NAD+ ; NAD(P)H:quinone oxidoreductase 1 (NQO1

KL1333 is NAD A powerful regulator within cells, NAD It is essential for the cellular energy awakening coenzyme. PGC-1α is activated by SIRT1 and AMPK, which are activated by elevated NAD+ levels[1].
Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), one of the most common maternally inherited mitochondrial diseases, is caused by mitochondrial DNA mutations that lead to mitochondrial dysfunction. Several treatment options exist, including supplementation with CoQ10, vitamins, and nutrients, but no treatment with proven efficacy is currently available. In this study, we investigated the effects of a novel NAD+ modulator, KL1333, in human fibroblasts derived from a human patient with MELAS. KL1333 is an orally available, small organic molecule that reacts with NAD(P)H:quinone oxidoreductase 1 (NQO1) as a substrate, resulting in increases in intracellular NAD+ levels via NADH oxidation. To elucidate the mechanism of action of KL1333, we used C2C12 myoblasts, L6 myoblasts, and MELAS fibroblasts. Elevated NAD+ levels induced by KL1333 triggered the activation of SIRT1 and AMPK, and subsequently activated PGC-1α in these cells. In MELAS fibroblasts, KL1333 increased ATP levels and decreased lactate and ROS levels, which are often dysregulated in this disease. In addition, mitochondrial functional analyses revealed that KL1333 increased mitochondrial mass, membrane potential, and oxidative capacity. These results indicate that KL1333 improves mitochondrial biogenesis and function, and thus represents a promising therapeutic agent for the treatment of MELAS. [1]

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Cisplatin (CP) is a chemotherapeutic drug used to treat cancerous solid tumors, but it causes serious side effects, including ototoxicity. The major cause of CP-induced ototoxicity is increased levels of mitochondrial reactive oxygen species (ROS). In this study, we examined the effect of 2-Isopropyl-3H-naphtho(1,2-d)imidazole-4,5-dione (KL1333), a β-lapachone derivative, on CP-induced ototoxicity using ex vivo organotypic culture system of cochlea. Hair cell damages in CP-treated cochlear explants with or without KL1333 were compared by immunohistochemistry. CP-induced oxidative stress and the preventive effect of KL1333 were analyzed by measuring intracellular ROS levels and depolarization of mitochondrial membrane potential. Activation of apoptosis signaling pathway was detected using TUNEL assay and immunostaining of cleaved caspase-3. As the results, it was found that KL1333 pretreatment significantly decreased stereocilia degeneration and hair cell loss, and prevented an increase in mitochondrial ROS levels in response to CP. Immunohistochemical examinations of cochlear explants revealed greater caspase-3 immunopositivity in the CP group than in controls, while the KL1333 + CP group showed significantly less immunopositivity than the CP group (P < 0.05). Thus, it appeared that KL1333 protected hair cells in the organ of Corti from CP-induced apoptosis by decreasing mitochondrial damages due to the production of mitochondrial ROS. This study is the first report showed the preventive effect of KL1333 against CP-induced ototoxicity. Although further studies should be performed to determine if KL1333 could maintain anticancer effect of CP, our data cautiously suggests that the antioxidant KL1333 can be used as an effective anti-apoptotic agent to prevent ototoxicity caused by CP-induced oxidative stress, and may prove useful in preventing hearing loss caused by CP [2].

Over the past two decades there has been increased interest in orphan drug development for rare diseases. However, hurdles to clinical trial design for these disorders remain. This phase 1a/1b study addressed several challenges, while evaluating the safety and tolerability of the novel oral molecule KL1333 in healthy volunteers and subjects with primary mitochondrial disease. KL1333 aims to normalize the NAD+:NADH ratio that is critical for ATP production. The trial incorporated innovative design elements with potential translatability to other rare diseases including patient involvement, adaptive design and exploratory objectives, all of which have subsequently informed the protocol of an ongoing phase 2, pivotal efficacy study of KL1333. Results indicate KL1333 is safe and well tolerated, with dose-dependent gastrointestinal side effects, and validate potential novel outcome measures in primary mitochondrial disease including the 30-s Sit to Stand, and the patient-reported fatigue scales. Importantly, the data from the trial support efficacy of KL1333 based on improvements in fatigue and functional strength and endurance. Furthermore, the study highlights the value in using phase 1 studies to capture data that helps optimize later phase efficacy trial design[3].

