Nicotinamide phosphoribosyltransferase (NAMPT) (IC50 = 0.09 nM)

By selectively killing MM cells, Nampt inhibition with (E)-Daporinad (FK866) causes a large intracellular decrease in NAD+. Higher baseline NAD+ levels in MM cells than in normal PBMCs give (E)-Daporinad (FK866) sensitivity. (E)-Daporinad (FK866)-induced cell death is related with inhibition of Nampt activity, rather than protein expression. The bone marrow microenvironment's survival advantage is eliminated by (E)-Daporinad (FK866)[1]. (E)-Daporinad (FK866) lowers the Ca2+ content of TG-responsive Ca2+ stores in Jurkat and activated PBLs and inhibits the [Ca2+]i rise brought on by various mitogens. The Ca2+ content of TG-responsive Ca2+ stores in Jurkat cells is decreased by (E)-Daporinad (FK866), but not in Bcl2-Jurkat cells[2]. Using the p53 acetylation route, suppression of NAMPT by (E)-Daporinad (FK866) or inhibition of SIRT by nicotinamide reduces proliferation and causes death of 293T cells[3].

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Nampt-specific chemical inhibitor Daporinad (FK866) triggered cytotoxicity in MM cell lines and patient MM cells, but not normal donor as well as MM patients PBMCs. Importantly, FK866 in a dose-dependent fashion triggered cytotoxicity in MM cells resistant to conventional and novel anti-MM therapies and overcomes the protective effects of cytokines (IL-6, IGF-1) and bone marrow stromal cells. Nampt knockdown by RNAi confirmed its pivotal role in maintenance of both MM cell viability and intracellular NAD(+) stores. Interestingly, cytotoxicity of FK866 triggered autophagy, but not apoptosis. A transcriptional-dependent (TFEB) and independent (PI3K/mTORC1) activation of autophagy mediated FK866 MM cytotoxicity.[1]

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Lowering [NAD(+)](i) by Daporinad (FK866)-mediated nicotinamide phosphoribosyltransferase inhibition decreased the mitogen-induced [Ca(2+)](i) rise in Jurkat cells and in activated T lymphocytes. Accordingly, the Ca(2+) content of thapsigargin-sensitive Ca(2+) stores was greatly reduced in these cells in the presence of FK866.[2]

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Using the 293T cell line (HEK293 cells transformed with large T antigen) as a model, we provide evidence that p53 is one of the critical downstream targets involved in Daporinad (FK866)-mediated killing of 293T cells. A reduced rate of apoptosis and an increased number of cells in S-phase was accompanied after knockdown of p53 in these cells. Inhibition of NAMPT by FK866, or inhibition of SIRT by nicotinamide decreased proliferation and triggered death of 293T cells involving the p53 acetylation pathway. Additionally, knockdown of p53 attenuated the effect of FK866 on cell proliferation, apoptosis, and cell cycle arrest. The data presented here shed light on two important facts: (1) that p53 in 293T cells is active in the presence of FK866, an inhibitor of NAMPT pathway; (2) the apoptosis induced by FK866 in 293T cells is associated with increased acetylation of p53 at Lys382, which is required for the functional activity of p53 [3].

In CB17-SCID mice, (E)-Daporinad (FK866) (30 mg/kg, ip) reduces the tumor burden and significantly reduces ERK phosphorylation and proteolytic cleavage of LC3 in the tumor tissue[1].

