NAMPT/Nicotinamide phosphoribosyltransferase (IC50 = 0.09 nM)

In vitro activity: APO866 at low concentrations ranging from 0.09-27 nM induces dose-dependent cytotoxicity in 41 hematologic malignant cells including acute myeloid leukemia [AML], acute lymphoblastic leukemia [ALL], mantle cell lymphoma [MCL], chronic lymphocytic leukemia [CLL], and T-cell lymphoma. APO866 at low concentrations ranging from 0-10 nM induces cell death, this effect is independent of caspase activation but is associated with depolarization of mitochondrial membrane. APO866 at concentrations ranging from 0-10 nM dose-dependently induces depletion of intracellular NAD and ATP contents and cell death in various hematologic cancer cells. APO866 at concentration of 10 nM inhibits PBEF-induced secretion of MMP-3, CCL2, and CXCL8 in HFFF2 cells.
Cell Assay: For MTT assays, 0.5 × 106 cells/mL is plated in triplicate on 96-well plates. APO866 (0.01 nM-100 nM) is added in 50 μL of culture medium, with culture medium alone serving as control. After 72 or 96 hours of incubation, 15 μL of dye solution is added to each well and cells are incubated for an additional 4 hours. Stop solution (100 μL/well) is added for 1 hour and the absorbance is read at 570 nm on a spectrophotometer. For trypan blue dye exclusion staining, 0.5 × 105 cells/well is grown in 6-well plates with 1 mL media in the absence or presence of APO866 for 96 hours. Cells from each sample are incubated with 10 μL trypan blue solution (at a 1:1 ratio [vol/vol] for 1 minute). Cell survival is determined by calculating proportion of live (unstained) cells.

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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].

Daporinad (FK866)/APO866 administered intraperitoneally at dose of 20 mg/kg twice a day for 4 days, repeat weekly over 3 weeks, prevents and abrogats tumor growth in C.B.-17 SCID mice xenograft models of human AML, lymphoblastic lymphoma, and leukemia. APO866 at dose of 0.12 mg/kg/hour prevents joint destruction and leukocyte infiltration through inhibition of PBEF in mice with CIA.

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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]
There are significant inter-individual differences in the pharmacokinetics of dapoxetine (FK866) and its metabolite FK866-N-oxide. Intra-individual differences were small between different treatment cycles in the same patient (Table 4 and Figure 2). Steady-state plasma concentrations (Css) of both FK866 and FK866-N-oxide were reached 48 hours after the start of infusion; the Css of the N-oxide metabolite was 10 times lower than that of FK866. Both were rapidly eliminated after the infusion was stopped. The steady-state plasma concentrations (Css) of both compounds increased with increasing dose, as shown in Tables 4 and 5.
The mean steady-state plasma concentrations (± standard deviation) and AUC_0-t of dapoxetine (FK866) at the maximum tolerated dose (MTD) were 5.51 ± 2.57 ng/ml (14 nM) and 505.9 ± 249.8 ng·hr/mL, respectively. The apparent terminal half-life (t_1/2) of FK866 is estimated to be 7.9–76.5 h. Steady-state plasma concentrations of FK866 are a suitable predictor of drug exposure because they are closely correlated with their AUC_0-t and the steady-state plasma concentrations and AUC_0-t of their metabolites. Based on the 95% confidence interval of the power equation multiplier between FK866 steady-state plasma concentration (Css) and infusion rate and absolute dose, the pharmacokinetics of FK866 are considered to be approximately dose-dependent. The relationship between the main dose-limiting toxicity—thrombocytopenia—and drug concentration is shown in Figure 3. This figure shows that platelet count decreases proportionally with increasing dapoxetine (FK866) concentration. Further comparison of hematological parameters (particularly lymphocyte count and hemoglobin) with FK866 levels confirms the dose-response relationship between FK866 and toxicity (data not shown). Daporene (FK866) is a highly specific inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), and its unique mechanism of action induces tumor cell apoptosis. This study developed a simple and sensitive liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-qTOF-MS) method to evaluate the drug metabolism and pharmacokinetic (DMPK) characteristics of daporene in mice. Sample pretreatment was performed using a simple protein precipitation method with acetonitrile (ACN), followed by separation using a C18 column. The calibration curve covered a concentration range of 1.02–2220 ng/mL, and was fitted using quadratic regression (weighted 1/concentration²), with a correlation coefficient ≥0.99. The accuracy and precision of the quality control samples were within ±25%, meeting the acceptance criteria. Dilution integrity was validated at 5-fold, 10-fold, and 30-fold dilutions, and the accuracy and precision of the diluted quality control samples were also within ±25% of the nominal values. Stability results showed that daporinad was stable under short-term (4 hours), long-term (2 weeks), and freeze-thaw cycles (3 times). This qualified method was successfully applied to intravenous pharmacokinetic (PK) studies of daporinad in mice at doses of 5, 10, and 30 mg/kg. Results showed that the pharmacokinetics of daporinad exhibited a linear trend in the 5–10 mg/kg dose range, while a non-linear trend was observed at 30 mg/kg. Furthermore, in vitro and in vivo metabolite identification (Met ID) studies were performed to understand the pharmacokinetic characteristics of daporinad. Results indicated that 25 metabolites involving 10 different metabolic types were identified under our experimental conditions. In summary, this study successfully developed an LC-qTOF-MS analytical method for the quantitative analysis of daporinad and its in vitro and in vivo metabolites in mouse plasma. [5]

