Cl- channel; NF-κB
Mitochondria (leading to decreased mitochondrial membrane potential, reduced ATP levels, and altered mitochondrial morphology) [3]
Chloride ion (Cl⁻) channels, including the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) Cl⁻ channel and the GABA_A receptor/Cl⁻ channel, by blocking their transport function [2]

Adjudin is a potent blocker of Cl- channels; it decreases the capacity and ability of human sperm to fertilize in vitro[1] by interfering with Cl- ion transport function. Adjudin (ADD) is an inhibitor of the mitochondria[2]. The Sertoli-germ cell interface is where adherens junction disruption is mediated by the molecule adjudin. More than ten different types of cancer cell lines from mice or humans are treated with increasing concentrations of adjudin to examine how it affects cancer cells. The proliferation of the cells is then measured using a modified MTT assay. Human gastric adenocarcinoma cell SGC-7901, human breast adenocarcinoma cell MDA-MB-231, human hepatoma cell Smmc-7721, and human pancreatic adenocarcinoma cell MIA Paca-2 exhibit dose-dependent inhibition of cell proliferation. After being administered for 24 hours, Adjudin's IC50 against SGC-7901, MDA-MB-231, Smmc-7721, and MIA Paca-2 cells were found to be 58.0 µM, 13.8 µM, 72.3 µM and 52.7 µM, respectively. Other cancer cell lines from humans and mice have produced results that are comparable. Adjudin's IC50 values in PC3 cells and A549 cells are 63.1 µM and 93.0 µM, respectively. The IC50 of Adjudin for WI-38 and BPH-1 cells can be observed at more than 300 µM and 200 µM, respectively. These values are approximately 5 times and 2 times higher than those for the cancer cell lines A549 and PC3, respectively[3].

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In MCF-7/ADR drug-resistant breast cancer cells, the combination of Adjudin and Doxorubicin (as ADD-DOX(M) micelles) exhibited an excellent anti-multidrug resistance (MDR) effect. The IC50 value of ADD-DOX(M) was 10.1 μM, which was about 12.1-fold lower than that of free ADD-DOX conjugates (IC50, 121.4 μM). The IC50 for the mixture of free ADD and DOX (1:1 molar ratio) was 4.3 μM, which was 9.1 and 17.1 fold lower than free ADD (39.1 μM) and DOX (73.7 μM), respectively. The resistance factor (RF) for ADD-DOX(M) was 2.7, about 29.9-fold lower than that of free DOX (80.1) [1].
In A549 lung adenocarcinoma cells, Adjudin inhibited cell proliferation in a dose-dependent manner with an IC50 of 63.1 μM after 24h treatment. It induced apoptosis, increasing the percentage of apoptotic cells from 18.7% (30 μM) to 24.6% (60 μM). Adjudin treatment (24h) led to a concentration-dependent increase in cleaved Caspase-3, release of cytochrome C from mitochondria, and activation/cleavage of Caspase-9. It also reduced BCL-2 mRNA and protein expression in a concentration-dependent manner. Adjudin (24h) decreased mitochondrial membrane potential (from 20% in control to 5% with 100 μM Adjudin) and significantly reduced intracellular ATP levels. It also reduced mitochondrial mass in a concentration- and time-dependent manner and caused mitochondrial morphology to become abnormal and diffused. In a colony formation assay, treatment with 300 μM Adjudin almost eradicated A549 cells, resulting in zero clone formation [3].
In PC3 prostate cancer cells, Adjudin inhibited cell proliferation with an IC50 of 93.0 μM after 24h treatment. It induced apoptosis, increasing the percentage of apoptotic cells from a basal level of 1% to 31% with 60 μM Adjudin. The Caspase-3 inhibitor Ac-DEVD-CHO significantly reduced the ratio of apoptotic cells induced by Adjudin. In a colony formation assay, treatment with 300 μM Adjudin almost eradicated PC3 cells, resulting in zero clone formation [3].
In SGC-7901, MDA-MB-231, Smmc-7721, and MIA Paca-2 cancer cell lines, Adjudin inhibited cell proliferation in a dose-dependent manner with IC50 values of 58.0 μM, 13.8 μM, 72.3 μM, and 52.7 μM, respectively, after 24h treatment [3].
In human spermatozoa, Adjudin (10 nM – 10 μM) significantly inhibited progesterone-elicited capacitation in a dose-dependent manner. It also inhibited the acrosome reaction induced by rhuZP3β or progesterone in a dose-dependent manner (10 nM – 10 μM). Adjudin (10 μM) significantly inhibited sperm hyperactivation but did not affect sperm motility. The inhibition of capacitation by Adjudin (10 μM) was reversible. Adjudin (10 μM) blocked forskolin- and HCO₃⁻-stimulated increases in intracellular cAMP levels. Adjudin (10 and 100 μM) did not inhibit capacitation-dependent protein tyrosine phosphorylation but did inhibit the increase in serine (70 kDa) and threonine (60, 72, 95, 110, and 130 kDa) phosphorylation. Adjudin (10 μM) significantly blocked sperm capacitation in complete Cl⁻-HTF medium but not in Cl⁻-deficient HTF medium. The addition of dbcAMP rescued Adjudin-induced inhibition of hyperactivation and capacitation in Cl⁻-deficient medium [2].

