A phase I/II study of arfolitixorin and 5-fluorouracil in combination with oxaliplatin (plus or minus bevacizumab) or irinotecan in metastatic colorectal cancer.

BACKGROUND: 5-fluorouracil (5-FU) combined with a folate remains an essential treatment component for metastatic colorectal cancer (mCRC). Leucovorin is the folate most often used, but requires intracellular conversion to a reduced folate, and has high pharmacokinetic variability and limited bioavailability in patients with low folate pathway gene expression. Arfolitixorin is an immediately active form of folate, [6R]-5,10-methylenetetrahydrofolate ([6R]-MTHF), and may improve outcomes. PATIENTS AND METHODS: This open-label, multicenter, phase I/II study in patients with mCRC (NCT02244632) assessed the tolerability and efficacy of first- or second-line arfolitixorin (30, 60, 120, or 240 mg/m2 intravenous) with 5-FU alone, or in combination with oxaliplatin (plus or minus bevacizumab) or irinotecan, every 14 days. Safety, efficacy, and pharmacokinetics were assessed before and after four cycles (8 weeks) of treatment. RESULTS: In 105 treated patients, investigators reported 583 adverse events (AEs) in 86 patients (81.9%), and 256 AEs (43.9%) were potentially related to arfolitixorin and 5-FU. Dose adjustments were required in 16 patients (15.2%). At 8 weeks, 9 out of 57 patients assessed for efficacy achieved an objective response (15.8%), and all 9 achieved a partial response. Six of these nine patients had received arfolitixorin as a first-line treatment. A further 33 patients (57.9%) achieved stable disease. Pharmacokinetics were assessed in 35 patients. The average tmax was 10 min, and area under the plasma concentration-time curve from time 0 to 1 h increased linearly between 30 and 240 mg/m2. No accumulation was observed for [6R]-MTHF following repeated administration, and there were no major pharmacokinetic differences between cycle 1 and cycle 4 at any dose. CONCLUSIONS: Arfolitixorin is a well-tolerated moderator of 5-FU activity. It is suitable for further investigation in mCRC and has the potential to improve treatment outcomes in patients with low folate pathway gene expression. Arfolitixorin can easily be incorporated into current standard of care, requiring minimal changes to chemotherapy regimens.

Digitoxin medication and cancer; case control and internal dose-response studies.

BACKGROUND: Digitoxin induces apoptosis in different human malignant cell lines in vitro. In this paper we investigated if patients taking digitoxin for cardiac disease have a different cancer incidence compared to the general population. METHODS: Computer stored data on digitoxin concentrations in plasma from 9271 patients with cardiac disease were used to define a user population. Age and sex matched controls from the Norwegian Cancer Registry were used to calculate the number of expected cancer cases. RESULTS: The population on digitoxin showed a higher incidence of cancer compared to the control population. However, an additional analysis showed that the population on digitoxin had a general increased risk of cancer already, before the start on digitoxin. Leukemia/lymphoma were the cancer types which stood out with the highest risk in the digitoxin population before starting on digitoxin. This indicates that yet unknown risk factors exist for cardiovascular disease and lymphoproliferative cancer. An internal dose-response analysis revealed a relationship between high plasma concentration of digitoxin and a lower risk for leukemia/lymphoma and for cancer of the kidney/urinary tract. CONCLUSION: Morbidity and mortality are high in the population on digitoxin, due to high age and cardiac disease. These factors disturb efforts to isolate an eventual anticancer effect of digitoxin in this setting. Still, the results may indicate an anticancer effect of digitoxin for leukemia/lymphoma and kidney/urinary tract cancers. Prospective clinical cancer trials have to be done to find out if digitoxin and other cardiac glycosides are useful as anticancer agents.

Digitalis; impinges on more than just the (ion-) pump.

Some forms of digitalis, such as digitoxin, inhibits proliferation and induces apoptosis in cancer cells in clinically relevant concentrations. Recently, it has been demonstrated that digitalis, in addition to inhibiting the Na(+)/K(+)-ATPase, also signals through the pathways of the epidermal growth factor receptor (EGFR). Digitalis has complex dose-dependent mechanisms of action involving many signaling systems and the relevance of this for the anticancer effects are discussed.

Digitoxin is a potential anticancer agent for several types of cancer.