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In vivo anti-MM efficacy of Daporinad/FK866 [1]
To investigate whether Nampt inhibition by Daporinad/FK866 could inhibit MM cell growth in vivo, we used CB17-SCID mice xenografted subcutaneously with MM.1S cells. Fourteen tumor-bearing mice were randomly assigned to receive either 30 mg/kg of Daporinad/FK866 administered by intraperitoneal injection (twice a day for 4 days consecutively followed by 3 days off therapy in each cycle, repeated over 3 weeks) or vehicle control. Figure 7A shows the comparison of MM.1S growth in cohorts receiving vehicle control (n = 7) versus treatment (n = 7). A significantly decreased tumor burden was observed as early as day 7 of treatment and was confirmed at day 21, in treated compared with control mice. (P = .0061 and P = .0067, respectively). Importantly, the treatment was well tolerated, without significant weight loss or neurologic changes, and resulted in a significant prolongation in overall survival (Figure 7B; P = .0014). The median overall survival was 19.5 days in control group versus 45 days in the FK866-treated group. Tumors were harvested, and lysates were subjected to Western blot analysis to evaluate phosphorylation of ERK and LC3B (Figure 7C). Consistent with our in vitro results, tumor tissue from FK866-treated mice demonstrated a significant decrease in ERK phosphorylation and proteolytic cleavage of LC3 compared with control. These results establish in vivo proof-of-concept for the investigational study of FK866 in the treatment of MM.

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Antitumor activity [4]
No objective responses were noted. Out of 24 patients treated, 4 had stable disease for at least 3 months (prostate 4 months, melanoma 5 months, mesothelioma 3 months, and oropharynx 5 months). The patient with prostate cancer had stable disease by imaging but was removed from study after 4 months due to a rise in the PSA. The patient with oropharynx carcinoma had some clinical benefit with a decrease in pain. The patient’s CT scans were stable on first disease evaluation; however there was progression at 5 months.

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VEGF measurements [4]
VEGF serum levels were measured in patients in the second cohort and higher. Levels were recorded prior to dosing, at 48 h into the infusion and again at 96 h. The levels of VEGF decreased in five of six patients treated at the MTD. Due to the small numbers of samples collected, the decrease in VEGF did not meet statistical significance (Fig. 1).

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Application for Intravenous (IV) Pharmacokinetic (PK) Studies in Mouse [5]
The developed LC-qTOF-MS method was successfully applied to obtain the PK parameters of Daporinad (FK866) in mouse plasma following IV administration at the doses of 5, 10 and 30 mg/kg. The concentrations of most PK samples were within the qualified calibration curve except some PK samples at early time point samples. Therefore, early time point PK samples were diluted appropriately with blank mouse plasma to ensure that every sample concentration was within the calibration curve range. The time to concentration profile of Daporinad (FK866) is shown in Figure 3. The PK parameters were calculated with the non-compartmental analysis (NCA) using WinNonlin (version 8.0.0) and the results are summarized in Table 4.

Sample Preparation for In Vitro Met ID In Mouse and Human Liver Microsomes [5]
First, a cofactor-compound mixture was prepared by mixing NADPH solution A and B, UDPGA, GSH and a working solution (2 mg/mL) of Daporinad (FK866). The above cofactor-compound mixture was pre-incubated at 37 °C for 5 min. 380 µL of the pre-incubated mixture was then transferred to 1.7 mL polypropylene tube, and 20 µL of 20 mg/mL mouse or human liver microsomes were applied. The microsomes and cofactor-compound mixture were incubated at 37 °C for 0 and 120 min and the reaction was stopped by adding 450 µL of ACN for protein precipitation. After centrifugation at 8000× g rpm for 10 min, 550 µL of supernatants was transferred to a fresh tube and evaporated to dryness under vacuum using rotary evaporator. Completely dried tubes were reconstituted with 110 µL of 30% ACN in DW with 0.1% FA. Reconstituted samples were centrifuged at 12,000× g rpm for 5 min, and its supernatant was transferred to an LC vial for the in vitro Met ID analysis.

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Sample Preparation for In Vivo Met ID in Mouse PK Samples [5]
For the in vivo Met ID analysis, plasma samples from 30 mg/kg IV PK groups were pooled according to the Hamilton pooling method, respectively. 210 µL of the pooled plasma sample was transferred to a polypropylene tube and 800 µL of ACN was added for protein precipitation. Then, the above samples were centrifuged at 10,000× g rpm for 10 min and 900 µL of the supernatant was evaporated to dryness under vacuum in a rotary evaporator. Completely dried tubes were reconstituted with 110 µL of 30% ACN in DW with 0.1% FA. The reconstituted sample was vortexed and centrifuged at 12,000× g rpm for 5 min and the supernatant was transferred into a LC vial for the in vivo Met ID analysis.