The effectiveness of Adjudin is examined in a subcutaneous lung and prostate cancer model to ascertain whether it can inhibit the growth of lung and prostate cancer in vivo. A549 human lung cancer cells and PC3 prostate cancer cells are subcutaneously injected into athymic nude mice at different sites on the lower back. Then, mice are randomly divided into two treatment groups—adjudin and vehicle (control), which have comparable mean tumor sizes. Adjudin is administered intraperitoneally once every three days in lung carcinoma cells and once every other day in prostate carcinoma cells, beginning about two weeks after tumor inoculation. In rodents, adjuvant therapy is generally well tolerated. Additionally, mice treated with Adjudin exhibit a notable inhibition of tumor growth when compared to the control group(P<0.0001 in the human lung carcinoma cells A549 and P=0.006 in the prostate carcinoma cells PC3)[3].

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In a subdermal tumor inoculation model with A549 cells in male BALB/C nude mice, intraperitoneal (i.p.) injection of Adjudin (100 mg/kg, dissolved in corn oil from a DMSO stock solution) every three days for two weeks significantly slowed down tumor volume increase compared to the vehicle control group (p < 0.0001) [3].
In a subdermal tumor inoculation model with PC3 cells in male BALB/C nude mice, intraperitoneal (i.p.) injection of Adjudin (100 mg/kg, dissolved in corn oil from a DMSO stock solution) every other day for two weeks reduced tumor growth compared to the vehicle control group (p = 0.006) [3].

The hydrolysis profile of the ADD-DOX conjugate was investigated. The conjugate was dissolved in a water-methanol (75/25, v/v) solution at different pH values (4.0, 5.2, 6.5, and 7.4) and incubated at 37°C for periods ranging from 0.5 to 48 hours. At predetermined time points, an aliquot was analyzed by HPLC to determine the hydrolysis ratio, confirming that the conjugate hydrolyzes into free ADD and DOX, with a faster rate at more acidic pH [1].
The ability of Adjudin to inhibit forskolin- and HCO₃⁻-stimulated intracellular cAMP levels in human spermatozoa was measured using an enzyme immunoassay (EIA) kit. Spermatozoa were incubated for 0 and 5 hours in HTF medium with or without 10 μM forskolin and/or 10 μM Adjudin. Incubations were terminated by adding ice-cold 100 mM HCl in 100% ethanol, samples were lyophilized, and then assayed for cAMP levels according to the kit's protocol [2].
To measure ATP levels in A549 cells treated with Adjudin, the Roche ATP Bioluminescence Assay Kit (HS II) was used following the standard protocol. Cells were washed with PBS, lysed with Cell Lysis Reagent for 20 minutes, and then homogenates were mixed with Luciferase Reagents. Luminescence was detected using a plate reader. ATP concentration was calculated using an ATP standard and normalized against total protein quantity determined by BCA assay [3].