The ability of digitalis to block cell proliferation has been well established for some time. Recently, digitalis in non-toxic concentrations has been showed to induce apoptosis in different malignant cell lines. In light of the pivotal role of apoptosis in cancer development and progression and this new experimental finding concerning digitalis, it seems probable that the apoptosis-inducing capability is explained by mechanisms other than just Na+/K+ ATPase inhibition. In this article, features of the cardiac glycosides which make them interesting to evaluate further as potential anticancer drugs are discussed. Some new data concerning inhibition and apoptosis in three human glioblastoma cell lines by digitoxin are also presented.

Specificity of ethephon as a butyrylcholinesterase inhibitor and phosphorylating agent.

Butyrylcholinesterase (BChE) is inhibited by the plant growth regulator (2-chloroethyl)phosphonic acid (ethephon) as observed 25 years ago both in vitro and in vivo in rats and mice and more recently in subchronic studies at low doses with human subjects. The proposed mechanism is phosphorylation of the BChE active site at S198 by ethephon dianion. The present study tests this hypothesis directly using [(33)P]ethephon and recombinant BChE (rBChE) with single amino acid substitutions and further evaluates if BChE is the most sensitive esterase target in vitro and with mice in vivo. [(33)P]Ethephon labels purified rBChE but not enzymatically inactive diethylphosphoryl-rBChE (derivatized at S198 by preincubation with chlorpyrifos oxon) or several other esterases and proteins. Amino acid substitutions that greatly reduce rBChE sensitivity to ethephon are G117H and G117K in the oxyanion hole (which may interfere with hydrogen bonding between glycine-N-H and ethephon dianion) and A328F, A328W, and A328Y (perhaps by impeding access to the active site gorge). Other substitutions that do not affect sensitivity are D70N, D70K, D70G, and E197Q which are not directly involved in the catalytic triad. The effect of pH and buffer composition on inhibition supports the hypothesis that ethephon dianion is the actual phosphorylating agent without activation by divalent cations. Human plasma BChE in vitro and mouse plasma BChE in vitro and in vivo are more sensitive to ethephon than any other esterases detected by butyrylthiocholine or 1-naphthyl acetate hydrolysis in native-PAGE. All mouse liver esterases observed are less sensitive than plasma BChE to ethephon in vitro and in vivo. More than a dozen other esterases examined are 10-100-fold less sensitive than BChE to ethephon. Thus, BChE inhibition continues to be the most sensitive marker of ethephon exposure.

Phosphobutyrylcholinesterase: phosphorylation of the esteratic site of butyrylcholinesterase by ethephon [(2-chloroethyl)phosphonic acid] dianion.

Ethephon [(2-chloroethyl)phosphonic acid] has two seemingly unrelated types of biological activity. It is a major agrochemical absorbed by crops, slowly releasing ethylene as a plant growth regulator. Ethephon also inhibits the activity of plasma butyrylcholinesterase (BuChE) in humans, dogs, rats, and mice. This is totally unexpected for an ionized phosphonic acid (mostly the dianion at physiological pH), in contrast to the classical inhibitors (nonionized triester phosphates) which phosphorylate serine at the active site. This study tests the hypothesis that ethephon (as the dianion) also acts as a phosphorylating agent in inhibiting BuChE activity. The sensitivity of plasma BuChE to ethephon (90 min preincubation at 25 degrees C) is greatest for humans, dogs, and mice (IC(50) = 6-23 microM), intermediate for chickens, rabbits, rats, and guinea pigs (IC(50) = 26-53 microM), and lowest for pigs and horses (IC(50) = 92-172 microM). The IC(50) decreases linearly with time on a log-log scale to values of 0.15-0. 3 microM for human, dog, and horse BuChE at 24 h. The inhibition rate is generally related to ethephon concentration, consistent with a bimolecular reaction, e.g., phosphorylation. The extent of inhibition of the esteratic activity of BuChE by ethephon is directly proportional to the extent of inhibition of [(3)H]diisopropyl phosphorofluoridate ([(3)H]DFP) postlabeling which is not reversible on removing the ethephon, either directly or after further incubation for 24 h at 25 degrees C. These observations strongly suggest that ethephon, as DFP, phosphorylates human plasma BuChE at Ser-198 of the esteratic site, or more generally, the formation of a phosphobutyrylcholinesterase. With human plasma BuChE, (2-bromoethyl)- and (2-iodoethyl)phosphonic acids have lower affinities for the site than ethephon but higher phosphorylation rate constants, consistent with their relative hydrolysis rates at pH 7.4 (phosphorylation of water). (2-Chlorohexyl)phosphonic acid is a poor inhibitor, perhaps being too reactive with water. Thus, potency differences for ethephon and its analogues with BuChE of various species depend on both the affinities and phosphorylation rates, i.e., the binding and reactivity of the (2-haloalkyl)phosphonic acid dianion in the esteratic site.