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Fluorimetric Determination of Intracellular Calcium Levels [2]
PBL or Jurkat cells (2 × 106/ml), stimulated or not with phytohemagglutinin (PHA) (5 μg/ml) and/or treated for 24 h in the presence or absence of 33 nm Daporinad (FK866) or 0.1 mm NAM, NA, or NMN, were loaded with 10 μm FLUO-3AM or Fura-2AM for 45 min at 37 °C in RPMI medium, washed with Ca2+-containing Hanks' balanced salt solution (HBSS), and resuspended in the same solution at 2 × 106 cells/ml. Alternatively, in some experiments, cells were washed and resuspended in Ca2+-free HBSS before thapsigargin (TG) addition. [Ca2+]i measurements with Fluo-3-loaded cells were performed in 96-well plates (105 cells/well). The basal fluorescence (excitation, 485 nm; emission, 520 nm) was adjusted to have a comparable (within a ±10% range) basal intensity in each well. Fluorescence was measured every 3 s with a fluorescence plate reader. The intensity of emitted light was plotted as a function of time. Calcium changes were calculated for each trace using the formula Δ/basal × 100, where Δ is the difference between the maximal fluorescence upon the addition of stimulus and the basal fluorescence (basal), normalized to the basal fluorescence (basal). Fura-2-loaded cells were seeded on poly-l-lysine-coated, glass bottom, cell culture dish and incubated for 20 min at 37 °C. [Ca2+]i measurements and calibrations were performed with a microfluorimetric system.

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Determination of Intracellular NAD+ and cADPR Levels [2]
PBL or Jurkat cells were cultured as described above for 24 h and stimulated or not with PHA (5 μg/ml) in the presence or absence of Daporinad (FK866) (33 nm), NAM (0.1, 1, or 10 mm), NA (0.1 mm), or NMN (0.1 mm). At the end of each incubation, 1 ml of cells were withdrawn and centrifuged for 15 s at 16,000 × g. Cell pellets were lysed in 0.3 ml of 0.6 m perchloric acid at 4 °C. Cell extracts were centrifuged for 3 min at 16000 × g; the supernatants were collected, and an aliquot was diluted 200-fold in 100 mm sodium phosphate buffer, pH 8.0, for determination of NAD+ content. Perchloric acid was removed from the rest of the supernatant by mixing the aqueous sample with 4 volumes of a solution containing 1,1,2-trichlorotrifluoroethane and tri-n-octylamine, and the cADPR content was determined. NAD+ and cADPR values were normalized on protein concentrations, determined by the Bradford assay.

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Determination of Intracellular ATP Content [2]
PBLs were cultured as described above for 24 h, stimulated or not with PHA (5 μg/ml), in the presence or absence of Daporinad (FK866) (33 nm), NAM (0.1 mm), NA (0.1 mm), or NMN (0.1 mm). At the end of the incubation, 1 ml of cells was withdrawn and centrifuged for 15 s at 16,000 × g. Cell pellets were lysed in 0.3 ml of 0.6 m perchloric acid at 4 °C, and the neutralized extracts were analyzed by HPLC (11). ATP values were normalized on protein concentrations, determined by the Bradford assay.

Effect of Daporinad (FK866) on paracrine MM cell growth in the BM [1]
MM1S cells (2 × 104 cells/well) were cultured for 72 and 96 hours in BMSC-coated 96-well plates n the presence or absence of drug. DNA synthesis was measured by (3H)–thymidine uptake, with (3H)–thymidine added (0.5 μCi/well) during the last 8 hours of cultures.

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Characterization of cell death [1]
Cells were cultured with Daporinad (FK866) (0.1nM-33nM) for 96 hours. For caspase inhibition assays, cells were pretreated with pan caspase inhibitor (zVAD-fmk), caspase 3-(zDEVD-fmk) and caspase 9-(zLEHD-fmk) inhibitors for at least 2 hours before addition of FK866. For inhibition of autophagy, cells were incubated with the inhibitors wortmannin (0.25μM), LY294002 (5μM), 3-methyladenine (3MA 100μM), and chloroquine (20μM) for at least 30 minutes before FK866 treatment. Apoptosis was quantitated using annexin-V–FITC staining and flow cytometric analysis, according to the manufacturer's protocol.

