Translocator protein (TSPO) [1]

For PK 11195, the median IC50 values were 14.2 μM for L. amazonensis, L at 8.2 μM. major, and L at 3.5 μM. braziliensis. The value of L's selectivity index. amazonensis was 13.7, suggesting that PK 11195 is safe for use in upcoming tests on mammals. There were observed time- and dose-dependent decreases in the number of live intracellular parasites, the number of parasites per infected macrophage, and the proportion of infected macrophages. Several morphological alterations suggestive of autophagy were detected by electron microscopy. It's interesting to note that L had lower MCP-1 and superoxide levels. macrophages infected with Amazonensis and treated with PK 11195 [1].

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PK11195 reduced viability of axenic Leishmania amazonensis promastigotes with median IC50/48h value of 14.22 µM (interquartile range 10.18-18.02) after 48h treatment at concentrations ranging from 0.20 to 400 µM [1]
- PK11195 reduced viability of axenic Leishmania braziliensis promastigotes with median IC50/48h value of 3.51 µM (IQR 2.34-5.89) under same conditions [1]
- PK11195 reduced viability of axenic Leishmania major promastigotes with median IC50/48h value of 8.23 µM (IQR 6.17-9.83) under same conditions [1]
- PK11195 caused time- and dose-dependent reduction in percentage of infected macrophages at early stages of infection: 97.75% reduction (IQR 98.69-97.58) with 100 µM treatment for 24h; after 48h, percentage of infected cells was 1.37% (IQR 0.68-2.05) vs control 86.88% (IQR 81.5-90.94) [1]
- PK11195 reduced number of parasites per infected macrophage: median values of 1.41 (IQR 1.28-2.77) at 75 µM and 1.16 (IQR 1.00-2.22) at 100 µM vs control 6.37 (IQR 5.95-6.65) [1]
- Intracellular amastigote IC50/48h value for PK11195 was 46.55 ± 11.88 µM (concentrations ranging from 6.25 to 175 µM) [1]
- At early infection stage, 100 µM PK11195 for 24h reduced viable intracellular parasites by 91.08%; 48h treatment with 75 µM and 100 µM reduced viable intracellular parasites by 99.09% and 100%, respectively [1]
- At later infection stage (96h post-infection, all parasites transformed to amastigotes), 75 µM PK11195 for 48h caused 100% reduction in viable intracellular parasites; 50 or 75 µM for 72h also caused 100% reduction [1]
- The effect of PK11195 (75 µM) on parasite viability was irreversible: 97.15% irreversible reduction after 24h treatment followed by 48h drug-free culture; 100% irreversible reduction after 48h treatment [1]
- PK11195 (75 µM pre-treatment for 24h) reduced superoxide (O2•−) production by plasma-membrane NADPH-dependent oxidase during phagocytosis by 3.5- to 5.0-fold compared to untreated controls; in LPS-pre-treated macrophages (500 ng/mL), PK11195 reduced O2•− production by 5.0-fold compared to LPS alone [1]
- PK11195 (50 µM for 24 or 48h) significantly reduced MCP-1 production in infected macrophages: 55.69% reduction at 24h (p<0.0001) and 39.39% at 48h (p<0.0001); in IFN-γ-primed infected macrophages, reductions were 59.69% at 24h and 56.82% at 48h (p<0.0001) [1]
- No changes in NO production or levels of IL-6, IL-10, TNF-α, IL-12, IFN-γ were detected in infected macrophages treated with PK11195 for 24 or 48h [1]
- Ultrastructural alterations observed by electron microscopy in intracellular L. amazonensis after PK11195 treatment (75 µM for 24 or 48h): double membrane vesicles (suggestive of autophagy), enhanced mitochondrial size, marked cytosolic disorganization, multivesicular bodies, high electrodensity in cytosol, and debris of dead parasites inside parasitophorous vacuoles [1]