NQO1 oxidation assay [1]
NADH oxidation assays were performed with rhNQO1. NQO1 protein (2.5 mU) was mixed with KL1333, CoQ10, or idebenone at various concentrations (0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, and 100 μM) in 50 mM Tris-HCl (pH 7.5) buffer containing 0.14% BSA. Reactions were initiated by addition of 200 μM NADH, and the change in absorbance at 340 nm was measured over time for 3 min at 25°C (extinction coefficient for NADH [εNADH] = 6,220 M−1 · cm−1).

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Cytochrome c reduction assay [1]
The reaction medium consisted of 77 μM cytochrome c, 200 μM NADH, and each compound (KL1333, CoQ10, or idebenone; 0.1–100 μM range) in 50 mM Tris-HCl (pH 7.5) buffer containing 0.14% BSA. Cytochrome c reduction activity was measured at 30°C using NADH as the immediate electron donor and cytochrome c as the terminal electron acceptor. Reactions were initiated by the addition of rhNQO1 (5 mU). Activity was calculated as μmol of cytochrome c reduced/mg/min of protein, based on the initial rate of change in OD at 550 nm and the extinction coefficient for cytochrome c (21.1 mM−1 · cm−1).

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Measurement of NAD+/NADH ratio [1]
Intracellular NAD+ and NADH levels were measured using the EnzyChrom NAD+/NADH Assay Kit. Briefly, cells were homogenized in either 100 μl of NAD+ extraction buffer (for NAD+ determination) or 100 μl of NADH extraction buffer (for NADH determination). Samples were heated at 60°C for 5 min, and then mixed with 20 μl of assay buffer and 100 μl of the opposite extraction buffer to neutralize the extracts. Next, samples were briefly vortexed, and centrifuged at 14,000 rpm for 5 min. Supernatants were subjected to NAD+/NADH assays based on the lactate dehydrogenase cycling reaction, in which the generated NADH reduces a tetrazolium salt to a purple colored formazan product. NAD+ and NADH levels were quantified by measuring the increase in formazan at 570 nm using a microplate reader. To prepare the cell-free enzyme system, 1 μM KL1333 was mixed with 200 μM NADH and 200 μM NAD in 50 mM Tris-HCl (pH 7.5) buffer containing 0.14% BSA (total volume, 200 μl). Reactions were initiated by addition of rhNQO1 (10 mU), and assay mixtures were incubated for 1 h at 37°C. Twenty microliters of reaction mixture were mixed with either 100 μl of NAD+ extraction buffer or 100 μl of NADH extraction buffer. Samples were heated at 60°C for 5 min, and then mixed with 20 μl of assay buffer and 100 μl of the opposite extraction buffer to neutralize the extracts. Next, samples were briefly vortexed, and centrifuged at 14,000 rpm for 5 min. Supernatants were subjected to NAD+/NADH assays, and NAD+ and NADH levels were measured on a microplate reader by monitoring absorbance at 570 nm.

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Measurement of SIRT1 activity [1]
SIRT1 activity was measured using the SIRT1 Activity Assay Kit. Cells were seeded into 6 well plates (2 × 105 cells/well). The day after, the medium was removed and cells were treated with 1 or 2 μM KL1333. After 1 h, cells were harvested and lysed in lysis buffer. Reactions were initiated by adding cell lysates to the reaction mixture containing SIRT1 assay buffer, fluoro-substrate peptides (100 mM), and NAD+ (100 mM). Fluorescence intensity was measured for 30 min at 2–3 min intervals on a microplate fluorometer (excitation, 350 nm; emission, 460 nm). SIRT1 activity was calculated within the linear range of reaction velocity, and normalized against the protein concentration in WT control cells.

Luciferase assay [1]
C2C12 myoblasts were seeded into 6 well plates (2 × 105 cells/well). The day after, cells were co-transfected with pGL3-mouse PGC-1α luciferase reporter and pRL-SV40 encoding Renilla luciferase using Turbofect transfection reagent. The transfected cells were allowed to stabilize for 24 h, and then treated with 1 μM KL1333 for 24 h. Cell lysates were subjected to luciferase assay using the Dual-Luciferase Reporter Assay System. Luciferase activity was measured using a luminometer. Firefly luciferase activity was normalized against Renilla luciferase activity.