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Immunofluorescence [1]
U266 and RPMI8226/S cells were transiently transfected with pEGFP-LC3B, and treated with either vehicle or Daporinad (FK866) 10nM for 24 and 48 hours. The GFP fluorescence was recorded using a Nikon E800 epifluorescence microscope equipped with a Coolsnap CF color camera. For quantification, 10 fields, each consisting of 40-100 GFP-positive cells, were used to calculate the number of cells with GFP-LC3B puncta relative to the total number of GFP-positive cells.

Murine xenograft model of human MM [1]
CB17-SCID mice (28-35 days old) were used. All animal studies were conducted according to protocols approved by the Animal Ethics Committee of the Dana-Farber Cancer Institute. Mice were irradiated (200 cGy), and then inoculated subcutaneously in the right flank with 3 × 106 MM1S cells in 100 μL RPMI 1640. After detection of tumor (∼ 2 weeks after the injection), 7 mice were treated intraperitoneally with either vehicle or Daporinad (FK866) (30 mg/kg body weight) twice a day for 4 days, repeated weekly over 3 weeks. Caliper measurements of the longest perpendicular tumor diameters were performed twice a week to estimate the tumor volume using the following formula: length × width2 × 0.5. Tumor growth inhibition (TGI) was calculated using the formula (Δcontrol average volume − Δ treated average volume) × 100 (Δcontrol average volume). Animals were killed when tumors reached 2 cm3 or the mice appeared moribund. Survival was evaluated from the first day of treatment until death. The images were captured with a Canon IXY digital 700 camera. Excised tumors from mice were collected and assessed by Western blotting.

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Male ICR mice (30 ± 3 g) were housed in groups of 6~8 per cage and given standard rodent chow. The animals were fasted overnight with free access to water for at least 12 h before administration. Mice were distributed into three different dosing groups (n = 3 for each dosing group; 5, 10 and 30 mg/kg). The blood sampling time points were 2, 10, 30, 60, 120, 240 and 420 min after administration. The sampling blood samples were centrifuged at 12,000× g rpm for 5 min and the plasma was transferred to another tube and then stored at −20 °C until further analysis. [5]

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Pharmacokinetic analysis [4]
Plasma samples were obtained prior to dosing and serially up to 152 h after the initiation of the infusion to determine the concentration and clearance of Daporinad (FK866) both during the continuous infusion and after the completion of the infusion. The concentrations of FK866 and its metabolite, FK866-N-oxide (FR247684), were measured in plasma samples using HPLC (high performance liquid chromatography) with tandem mass spectroscopy (LS/MS/MS) by BioProof AG for DL 1 and 2, and by Japan Clinical Laboratories, Inc. for DL three to 6. The lower limits of quantification (LLOQ) of FK866 and FR247684 in plasma were 0.04 ng/ml. The mean and the standard deviation (SD) of concentrations of FK866 and FR247684 in plasma were calculated at each dose levels. The concentrations below LLOQ were treated as zero. If the number of measurable concentration data was less than two at each treatment group, SD was not calculated. Plasma concentration data were excluded from all pharmacokinetic calculations if they were clearly recognized as outliers due to a fairly large deviation from the individual concentration-time curve.
The following pharmacokinetic parameters of Daporinad (FK866) and FR247684, except for plasma concentration at steady-state (CSS), were calculated for each subject based on a model-independent approach by the computer program WinNonlin® Standard version 4.1. All statistical analyses were performed by SAS® software release 8.02 .

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Dissolved in 0.9% saline; 20 mg/kg; i.p. injection

C.B.-17 SCID mice xenograft models of human AML, lymphoblastic lymphoma, and leukemia.