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This study developed and validated an LC-qTOF-MS analytical method for the quantitative analysis of daporinad (FK866) in mouse plasma. Using quadratic regression, calibration curves for daporinad were acceptable across a concentration range of 1.02 to 2200 ng/mL, with correlation coefficients ≥0.99. Daporinad remained stable in mouse plasma and maintained dilution integrity under several preliminary stability test conditions, including short-term (4 hours), freeze-thaw cycles (3 times), and long-term (2 weeks). This method has been successfully applied to the quantitative analysis of pharmacokinetic (PK) samples from mouse plasma after intravenous administration of daporinad (IV).
Pharmacokinetic results showed that daporinad clearance was low to moderate, depending on the dose range of 5 to 30 mg/kg. Interestingly, Cmax was dose-proportional in the dose range of 10 to 30 mg/kg, but AUC appeared to increase disproportionately. Several possibilities exist regarding this result. Based on the clearance mechanism, we hypothesized that metabolic enzymes or transporters associated with the dapoxetine elimination pathway might play a role. This prompted us to conduct in vitro and in vivo metabolite identification (MetID) studies, which resulted in the identification of 25 new metabolites under the current experimental conditions. The results showed that dapoxetine has 10 different metabolic pathways, most of which are phase I metabolic reactions, such as amide hydrolysis, oxidation and desaturation. Although many interesting new metabolites were identified in this study, no significant differences were observed between different dose levels from the perspective of in vivo metabolites (data not shown). Therefore, other mechanisms, such as transporter saturation, may play a role in the clearance changes we observed from in vivo mouse pharmacokinetics. Further experiments such as half-mass balance studies or in vitro transporter analysis are needed to understand the elimination pathway. [5] In summary, we developed a sensitive, simple and reproducible LC-qTOF-MS method to evaluate dapoxetine in mouse pharmacokinetic samples and evaluated the in vitro and in vivo metabolic profiles of dapoxetine and several of its novel metabolites. Further experiments are needed to better understand the in vivo clearance mechanism of dapoxetine.

Toxicity[4]
Toxicity is defined as an adverse event that is at least likely to be associated with the study drug. Dapoxetine (FK866) was generally well tolerated, with the only dose-limiting toxicity (DLT) being thrombocytopenia (below 25,000/μl). This toxicity occurred in two patients in the 0.144 mg/m²/h dose group and one patient in the 0.126 mg/m²/h dose group. In addition, a persistent decrease in lymphocyte count was observed. However, lymphopenia never exceeded grade 3, and no opportunistic infections occurred. No other neutropenia was observed except in one patient with grade 1 neutropenia. Non-hematologic toxicities were relatively rare and mild. One patient experienced grade 3 fatigue, and two patients experienced grade 3 nausea/vomiting. Nausea and vomiting were well controlled after administration of five HT3 receptor antagonists. The only other grade 3 toxicity was hyperglycemia. All grade 2 or higher adverse events are listed in Table 3 by cohort. One patient experienced a cerebrovascular accident during the study; the event was assessed to be unrelated to the treatment. The patient did not complain of visual impairment, and repeat ophthalmological examinations (including ERG) showed no signs of retinopathy. No treatment-related deaths occurred during the study.