Viability of axenic Leishmania promastigotes: Stationary-phase promastigotes of L. amazonensis, L. braziliensis, and L. major were cultured at 2×10⁶ cells/mL in Schneider's complete medium in 96-well plates at 24°C. Parasites were treated with 12 two-fold serial dilutions of PK11195 (400, 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.20 µM) or diluent (ethanol) for 48h. AlamarBlue cell viability reagent was added to 10% v/v final concentration and incubated at 24°C for 4h. Absorbance was measured at 570 and 600 nm [1]
- Macrophage viability (cytotoxicity): Uninfected thioglycolate-elicited peritoneal macrophages from CBA mice were cultured at 2×10⁵ cells/mL in complete DMEM in 96-well plates at 37°C, then treated with PK11195 for 48h at concentrations 400, 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.20 µM, alongside diluent (ethanol) negative control. AlamarBlue was added to 10% v/v, incubated at 37°C for 4h, and absorbance measured at 570 and 600 nm. CC50/48h value was 194.4 µM (95% CI 151.4-249.6) [1]
- Infection and treatment (early stage): CBA mouse macrophages were infected with stationary-phase L. amazonensis promastigotes at 10:1 ratio for 6h, washed to remove non-internalized parasites, then treated with PK11195 at 25, 50, 75, or 100 µM for 6, 24, or 48h. Control cells received ethanol diluent or remained untreated. Cells were fixed and stained with hematoxylin and eosin, and at least 400 cells per condition were counted under light microscopy at 1000× magnification to determine percentage of infected cells and number of parasites per infected macrophage [1]
- IC50 determination for intracellular amastigotes: Infected macrophages (6h infection) were treated with PK11195 at 6.25, 12, 25, 50, 75, 100, 125, 150, and 175 µM for 48h, then fixed and stained with DAPI. Percentage of infected cells was determined by fluorescence microscopy [1]
- Intracellular parasite viability assay (early and late stages): After treatment, macrophages were washed and medium replaced with Schneider's complete medium to release amastigotes. If viable, amastigotes transformed into promastigotes. Cells were incubated at 24°C for five days, and viable promastigotes were counted in a Neubauer chamber. For late stage, cells were infected for 6h, washed, incubated in fresh DMEM for 96h (allowing complete transformation to amastigotes), then treated with 50 or 75 µM PK11195 or 2.1 µM amphotericin B for 24, 48, or 72h [1]
- Reversibility assay: Infected macrophages were treated with 75 µM PK11195 for 6, 12, 24, or 48h, then washed and incubated in PK11195-free complete DMEM for additional 48h. Viable parasites were counted as above [1]
- Superoxide production assay: Lucigenin-enhanced chemiluminescence was used. Untreated inflammatory peritoneal CBA macrophages or cells pre-treated for 24h at 37°C with 75 µM PK11195, 500 ng/mL LPS, or both, were placed in a luminometer. Baseline O2•− release was measured for 2 min before adding L. amazonensis promastigotes, then photon emissions measured for 20 min, followed by addition of 2.5 U/mL superoxide dismutase [1]
- Cytokine and NO production assay: Thioglycolate-elicited peritoneal macrophages (10⁶ cells) were primed with 50 U/mL IFN-γ for 24h, infected, then treated with 50 µM PK11195 for 24 or 48h with or without IFN-γ. Culture supernatants were collected. Cytokine levels (IL-6, IL-10, TNF-α, IL-12, IFN-γ, MCP-1) were quantified using a flow cytometric multiplexed bead-based immunoassay. NO production was measured by nitrite accumulation using Griess reaction [1]

Cytotoxicity to uninfected macrophages: CC50/48h = 194.4 µM (95% CI 151.4-249.6) [1]
- Selectivity index (CC50:IC50) for L. amazonensis = 13.7 [1]

[1]. Guedes CES, et al. In vitro evaluation of the anti-leishmanial activity and toxicity of PK-11195. Mem Inst Oswaldo Cruz. 2018 Feb 5;113(4):e170345.

PK-11195 is a monocarboxylic acid amide, formed by the condensation of the carboxyl group of 1-(2-chlorophenyl)isoquinoline-3-carboxylic acid with the amino group of sec-butylmethylamine. It is an antitumor drug. PK-11195 belongs to the isoquinoline class, monocarboxylic acid amide class, aromatic amide class, and monochlorobenzene class of compounds.

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PK11195 is a ligand of translocator protein (TSPO), previously known as peripheral benzodiazepine receptor (PBR) [1]
- TSPO levels are reduced in L. amazonensis-infected CBA mouse macrophages compared to L. major-infected cells, suggesting TSPO as a potential chemotherapeutic target for leishmaniasis [1]
- PK11195 has been used as a marker of neuroinflammation in PET imaging and exhibits anticancer and immunomodulatory activities; it has also been shown to reduce proliferation of Plasmodium falciparum and Toxoplasma gondii [1]
- The anti-leishmanial effect of PK11195 may be independent of TSPO interaction, as no TSPO homologues have been found in Leishmania genome; PK11195 may act by altering lipid bilayer fluidity [1]
- PK11195 is hydrophobic and binds to alpha-1-acid glycoprotein with high affinity and to albumin with low affinity, which may explain the need for micromolar concentrations for therapeutic effects [1]
- The observed ultrastructural alterations (double-membrane vesicles, mitochondrial swelling, multivesicular bodies) suggest cell death via multiple mechanisms including autophagy [1]

C21H21CLN2O

352.862

352.134

85532-75-8

1345

Off-white to light brown solid powder

1.2±0.1 g/cm3

511.7±45.0 °C at 760 mmHg

74-75

263.3±28.7 °C

0.0±1.3 mmHg at 25°C

1.611

4.58

0

2

4

25

457

0

O=C(N(C(CC)C)C)C1C=C2C(C=CC=C2)=C(C2C(Cl)=CC=CC=2)N=1

RAVIZVQZGXBOQO-UHFFFAOYSA-N

InChI=1S/C21H21ClN2O/c1-4-14(2)24(3)21(25)19-13-15-9-5-6-10-16(15)20(23-19)17-11-7-8-12-18(17)22/h5-14H,4H2,1-3H3

N-(sec-butyl)-1-(2-chlorophenyl)-N-methylisoquinoline-3-carboxamide

PK-11195 PK 11195PK11195

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 : ~25 mg/mL (~70.85 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.89 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 20.8 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: ≥ 2.08 mg/mL (5.89 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 20.8 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.)