Pharmacokinetics [4]
Pharmacokinetics of Daporinad (FK866) and its metabolite FK866-N-Oxide had a large inter-individual variability. Intra-individual variability between cycles for each patient was rather limited (Table 4 and Fig. 2). The Css of FK866 and FK866-N-Oxide was reached 48 h after the start of the infusion; the Css of the N-Oxide metabolite was 10-fold lower than that of FK866. Both were eliminated rapidly after stopping the infusion. The Css of both compounds increased with dose escalation, see Tables 4 and 5.
The mean Css (+SD) and AUC0–t of Daporinad (FK866) at MTD were 5.51 + 2.57 ng/ml (14 nM) and 505.9 + 249.8 ng.hr/mL, respectively. The apparent terminal t 1/2 of FK866 was estimated to be 7.9–76.5 h. The Css of FK866 can be regarded as an appropriate predictor of drug exposure, because it closely correlated with its AUC0–t and the Css and AUC0–t of its metabolite. The pharmacokinetics of FK866 was considered to be roughly dose-dependent, based upon the 95% confidence intervals of the multiplier in the power equation for Css of FK866 against infusion rate and absolute amount used.
The major dose limiting toxicity, thrombocytopenia, was compared to drug levels in Fig. 3. This figure shows a proportional decline in platelets as the concentration of Daporinad (FK866) increases. Further comparisons between hematologic parameters, specifically lymphocyte count and hemoglobin, and FK866 levels confirmed a dose relationship of FK866 to toxicity (data not shown).

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Daporinad (FK866) is one of the highly specific inhibitors of nicotinamide phosphoribosyl transferase (NAMPT) and known to have its unique mechanism of action that induces the tumor cell apoptosis. In this study, a simple and sensitive liquid chromatography–quadrupole-time-of-flight–mass spectrometric (LC-qTOF-MS) assay has been developed for the evaluation of drug metabolism and pharmacokinetics (DMPK) properties of Daporinad in mice. A simple protein precipitation method using acetonitrile (ACN) was used for the sample preparation and the pre-treated samples were separated by a C18 column. The calibration curve was evaluated in the range of 1.02~2220 ng/mL and the quadratic regression (weighted 1/concentration2) was used for the best fit of the curve with a correlation coefficient ≥ 0.99. The qualification run met the acceptance criteria of ±25% accuracy and precision values for QC samples. The dilution integrity was verified for 5, 10 and 30-fold dilution and the accuracy and precision of the dilution QC samples were also satisfactory within ±25% of the nominal values. The stability results indicated that Daporinad was stable for the following conditions: short-term (4 h), long-term (2 weeks), freeze/thaw (three cycles). This qualified method was successfully applied to intravenous (IV) pharmacokinetic (PK) studies of Daporinad in mice at doses of 5, 10 and 30 mg/kg. As a result, it showed a linear PK tendency in the dose range from 5 to 10 mg/kg, but a non-linear PK tendency in the dose of 30 mg/kg. In addition, in vitro and in vivo metabolite identification (Met ID) studies were conducted to understand the PK properties of Daporinad and the results showed that a total of 25 metabolites were identified as ten different types of metabolism in our experimental conditions. In conclusion, the LC-qTOF-MS assay was successfully developed for the quantification of Daporinad in mouse plasma as well as for its in vitro and in vivo metabolite identification.[5]