The role of interleukin-2 in regulating the sensitivity of natural killer cells for Fas-mediated apoptosis.

The Fas/Fas-ligand (FasL) system seems to play a key role in regulating immunoresponses. Highly purified CD56+CD3- natural killer (NK) cells were found to be resistant to the apoptosis-inducing Fas mAb CH11 in the absence or in the presence of interleukin-2 (IL-2) for up to 3 days. However, NK cells activated with IL-2 for 3 days became apoptotic following combined treatment with CH11 and actinomycin D, suggesting the presence of an intact apoptotic machinery. In contrast, NK cells cultivated in IL-2 for 6 days became sensitive to CH11-induced apoptosis without addition of actinomycin D. At this time, a pronounced up-regulation of the Fas protein on the NK cell membrane was detected. By using reverse transcription/polymerase chain reaction it was found that the anti-apoptotic gene FLIP was strongly expressed in NK cells for up to 6 days of IL-2 stimulation. After day 6, a time-dependent decrease in the expression of FLIP was observed concomitantly with increased sensitivity for Fas-mediated apoptosis. The amount of apoptotic and necrotic NK cells in the presence of IL-2 increased in a time-dependent manner, reaching 40% at day 6 of culture. The amount of apoptotic and necrotic NK cells was reduced in the presence of Fas-Fc protein. In addition, IL-2 stimulated the NK cells to release soluble FasL in a time-dependent manner, whereas membrane FasL did not seem to increase in a similar manner. These results indicate that Fas/FasL interactions are involved in the down-regulation of IL-2-activated human NK cells.

Regulation of APO-2 ligand/trail expression in NK cells-involvement in NK cell-mediated cytotoxicity.

Apo-2L is a new member of the tumour necrosis factor (TNF) family shown to induce apoptosis in a number of tumour cell lines. Apo-2L mRNA is expressed by numerous human tissues. Here we report that Apo-2L is expressed and utilized by human Natural Killer (NK) cells. NK cells were shown to express surface Apo-2L in response to interleukin 2 (IL-2) activation, and this response was restricted to the CD3(-)population of the NK cells. Apo-2L mRNA and intracellular Apo-2L were present in both CD3(-)and CD3(+)NK cells; however, increased expression in response to IL-2 was only observed in CD3(-)CD56(+)cells. Also, IL-2-activated NK cells were shown to utilize membrane-bound Apo-2L in mediating lysis of Jurkat cells. Furthermore, Apo-2L-induced apoptosis of Jurkat cells was more rapid than FasL-induced apoptosis, indicating an important and distinct role for Apo-2L in apoptotic cell destruction. In conclusion, we report that NK cells express Apo-2L and that IL-2 activated CD3(-)NK cells utilize the Apo-2L pathway in mediating target cell lysis.

Regulation of Fas and Fas-ligand expression in NK cells by cytokines and the involvement of Fas-ligand in NK/LAK cell-mediated cytotoxicity.

This study demonstrates cytokine-mediated regulation of Fas and Fas-ligand (Fas-L) expression in human NK cells and the involvement of the Fas-L pathway in NK/LAK cytotoxicity. Freshly isolated, high purified human CD56+CD3- NK cells were found to express Fas and Fas-L. Cytokines further increased the Fas expression in the CD56+CD3- NK cells, with interleukin (IL)-2 being the most potent stimulus followed by IL-12, while IL-7 had no effect. IL-2 and IL-7 equally enhanced the Fas expression in the CD56+CD3+ population, while IL-12 had a less pronounced effect. Incubation of the CD56+CD3- NK cells with IL-2, but not with IL-12 and IL-7, led to an upregulation at the Fas-L expression, whereas neither of the cytokines affected the Fas-L expression in the CD56+CD3+ cells. Antagonistic Fas mAb M3 and Fas-IgG1 fusion protein significantly inhibited NK cytotoxicity towards Fas-expressing Jurkat cells, while non-antagonistic Fas mAb M31 and irrelevant CD14-IgG1 fusion protein had no effect. IL-2-generated LAK cells were much more potent than NK cells in exerting the cytotoxic effect on Jurkat cells, which was also partially inhibited to M3 and Fas-IgG1. Thus, human NK and LAK cells express Fas and Fas-L, utilize the Fas-L cytotoxic pathway and enhance the expression of these molecules in response to cytokines.