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Toxicity and Maximum Tolerated Dose [4]
Two of the five patients treated with a dose of 0.144 mg/m²/h experienced dose-limiting toxicity (DLT) related to thrombocytopenia. Both patients received platelet transfusions and recovered well. Following a de-escalation protocol, another cohort of patients was subsequently treated with a dose of 0.108 mg/m²/h. No DLT occurred in this cohort, so an intermediate dose of 0.126 mg/m²/h was tested in a subsequent dose escalation trial. At this dose level, one in six patients experienced dose-limiting toxicity (thrombocytopenia). Therefore, this dose was the maximum tolerated dose.

[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 belongs to the benzamide and N-acylpiperidine derivatives. Dapoxetine sodium has been used in clinical trials for the treatment of melanoma, cutaneous T-cell lymphoma, and B-cell chronic lymphocytic leukemia. Dapoxetine sodium is a small molecule compound with potential antitumor and anti-angiogenic activities. Dapoxetine sodium binds to and inhibits the activity of nicotinamide phosphoribosyltransferase (NMPRTase), thereby inhibiting the synthesis of nicotinamide adenine dinucleotide (NAD+) from nicotinamide (vitamin B3), which may deplete the energy reserves of metabolically active tumor cells and induce tumor cell apoptosis. Furthermore, this drug may also inhibit the production of vascular endothelial growth factor (VEGF) by tumor cells, thereby inhibiting tumor angiogenesis. The coenzyme NAD+ plays a crucial role in cellular redox reactions, including linking the citric acid cycle and oxidative phosphorylation. The turnover rate of nicotinamide adenine dinucleotide (NAD+) in malignant cells is higher than in normal cells, making this biosynthetic pathway an ideal target for cancer therapy. This study investigated the biological role of Nampt, the rate-limiting enzyme in NAD+ synthesis, in multiple myeloma (MM). The Nampt-specific chemopreventive inhibitor FK866 induced cytotoxicity in MM cell lines and MM patient cells, but not in peripheral blood mononuclear cells (PBMCs) from normal donors and MM patients. Importantly, FK866 induced cytotoxicity in MM cells resistant to traditional and novel anti-MM therapies in a dose-dependent manner, overcoming the protective effects of cytokines (IL-6, IGF-1) and bone marrow stromal cells. Nampt knockdown via RNAi confirmed its crucial role in maintaining MM cell viability and intracellular NAD(+) storage. Interestingly, FK866 cytotoxicity induced autophagy, rather than apoptosis. The MM cytotoxicity of FK866 was mediated through transcription-dependent (TFEB) and transcription-independent (PI3K/mTORC1) autophagy activation. Finally, FK866 showed significant anti-MM activity in xenograft mouse MM models, which was associated with downregulation of ERK1/2 phosphorylation and proteolytic cleavage of LC3 in tumor cells. Therefore, our data reveal the key role of Nampt in MM biology and lay the foundation for a new targeted therapy. [1] Intracellular NAD(+) levels ([NAD(+)](i)) play an important role in regulating human T lymphocyte survival, cytokine secretion and responsiveness to antigen stimulation. NAD⁺-derived Ca²⁺ generated by CD38 mobilizes second messengers that play a key role in T cell activation. This study demonstrates that alterations in NAD+ in T lymphocytes affect intracellular Ca2+ homeostasis, including mitogen-induced increases in Ca2+ and replenishment of endoplasmic reticulum Ca2+ stores. Inhibition of nicotinamide phosphoribosyltransferase mediated by FK866 reduces NAD+, thereby decreasing mitogen-induced increases in Ca2+ in Jurkat cells and activated T lymphocytes. Correspondingly, in the presence of FK866, the content of thapsigargin-sensitive Ca2+ stores in these cells is significantly reduced. When NAD(+) levels were increased by supplementing peripheral blood lymphocytes with the NAD(+) precursor nicotinamide, nicotinic acid, or nicotinamide mononucleotide, the Ca(2+) content of the carotenoid-sensitive Ca(2+) reservoir and the cellular responsiveness to mitogens (indicated by an increase in [Ca(2+)](i)) were both upregulated. Using specific siRNAs, it was shown that the changes in Ca(2+) homeostasis induced by NAD(+) precursors were mediated by CD38 and subsequently acted through ADPR-mediated TRPM2 gating. Finally, the presence of NAD(+) precursors upregulated important T cell functions, such as mitogen proliferation and IL-2 release. [2]