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In this study, an LC-qTOF-MS assay was developed and qualified for the quantification of Daporinad (FK866) in mouse plasma. The calibration curves were acceptable over the concentration range from 1.02 to 2200 ng/mL for Daporinad using the quadratic regression with a correlation coefficient ≥ 0.99. Daporinad was stable in mouse plasma under the several preliminary stability test conditions, such as short-term (4 h), freeze–thaw (three cycles), and long-term (2 weeks), and achieved the dilution integrity. This method was successfully applied to quantify the in vivo IV PK mouse plasma samples.
The PK results suggest that Daporinad has low to moderate clearance values depending on the 5 to 30 mg/kg administered dose range. It was interesting that in the dose range from 10 to 30 mg/kg, the Cmax was dose-proportional, but the AUC appeared to increase supra proportions. There are several possibilities regarding this result and based on the clearance mechanism, we hypothesize that either metabolic enzymes or transporters related to the elimination pathway of Daporinad might play a role. This triggers in vitro and in vivo MetID studies, and as a result, twenty-five metabolites were newly identified under the current experimental conditions. The results suggest that ten different metabolic pathways were identified for Daporinad, and most of them were phase Ⅰ metabolic reactions, such as amide hydrolysis, oxidation and desaturation. Although many interesting metabolites were newly identified in this experiment, no significant difference from in vivo metabolites perspectives were observed from different dose levels conducted in this study (data not shown) and, therefore, it appeared that other mechanism such as saturation of transporters etc might likely play a role for this phenomenon of clearance change we observed from the in vivo mouse PK. Further experiments such as semi-mass balance study to understand the elimination pathway or in vitro transporter assays to identify the responsible transporters would be necessary.[5] In conclusion, we developed a sensitive, simple and reproducible LC-qTOF-MS assay to evaluate Daporinad in mouse PK samples, and also evaluated the in vitro and in vivo metabolite profiling of Daporinad with several novel metabolites. This research would warrant further experiments to better understand the in vivo clearance mechanism of Daporinad.

Toxicity [4]
A toxicity was defined as an adverse event that was at least possibly related to the study drug. Daporinad (FK866) was generally well tolerated with thrombocytopenia less than 25,000/μl being the unique dose limiting toxicity (DLT). This occurred in two patients in the cohort at 0.144 mg/m2/h and in one patient at 0.126 mg/m2/h There was also a consistent drop in the lymphocyte counts. This lymphopenia, however, was never greater than grade 3 and there were no cases of opportunistic infections. Neutropenia, other than one patient with grade 1, was not observed. Non-hematology toxicities were relatively infrequent and mild. There was one grade 3 fatigue and two patients with grade 3 nausea/vomiting. The nausea and vomiting was well controlled with five HT3 antagonists. The only other grade 3 toxicity was hyperglycemia. All adverse events grade 2 or higher are listed by cohort in Table 3. One patient had a cerebral vascular accident while on study; this was assessed as not related to therapy. There were no complaints of visual acuity loss nor did repeated ophthalmologic evaluation, including ERG, show signs of retinopathy. There were no treatment-related deaths on study.

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LTs and the MTD [4]
Two of the five patients treated at 0.144 mg/m2/h had thrombocytopenia-related DLTs. Both of these patients were transfused with platelets and recovered appropriately. According to the de-escalation design, a cohort was then treated at 0.108 mg/m2/h. There were no DLTs at this cohort and therefore the subsequent escalation tested an intermediate dose at 0.126 mg/m2/h. One out of six patients at this dose level experienced a DLT (thrombocytopenia). This dose is therefore the MTD.

[1]. Targeting NAD+ salvage pathway induces autophagy in multiple myeloma cells via mTORC1 and extracellular signal-regulated kinase (ERK1/2) inhibition. Blood. 2012 Oct 25;120(17):3519-29.

[2]. NAD+ levels control Ca2+ store replenishment and mitogen-induced increase of cytosolic Ca2+ by Cyclic ADP-ribose-dependent TRPM2 channel gating in human T lymphocytes. J Biol Chem. 2012 Jun 15;287(25):21067-81.

[3]. Inhibition of NAMPT pathway by FK866 activates the function of p53 in HEK293T cells. Biochem Biophys Res Commun. 2012 Aug 3;424(3):371-7.

[4]. The pharmacokinetics, toxicities, and biologic effects of FK866, a nicotinamide adenine dinucleotide biosynthesis inhibitor. Invest New Drugs. 2008 Feb;26(1):45-51.

[5]. Quantitative Analysis of Daporinad (FK866) and Its In Vitro and In Vivo Metabolite Identification Using Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometry. Molecules. 2022 Mar 21;27(6):2011.