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Quantification of ATP levels [1]
To measure intracellular ATP levels, the ATP Determination Kit was used. Human fibroblasts were seeded into 6 well plates (1.5 × 105 cells/well). The day after, cells were treated with 1 μM KL1333 or 1 μM idebenone for 24 h, and then lysed in 100 μl of cell lysis buffer. Cell lysates were centrifuged at 13,000 rpm for 10 min. ATP levels in the supernatant were measured using a luminometer.

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Determination of intracellular lactate levels [1]
Intracellular lactate levels were measured using the Lactate Colorimetric Assay Kit. Human fibroblasts were treated with 1 μM KL1333 for 24 h, and then homogenized in assay buffer. Cell lysates were reacted with reaction mixture containing assay buffer, substrate mix, and enzyme mix, and then incubated for 30 min at room temperature. Lactate levels in each sample were analyzed by monitoring optical density at 450 nm.

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Measurement of intracellular ROS levels [1]
Human fibroblasts were treated with 1 μM KL1333 for 24 h, and then incubated with 2 μM CM-H2DCFDA for 30 min at 37°C. The cells were then washed twice with phosphate-buffered saline (PBS) and resuspended in 500 μl of PBS. ROS levels in each sample were analyzed using a flow cytometer (excitation, 488 nm; emission, 530 nm).

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Measurement of mitochondrial mass and mitochondrial membrane potential [1]
Human fibroblasts were treated with 1 μM KL1333 for 24 h. After treatment, cells were washed with PBS and trypsinized, centrifuged at 400 g for 5 min, and resuspended in PBS. Cell suspensions were mixed with 200 nM MitoTracker Green FM (excitation, 488 nm; emission, 530 nm) for assessment of mitochondrial mass, or 200 nM TMRM (excitation, 488 nm; emission, 585 nm) for measurement of mitochondrial membrane potential. The cells were then stained at 37°C in a CO2 incubator for 30 min, washed with PBS, and analyzed using a flow cytometer.

This double-blind, randomized, placebo-controlled, single and multiple oral dose phase 1a/1b study was conducted in four parts (A, B, C and D). Parts A, B and D included a total of eight cohorts of healthy volunteers, while Part C, conducted at completion of Parts A and B, included one cohort of subjects with genetically confirmed PMDs. The primary objective of the study was to evaluate the safety and tolerability of KL1333 in healthy subjects and people with PMD. Other objectives were to explore pharmacokinetics (PK), food effect and pharmacodynamics (PD) of KL1333. An overview of the study design is shown in Fig. 1, Supplementary Table 1 and the Supplementary material. The investigational medicinal products were provided as 25 and 100 mg KL1333 encapsulated tablets and matching placebo tablets. The two healthy cohorts in Part D, receiving the same daily dose of 150 mg divided in two or three doses, were added later to refine the tolerability profile of KL1333, after reviewing preliminary results from Parts B and C.[3]

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Culture of mouse cochlear explants [2]
Cochlear explant and culture were performed as previously described. The cultured organs of Corti were divided into four treatment groups: untreated control (CT, n = 5), CP alone (n = 5), KL1333 pretreatment plus CP (KL1333 + CP, n = 5), and KL1333 alone (n = 5). After 16 h equilibration in a humidified atmosphere of 5 % CO2 at 37°C, the appropriate organs were treated with 1 μM KL1333 diluted into culture medium from DMSO solution. After 1 h incubation, 30 μM CP, or vehicle control, was added. Incubation continued for 30 h before collecting the explants for analyses.

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Histological evaluation [2]
To evaluate the protective effects of KL1333 on CP-induced ototoxicity, we examined the morphology of inner hair cells (IHCs) and outer hair cells (OHCs) within the treated organs of Corti. At the end of incubation, the organs were washed with phosphate-buffered saline (PBS), fixed for 15 min with 4 % paraformaldehyde (PFA, pH 7.4) in PBS, and washed with PBS three times. All samples were stained with Alexa Fluor 488-conjugated phalloidin (1:1000) in PBS-Tx, then washed three times with PBS, and mounted on glass slides using Fluoromount. For immunohistochemical quantification, IHCs and OHCs were each counted in three fields near the apex, middle and basal of each cochlear explant. Perfectly shaped hair cells were counted over a 200-μm length of basilar membrane at 30 % (apical), 50 % (middle), and 70 % (basal) positions from the apical end of the cochlear duct, respectively. Each experiment was performed independently and repeated at least five times (n = 5). Images were captured using a Zeiss Axio Imager A2 fluorescence microscope.