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Histone deacetylase inhibitors (HDACi) are a new class of therapeutic agents because they can induce cytotoxicity in a variety of cancer cells. Remodeling gene expression programs by inhibiting HDACs is a potential mechanism by which HDAC inhibitors exert their therapeutic effects. HDAC inhibitors such as SAHA and VPA primarily inhibit class I and II deacetylases, exhibiting potent antitumor activity, and are currently in phase II clinical trials. Increasing evidence suggests that SIRTs are key regulators of major tumor suppressor proteins such as p53 and FOXO3a, prompting the development of drugs that specifically target SIRTs. The fact that cancer cells require high-turnover NAD+ to sustain their growth, and that SIRTs also require NAD+ to maintain their activity, further highlights the importance of FK866 and its ability to specifically target cancer cells. The data presented in this article contribute to understanding the mechanism by which FK866 exerts its anticancer effect. In a recent paper, we found that FK866 upregulates the acetylation level of FOXO3a protein in 293T cells, thereby inducing apoptosis. However, this is the first evidence of functional p53 found in 293T cells. The 293T cell line suffers from p53 dysfunction due to its interaction with the large T antigen. In summary, enhancing p53 acetylation by inhibiting the NAMPT/SIRT pathway can improve the functional activity of p53 in cells transformed with large T antigens, which has broad implications for malignancies characterized by p53 inactivation. [3]

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Background: FK866 is a potent NAD synthesis inhibitor. This first-in-human study aimed to determine the maximum tolerated dose, toxicity profile, and pharmacokinetics under a 96-hour continuous infusion regimen. Materials and Methods: Twenty-four patients with advanced solid tumors who had failed standard therapy received escalating doses of FK866 via a 96-hour continuous infusion every 28 days. A series of plasma samples were collected to characterize the pharmacokinetics of FK866. Blood samples were also collected to measure plasma VEGF levels. Results: A total of 12 women and 12 men, with a median age of 61 years (range 34–78 years) and a median KPS score of 80%, received 4 days of FK866 infusions 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 a dose-limiting toxicity observed in two patients in the highest dose group and one patient at the recommended phase II dose of 0.126 mg/m²/h. No other hematologic toxicities were observed except for mild lymphopenia and anemia. Patients experienced mild fatigue and grade 3 nausea; the latter could be controlled with antiemetics and was not considered a dose-limiting toxicity. Css (mean plasma concentrations at 72 and 96 hours) increased with increasing dose. The study drug had no significant effect on VEGF plasma concentrations. No objective remission was observed, but four patients were stable (treated for 3 months or longer). Conclusion: The recommended phase II dose is 0.126 mg/m²/h, administered continuously for 96 hours every 28 days. The dose-limiting toxicity of FK866 is thrombocytopenia. Pharmacokinetic data showed that plasma Css increased with increasing FK866 dose. [4]

C24H30CLN3O2

427.97

427.202

C, 67.36; H, 7.07; Cl, 8.28; N, 9.82; O, 7.48

1785666-54-7

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

78243733

Typically exists as solid at room temperature

2

3

8

30

534

0

Cl.O=C(C1C=CC=CC=1)N1CCC(CCCCNC(/C=C/C2C=NC=CC=2)=O)CC1

MULSIBUGDPOSHV-CALJPSDSSA-N

InChI=1S/C24H29N3O2.ClH/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)1H/b12-11+

N-[4-(1-Benzoyl-4-piperidinyl)butyl]-3-(3-pyridinyl)-2-propenamide hydrochloride

FK-866 Hydrochloride; APO-866 HCl; Daporinad HCl; Daporinad (hydrochloride); FK866 HCl; APO-866 hydrochloride; FK866 Hydrochloride; FK-866 HCl; FK 866 HCl

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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