FK-866 is a member of benzamides and a N-acylpiperidine.
Daporinad has been used in trials studying the treatment of Melanoma, Cutaneous T-cell Lymphoma, and B-cell Chronic Lymphocytic Leukemia.
Daporinad is a small molecule with potential antineoplastic and antiangiogenic activities. Daporinad binds to and inhibits nicotinamide phosphoribosyltransferase (NMPRTase), inhibiting the biosynthesis of nicotinamide adenine dinucleotide (NAD+) from niacinamide (vitamin B3), which may deplete energy reserves in metabolically active tumor cells and induce tumor cell apoptosis. In addition, this agent may inhibit tumor cell production of vascular endothelial growth factor (VEGF), resulting in the inhibition of tumor angiogenesis. The coenzyme NAD+ plays an essential role in cellular redox reactions, including the redox reaction linking the citric acid cycle and oxidative phosphorylation.

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Malignant cells have a higher nicotinamide adenine dinucleotide (NAD(+)) turnover rate than normal cells, making this biosynthetic pathway an attractive target for cancer treatment. Here we investigated the biologic role of a rate-limiting enzyme involved in NAD(+) synthesis, Nampt, in multiple myeloma (MM). Nampt-specific chemical inhibitor FK866 triggered cytotoxicity in MM cell lines and patient MM cells, but not normal donor as well as MM patients PBMCs. Importantly, FK866 in a dose-dependent fashion triggered cytotoxicity in MM cells resistant to conventional and novel anti-MM therapies and overcomes the protective effects of cytokines (IL-6, IGF-1) and bone marrow stromal cells. Nampt knockdown by RNAi confirmed its pivotal role in maintenance of both MM cell viability and intracellular NAD(+) stores. Interestingly, cytotoxicity of FK866 triggered autophagy, but not apoptosis. A transcriptional-dependent (TFEB) and independent (PI3K/mTORC1) activation of autophagy mediated FK866 MM cytotoxicity. Finally, FK866 demonstrated significant anti-MM activity in a xenograft-murine MM model, associated with down-regulation of ERK1/2 phosphorylation and proteolytic cleavage of LC3 in tumor cells. Our data therefore define a key role of Nampt in MM biology, providing the basis for a novel targeted therapeutic approach. [1]

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Intracellular NAD(+) levels ([NAD(+)](i)) are important in regulating human T lymphocyte survival, cytokine secretion, and the capacity to respond to antigenic stimuli. NAD(+)-derived Ca(2+)-mobilizing second messengers, produced by CD38, play a pivotal role in T cell activation. Here we demonstrate that [NAD(+)](i) modifications in T lymphocytes affect intracellular Ca(2+) homeostasis both in terms of mitogen-induced [Ca(2+)](i) increase and of endoplasmic reticulum Ca(2+) store replenishment. Lowering [NAD(+)](i) by FK866-mediated nicotinamide phosphoribosyltransferase inhibition decreased the mitogen-induced [Ca(2+)](i) rise in Jurkat cells and in activated T lymphocytes. Accordingly, the Ca(2+) content of thapsigargin-sensitive Ca(2+) stores was greatly reduced in these cells in the presence of FK866. When NAD(+) levels were increased by supplementing peripheral blood lymphocytes with the NAD(+) precursors nicotinamide, nicotinic acid, or nicotinamide mononucleotide, the Ca(2+) content of thapsigargin-sensitive Ca(2+) stores as well as cell responsiveness to mitogens in terms of [Ca(2+)](i) elevation were up-regulated. The use of specific siRNA showed that the changes of Ca(2+) homeostasis induced by NAD(+) precursors are mediated by CD38 and the consequent ADPR-mediated TRPM2 gating. Finally, the presence of NAD(+) precursors up-regulated important T cell functions, such as proliferation and IL-2 release in response to mitogens.[2]