In 96-well plates with complete growth medium, 0.5×104 A549 cells, WI-38 cells, BPH-1 cells, PC-12 cells, and other cell lines are seeded. The cells are then incubated for 24 hours. Adjudin is then serially diluted in growth medium (without serum) at concentrations of 300 µM, 100 µM, 30 µM, 10 µM, 3 µM and 0. After another 24 hours of incubation, 10 µl of the Cell Counting Kit-8 solution is added to each well. A microplate reader is used to measure the absorbance at 450 nm following 4 h of incubation at 37 °C in the cell incubator[3].

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The cytotoxicity of ADD-DOX(M) was assessed using a standard MTT assay. MCF-7/ADR or MCF-7 cells were seeded in 96-well plates and incubated for 24 hours. Cells were then treated with medium containing serial concentrations of drugs for 72 hours. After incubation, cells were washed with PBS and exposed to MTT-containing serum-free medium for 2 hours at 37°C. The medium was then discarded, and DMSO was added to dissolve the formazan. After 15 minutes of incubation, the absorbance at 570 nm was determined using a microplate reader. The half-maximal inhibitory concentrations (IC50) were calculated [1].
Cellular uptake of ADD-DOX(M) by MCF-7/ADR cells was assessed using confocal laser scanning microscopy (CLSM) and flow cytometry (FCM). Cells were incubated with ADD-DOX(M) for specified time periods (1, 4, 12, 24, 48 h). For CLSM, cells were fixed with paraformaldehyde, stained with Hoechst 33258 for nuclei, and imaged. For FCM, cells were trypsinized, collected, resuspended in ice-cold PBS, and analyzed. Energy-dependent endocytosis was investigated by comparing intracellular fluorescence at 4°C vs 37°C [1].
The anti-proliferative effect of Adjudin was determined using a modified MTT assay (or Cell Counting Kit-8). Cells were seeded in 96-well plates and incubated for 24 hours. The growth medium was then replaced with a serial dilution of Adjudin (300, 100, 30, 10, 3 μM) in serum-free medium. After 24 hours of incubation, CCK-8 solution was added to each well. After 4 more hours, the absorbance at 450 nm was measured using a microplate reader. The IC50 was determined from the graph [3].
Apoptosis was evaluated using the Annexin V/7-AAD Apoptosis Detection Kit. Cells were treated with Adjudin, then resuspended in binding buffer. Annexin V and 7-AAD were added and incubated at room temperature for 15 minutes in the dark, followed by analysis by flow cytometry. For Caspase-3 inhibition studies, cells were pre-incubated for 2 hours with the Caspase-3 inhibitor Ac-DEVD-CHO (20 μM) before Adjudin treatment [3].
For the colony formation assay, A549 and PC3 cells were cultured for 2 weeks on plates with a top agar layer made by mixing cells with a solution containing 0.7% Agar, 2x medium, 20% FBS, and different concentrations of Adjudin. The base agar was made with 1% agar and 2x medium with 20% FBS. Colonies were stained with 0.05% crystal violet for 1 hour, and colonies containing >50 cells were counted [3].
JC-1 staining was used to measure mitochondrial membrane potential (Δψm). A549 cells treated with Adjudin for 24h were collected, washed, and incubated with 2 μM JC-1 at 37°C for 30 minutes. Cells were then pelleted, resuspended in PBS, and analyzed on a flow cytometer using 488 nm excitation with 530 nm and 585 nm bandpass emission filters [3].
Mitochondrial mass was detected using MitoTracker Green. A549 cells treated with Adjudin were incubated with 200 nM MitoTracker probe at 37°C for 45 minutes. The staining solution was then replaced with PBS, and cells were analyzed by flow cytometry [3].
For quantification of mitochondrial morphology, A549 cells were stained with 200 nM MitoTracker Red, fixed, permeabilized, and blocked. Images were taken by a confocal microscope and analyzed using a custom macro in NIH ImageJ software. The mean area/perimeter ratio was used as an index of mitochondrial interconnectivity, and the inverse circularity was used as a measure of mitochondrial elongation [3].

Mice: Equal amounts of A549 cells (0.5×107 cells) and PC3 cells (1×106 cells) are hypodermically implanted into male BALB/C nude mice weighing under ~20g. After two weeks, the mice with palpable tumors are split into two groups (n = 4 per group in each experiment; repeated three times total): one group receives an i.p. injection of Adjudin, which is dissolved in corn oil from a DMSO stock solution and has a final administered quantity of 100 mg/kg (~300 µM used in vitro); the other group receives an injection of the same quantities of corn oil and DMSO as the equivalent vehicle control group. Adjudin or vehicles are given every three days to A549 patients and every other day to PC3 patients for up to two weeks. Calculated and determined tumor volumes.