[1]. KL1333, a Novel NAD+ Modulator, Improves Energy Metabolism and Mitochondrial Dysfunction in MELAS Fibroblasts. Front Neurol. 2018;9:552. Published 2018 Jul 5.

[2]. KL1333, a derivative of β-lapachone, protects against cisplatin-induced ototoxicity in mouse cochlear cultures. Biomed Pharmacother. 2020;126:110068.

[3]. Optimizing rare disorder trials: a phase 1a/1b randomized study of KL1333 in adults with mitochondrial disease. Brain. 2025 Jan 7;148(1):39-46.

KL-1333 is a small molecule drug with a maximum clinical trial phase of II (across all indications) and has 2 investigational indications.

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Our results show that KL1333 augments mitochondrial biogenesis and functions by upregulating the major mitochondrial regulator PGC-1α, which is a well-investigated target in mitochondrial medicine research due to its roles in mitochondrial function and metabolism; specifically, it activates several transcription factors involved in mitochondrial and metabolic gene expression. The activity of PGC-1α can be regulated by post-translational modifications. As shown in Figure 4, we found that KL1333 induces dual activation of PGC-1α through deacetylation and phosphorylation, probably mediated by SIRT1 and AMPK, respectively. SIRT1 and AMPK are major metabolic sensors that play various roles in glucose and lipid metabolism, mitochondrial biogenesis, and transcriptional regulation. Therefore, the indication for KL1333, as an activator of the SIRT1/AMPK/PGC-1α signaling network, could be extended to aging-related and metabolic diseases such as neurodegeneration, diabetes, and non-alcoholic fatty liver diseases. Taken together, our results suggest that pharmacological modulation of NAD+ via the action of KL1333 on NQO1 improves energy balance, decreases oxidative stress, and restores mitochondrial functions and could be used to relieve the deleterious effects of mitochondrial diseases.[1]

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All these studies suggest that β-lapachone can induce cancer cell-specific apoptosis, without causing non-specific cytotoxicity in normal tissues. As a β-lapachone derivative, KL1333 has demonstrated β-lapachone-like functions in fibroblasts from a human patient with MELAS. It increased intracellular ATP levels and mitochondrial membrane potential, while it decreased ROS levels. Results in our study largely corroborate the findings of those studies. Although further confirmative studies should be performed to determine whether KL1333 could maintain anticancer effect of CP, we cautiously expect that KL1333 has a positive potential to be a useful drug candidate in preventing hearing impairment in patients undergoing CP-based chemotherapy, potentially maximizing the anticancer effects of CP at the same time. [2]

C14H12N2O2

240.257283210754

240.089

C, 69.99; H, 5.03; N, 11.66; O, 13.32

1800405-30-4

91820639

Reddish brown to red solid powder

2.3

1

3

1

18

380

0

O=C1C(C2C=CC=CC=2C2=C1NC(C(C)C)=N2)=O

AJFWITSBVLLDCC-UHFFFAOYSA-N

InChI=1S/C14H12N2O2/c1-7(2)14-15-10-8-5-3-4-6-9(8)12(17)13(18)11(10)16-14/h3-7H,1-2H3,(H,15,16)

2-(1-Methylethyl)-3H-naphth(1,2-d)imidazole-4,5-dione

KL-1333; KL 1333; 1800405-30-4; 2-isopropyl-3H-naphtho[1,2-d]imidazole-4,5-dione; NA2ZOL5UGM; UNII-NA2ZOL5UGM; 2-(1-Methylethyl)-3H-naphth(1,2-d)imidazole-4,5-dione; 2-Isopropyl-3H-naphtho(1,2-d)imidazole-4,5-dione; KL1333;

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)

DMSO : ~100 mg/mL (~416.22 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.41 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 (10.41 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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 (Please use freshly prepared in vivo formulations for optimal results.)