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Inhibitors of HDACs (HDACi) are a new class of therapeutic agents because they induce cytotoxicity in wide range of cancer cells. Reconstituting the gene expression programme by inhibition of HDACs is a potential underlying mechanism in the efficacy of HDACi. HDACi like SAHA and VPA, which primarily inhibit class I and II deacetylases, exhibit strong anti-tumor activity and have entered phase II clinical trials. The increasing evidence that SIRTs are critical regulators of major tumor-suppressor proteins, like p53 and FOXO3a, has recently lead to the development of drugs which can specifically target SIRTs. The fact that cancer cells require high turnover of NAD+ to maintain their growth, and SIRTs require NAD+ to maintain their activity, further highlights the importance of FK866 and its ability to specifically target cancer cells. The presented data contribute towards the understanding of mechanism(s) by which FK866 exerts its anti-cancer effects.
In a recent communication we have shown that FK866 upregulates the acetylation of FOXO3a protein in 293T cells, leading to apoptosis. However, this is the first evidence for the presence of functionally active p53 in 293T cells, a cell line known for the abnormal p53 function due to its interaction with large T-antigen. In conclusion, enhancing acetylation of p53 by inhibiting the NAMPT/SIRT pathway improves functional activity of p53 in cells transformed with large T-antigen, which has broad implications for malignancies characterized by p53 inactivation.[3]

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Background: FK866 is a potent inhibitor or NAD synthesis. This first-in-human study was performed to determine the maximum-tolerated dose, toxicity profile, and pharmacokinetics on a 96-h continuous infusion schedule. Materials and methods: Twenty four patients with advanced solid tumor malignancies refractory to standard therapies were treated with escalating doses of FK866 as a continuous, 96-h infusion given every 28 days. Serial plasma samples were collected to characterize the pharmacokinetics of FK866. Further blood samples were collected for the measurement of plasma VEGF levels. Results: There were 12 women and 12 men with a median age of 61 (range 34-78) and a median KPS of 80%, received a 4-day of infusion of FK866 at dose levels of 0.018 mg/m2/h (n=3), 0.036 mg/m2/h (n=3), 0.072 mg/m2/h (n=3), 0.108 mg/m2/h (n=4), 0.126 mg/m2/h (n=6), and 0.144 mg/m2/h (n=5). Thrombocytopenia was the dose limiting toxicity, observed in two patients at the highest dose level and one patient at the recommended phase II dose of 0.126 mg/m2/h No other hematologic toxicities were noted other than mild lymphopenia and anemia. There was mild fatigue and grade 3 nausea; the latter was controlled with antiemetics and was not a DLT. Css (the mean of the 72 and 96 h plasma concentrations) increased in relation to the dose escalation. The study drug did not significantly affect plasma concentrations of VEGF. There were no objective responses, although four patients had stable disease (on treatment for 3 months or greater). Conclusions: The recommended phase II dose is 0.126 mg/m2/h given as a continuous 96-h infusion every 28 days. The dose limiting toxicity of FK866 is thrombocytopenia. Pharmacokinetic data suggest an increase in the plasma Css in relation to the escalation of FK866.[4]

C24H29N3O2

391.51

391.225

C, 73.63; H, 7.47; N, 10.73; O, 8.17.

658084-64-1

658084-64-1;201034-75-5;1785666-54-7 (HCl);1198425-96-5 (deleted);

6914657

White to light yellow solid powder

1.1±0.1 g/cm3

629.9±51.0 °C at 760 mmHg

334.8±30.4 °C

0.0±1.8 mmHg at 25°C

1.589

2.45

1

3

8

29

534

0

C1CN(CCC1CCCCNC(=O)/C=C/C2=CN=CC=C2)C(=O)C3=CC=CC=C3

KPBNHDGDUADAGP-VAWYXSNFSA-N

InChI=1S/C24H29N3O2/c28-23(12-11-21-8-6-15-25-19-21)26-16-5-4-7-20-13-17-27(18-14-20)24(29)22-9-2-1-3-10-22/h1-3,6,8-12,15,19-20H,4-5,7,13-14,16-18H2,(H,26,28)/b12-11+

(E)-N-[4-(1-benzoylpiperidin-4-yl)butyl]-3-pyridin-3-ylprop-2-enamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.39 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.39 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.39 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 45% Propylene glycol (dissolve first)+5% Tween 80+ddH2O: 15mg/mL

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Solubility in Formulation 5: 4 mg/mL (10.22 mM) in 20% SBE-β-CD in Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)