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For the in vivo tumor model, male BALB/C nude mice were subdermally implanted with A549 cells (0.5 x 10⁷ cells) mixed with matrigel or PC3 cells (1 x 10⁶ cells). After 2 weeks, mice with palpable tumors were divided into two groups (n=4 per group, experiment repeated three times). The treatment group received intraperitoneal (i.p.) injection of Adjudin which was dissolved in corn oil from a DMSO stock solution, with a final administered quantity of 100 mg/kg. The vehicle control group received the same amount of corn oil and DMSO via i.p. injection. Adjudin or vehicles were administered every three days for the A549 model and every other day for the PC3 model, up to 2 weeks. Tumor volumes were determined and calculated using the formula 1/2 x L x W², where W is the smaller dimension [3].

In rat primary Sertoli cells and an organ culture model, Adjudin posed minimal toxicity to these normal cells. The IC50 of Adjudin in non-cancerous human lung fibroblast WI-38 cells and benign prostatic hyperplasia epithelial BPH-1 cells was >300 μM and >200 μM, respectively, which is about 5 times higher than that for cancer cell lines A549 (63.1 μM) and PC3 (93.0 μM). Adjudin (up to 300 μM) did not affect the growth of primary human endothelial progenitor cells (EPC) [3].
In a study on human spermatozoa, Adjudin at concentrations up to 10 μM did not affect sperm motility, indicating no cytotoxicity at these concentrations [2].

[1]. Inhibition of sperm capacitation and fertilizing capacity by adjudin is mediated by chloride and its channels in humans. Hum Reprod. 2013 Jan;28(1):47-59.

[2]. Combination delivery of Adjudin and Doxorubicin via integrating drug conjugation and nanocarrier approaches for the treatment of drug-resistant cancer cells. J Mater Chem B. 2015 Feb 28;3(8):1556-1564.

[3]. Male contraceptive Adjudin is a potential anti-cancer drug. Biochem Pharmacol. 2013 Feb 1;85(3):345-55.

Adjudin (formerly AF-2364) is a potential non-hormonal male contraceptive that was developed to mediate adherens junction disruption at the Sertoli-germ cell interface, causing premature release of spermatids from the seminiferous epithelium, thereby inducing reversible infertility in rats [2, 3].
It is an analog of lonidamine (LND) [1, 2, 3].
Adjudin is the first reported drug used in combination with Doxorubicin for treating MDR cancer cells via a co-delivery strategy involving drug conjugation and nanocarriers [1].
The study demonstrates that Adjudin possesses anti-cancer activity by targeting mitochondria and inducing apoptosis through a Caspase-3-dependent pathway [3].
Adjudin's inhibition of human sperm capacitation and fertilizing capacity is mediated by its effect on chloride ions and their channels, an effect that is reversible [2].

C15H12CL2N4O

335.19

334.038

C, 53.75; H, 3.61; Cl, 21.15; N, 16.72; O, 4.77

252025-52-8

252025-52-8

9819086

White to off-white solid powder

1.5±0.1 g/cm3

1.705

2.67

2

3

3

22

411

0

O=C(C1=NN(CC2=CC=C(Cl)C=C2Cl)C3=C1C=CC=C3)NN

VENCPJAAXKBIJD-UHFFFAOYSA-N

InChI=1S/C15H12Cl2N4O/c16-10-6-5-9(12(17)7-10)8-21-13-4-2-1-3-11(13)14(20-21)15(22)19-18/h1-7H,8,18H2,(H,19,22)

1-[(2,4-dichlorophenyl)methyl]indazole-3-carbohydrazide

Adjudin; AF-2364; AF2364; AF 2364

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: 16.67~67 mg/mL (49.7~199.9 mM)
Ethanol: 2 mg/mL (6.0 mM)

Solubility in Formulation 1: ≥ 1.67 mg/mL (4.98 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 16.7 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: ≥ 1.67 mg/mL (4.98 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 16.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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