MDCK models confirmed that amonafide is neither a substrate nor inhibitor of Pgp. These observations suggest that amonafide is a promising therapeutic candidate directed toward bypassing this common mechanism of drug resistance encountered in the treatment of patients with AML, and possibly in other resistant hematological malignancies as well.

Osimertinib for Previously Treated Patients With Advanced EGFR T790M Mutation-Positive NSCLC: Tolerability and Diagnostic Methods From an Expanded Access Program.

INTRODUCTION: The osimertinib (AZD9291) US Expanded Access Program (EAP) provided compassionate access to osimertinib prior to US Food and Drug Administration (FDA) approval for patients with advanced/metastatic epidermal growth factor receptor (EGFR) T790M-positive non-small cell lung cancer (NSCLC) following progression on tyrosine kinase inhibitors (TKIs) targeting EGFR. Here, we report the patient demographics, safety and tolerability, and diagnostic methods used for T790M testing in the EAP. METHODS: Adult patients with EGFR T790M-positive NSCLC following progression on prior EGFR-TKI therapy (irrespective of line of therapy) were enrolled in the EAP and treated with 80 mg osimertinib once daily until dose reduction, discontinuation, or completion of the EAP following FDA approval (November 2015). Various testing methods were allowed for the required T790M testing. RESULTS: In total, 248 patients from 25 centers throughout the USA were enrolled in the EAP. The starting dose of 80 mg osimertinib once daily was maintained for 96% (n = 238) of patients over the duration of the EAP (median duration of exposure 84 days). Most patients (overall 83% [n = 205/238]; patients aged ≥ 75 years 83% [n = 48/58]) completed the EAP and transitioned to commercially available osimertinib following FDA approval. Serious adverse events considered to be treatment related by investigators were reported in five patients (2%), all aged ≥ 65 years, and were dyspnea, deep vein thrombosis, femur fracture, alanine aminotransferase increase, and pneumonitis, respectively. A variety of biospecimen types were collected: solid tumor tissue (73%), blood (20%), cytology (6%), and urine (2%). PCR-based methods were most commonly used for determining EGFR mutation status (47%) followed by next-generation sequencing (33%). CONCLUSION: In a real-world setting, osimertinib was well tolerated, and most patients, including patients aged ≥ 75 years, transitioned to commercially available osimertinib following FDA approval. The EAP suggests there has been an uptake of minimally invasive T790M testing methods at some centers. FUNDING: AstraZeneca (Wilmington, DE, USA).

Taxol biosynthesis: Molecular cloning and characterization of a cytochrome P450 taxoid 7 beta-hydroxylase.

Biosynthesis of the anticancer drug Taxol in yew species involves eight cytochrome P450-mediated oxygenations and four coenzyme A-dependent acylations of the diterpenoid core. A family of cytochrome P450 genes, obtained from a yew cell cDNA library, were functionally expressed and screened with taxusin (taxa-4(20),11(12)-dien-5 alpha,9 alpha,10 beta,13 alpha-tetraol tetraacetate) as a surrogate substrate. One clone converted this substrate to an oxygenated derivative that was identified as 7 beta-hydroxytaxusin. The structure and properties of this 7 beta-hydroxylase are similar to those of other taxoid hydroxylases. Kinetic and binding assays indicated selectivity of the 7 beta-hydroxylase for polyoxygenated and acylated taxoid substrates, an observation consistent with the operation of this enzyme in the central portion of the Taxol biosynthetic pathway. Although the 7 beta-hydroxyl of Taxol is not essential for antimitotic activity, this functional group provides a convenient means for preparing taxoid derivatives.

Small molecule tyrosine kinase inhibitors for the treatment of intestinal inflammation.

BACKGROUND: We developed a series of dendritic cell autoimmune modulators (DCAMs) based on small molecule Flt3 receptor tyrosine kinase inhibitors (TKIs) for the inhibition of intestinal inflammation and oral delivery. METHODS: DCAMs were administered orally during and after induction of dextran sodium sulfate (DSS)-induced colitis. Dendritic cell recruitment and inflammatory responses were determined in the mucosal immune system during acute intestinal inflammatory responses and mucosal recovery. Bone marrow-derived macrophages were utilized to define the mechanisms by which DCAMs can modify responses to microbial signals. RESULTS: Oral doses of DCAMs prevented severe weight loss and mucosal inflammation associated with DSS colitis in mice. The presence of DCAMs increased the number of CD11c(+) PDCA1(+) dendritic cells, induced interleukin (IL)-10 expression, and reduced inflammatory cytokine expression in the mucosal immune system. Surprisingly, DCAMs regulated innate immune responses in macrophages resulting in the inhibition of tumor necrosis factor alpha (TNF-α) production and the induction of IL-10 expression during Toll-like receptor-mediated signaling. CONCLUSIONS: We identified two new imidazoacridinone derivatives that protect mice from severe colitis and promote mucosal recovery by enhancing protective cytokine production while inhibiting proinflammatory stimuli during microbial recognition. These compounds are promising candidates for further development into potent orally available drugs for the prevention of colitis and promotion of mucosal recovery.

Molecular cloning and characterization of a cytochrome P450 taxoid 2alpha-hydroxylase involved in Taxol biosynthesis.

The Taxol biosynthetic pathway, arising from the primary isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate in yew (Taxus), consists of approximately twenty steps, at least nine of which are thought to be cytochrome P450-mediated oxygenations. Several oxygenases involved in the early hydroxylation steps of the pathway have been identified and the corresponding genes have been cloned; however, defining the enzymes and their genes responsible for oxygenations in the central portion of the pathway is more difficult because neither the exact sequence of reactions nor the relevant intermediates are known. A surrogate substrate, (+)-taxusin (taxa-4(20),11(12)-dien-5alpha,9alpha,10beta,13alpha-tetraol tetraacetate), that was previously employed in the isolation of a taxoid 7beta-hydroxylase, was used here to functionally screen a family of cytochrome P450 oxygenases originating from a Taxus cell EST library. This in vivo screen in yeast led to the identification of a 1488bp cDNA clone (encoding a 495 residue protein) that was capable of producing 2alpha-hydroxytaxusin from taxusin with a K(m) value of 10.5 +/- 2.7 microM and k(cat) of about 0.05 s(-1) for the surrogate substrate. This structurally typical cytochrome P450 resembles most closely the previously isolated taxoid 7beta-hydroxylase, which also uses taxusin as a substrate, and both 2alpha- and 7beta-hydroxylases are capable of the reciprocal conversion of their respective pentaol tetraacetate products to the common hexaol tetraacetate. This C2-hydroxylase would appear to mediate the mid-pathway functionalization of the C2-position of the taxane core that ultimately bears a benzoyl group as an important Taxol pharmacophore. Overexpression of this cytochrome P450 taxoid 2alpha-hydroxylase in Taxus cells may improve Taxol yields and could prove useful in the production of other 2alpha-hydroxy taxoids as starting materials for subsequent acylation at this position.

Regioselectivity of taxoid-O-acetyltransferases: heterologous expression and characterization of a new taxadien-5alpha-ol-O-acetyltransferase.

In addition to the anticancer drug Taxol, yew (Taxus) species produce a large variety of other taxane diterpenoids which differ mainly in the type of acyl and aroyl groups appended to the many hydroxyl functions on the taxane core; acetate esters are particularly common. Taxol bears an acetate at C10 and another at C4 thought to originate by intramolecular migration of a C5 acetate function in the process of oxetane ring formation, but many other naturally occurring taxoids bear acetate groups at C1, C2, C7, C9, and C13, in addition to C5 and C10. cDNAs encoding a taxoid 5alpha-O-acetyltransferase (taxadien-5alpha-ol as substrate) and a taxoid 10beta-O-acetyltransferase (10-deacetylbaccatin III as substrate) have been acquired from a recently isolated family of Taxus acyl/aroyltransferase clones. To explore the origins of other acetylated taxoids, the group of recombinant Taxus acyltransferases was investigated with a range of polyhydroxylated taxoids as substrates. From this survey, a new acetyltransferase clone (denoted TAX19) was identified that was capable of acetylating taxadien-5alpha-ol with activity comparable to that of the previously identified 5alpha-O-acetyltransferase (clone TAX1). However, when these two recombinant enzymes were presented with taxadien-triol and tetraol substrates, they exhibited different regiospecificities. The TAX1 enzyme preferentially acetylates the "northern" hemisphere hydroxyls at C9 and C10, whereas the TAX19 enzyme preferentially acetylates the "east-west" pole positions at C5 and C13. The TAX1 enzyme possesses the lowest KM value with taxadien-5alpha-ol (an early pathway metabolite) as substrate, with much higher KM values for the polyhydroxylated taxoid substrates, whereas the TAX19 enzyme possesses lower KM values (than the TAX1 transferase) for all taxoid substrates tested. These results suggest that both TAX1 and TAX19 acyltransferases may function at the early C5 acetylation step of taxoid metabolism, and that the TAX19 acyltransferase, because of its broader specificity for polyhydroxylated taxoids, may also function later in metabolism and be responsible for the production of many other acetylated taxoids.

Random sequencing of an induced Taxus cell cDNA library for identification of clones involved in Taxol biosynthesis.

Biosynthesis of the anticancer drug Taxol involves 19 enzymatic steps from the universal diterpenoid progenitor geranylgeranyl diphosphate derived by the plastidial methylerythritol phosphate pathway for isoprenoid precursor supply. To gain further insight about Taxol biosynthesis relevant to the improved production of this drug and to draw inferences about the organization, regulation, and origins of this complex natural product pathway, random sequencing of a cDNA library derived from Taxus cuspidata cells (induced for taxoid biosynthesis with methyl jasmonate) was undertaken. This effort revealed surprisingly high abundances for transcripts of several of the 12 defined genes of Taxol biosynthesis, yielded cDNAs encoding two previously uncharacterized cytochrome P450 taxoid hydroxylases, and provided candidate genes for all but one of the remaining seven steps of this extended sequence of reactions.