In Saccharomyces cerevisiae, Zinc Pyrithione (ZPT) acts as a copper ionophore, facilitating copper influx across plasma and intracellular membranes. The primary cellular targets are iron-sulfur (Fe-S) cluster-containing enzymes, including aconitase, isopropylmalate isomerase (Leu1), and glutamate synthase. ZPT does not directly inhibit copper-containing enzymes (Sod1, Fet3), manganese-containing enzymes (Sod2), zinc-containing enzymes (alcohol dehydrogenase), or metal-independent enzymes (malate dehydrogenase) [1].

The gene ACE1 (copper resistance transcription factor) protects cells from ZPT; deletion of ACE1 increases ZPT sensitivity 11-fold, confirming that bioactive copper mediates ZPT toxicity [1].

Zinc Pyrithione (ZPT) inhibited growth of Saccharomyces cerevisiae with an IC50 that was modulated by copper concentration. In YPD medium, addition of 150 µM CuCl₂ increased ZPT potency, while the copper-specific chelator bathocuproine disulfonate (4.4 mM) attenuated ZPT effect [1].

ZPT (3 µM) caused 13% ± 4% growth inhibition and reduced aconitase specific activity to 7% ± 4% of untreated control. Aconitase activity was partially restored by adding ferrous ammonium sulfate and dithiothreitol (P < 0.01), indicating presence of inactivated enzyme [1].

ZPT treatment decreased specific activity of Fe-S enzymes: isopropylmalate isomerase (Leu1) and glutamate synthase. In contrast, activity of malate dehydrogenase (no Fe-S cluster) and alcohol dehydrogenase (zinc-containing) was unchanged [1].

In deletion library screening (∼4,700 haploid mutants), Zinc Pyrithione (ZPT) at 2.5, 5, and 10 µM identified 180 sensitive strains. The ninth most sensitive strain carried a deletion of ACE1. Among the 180 strains, deletions in genes involved in mitochondrial Fe-S cluster synthesis (IBA57, SSQ1, ISA1, GRX5, ISA2) were overrepresented. Five of seven known Fe-S transfer genes were ZPT sensitive; the other two are essential [1].

In Malassezia globosa, Zinc Pyrithione (ZPT) (tested in minimal medium with 1.2 mM CuCl₂) increased cellular copper levels (P < 0.02) and decreased iron (P ≤ 0.05) and zinc levels (P ≤ 0.025). The CTR1 homolog was the fourth most downregulated gene; iron starvation genes and a copper exporter homolog were upregulated [1].

1-hydroxy-2(1H)pyridinethione (sodium salt, MC 3277) showed antibacterial activity against a wide range of microorganisms in vitro. Minimal inhibitory concentrations (MICs) ranged from 0.006 µg/ml for Mycobacterium tuberculosis var. bovis BCG to 4.0 µg/ml for Pseudomonas aeruginosa. Gram-positive organisms were most sensitive, but gram-negative organisms also showed high susceptibility (e.g., Klebsiella pneumoniae 1.5 µg/ml, Salmonella typhosa 1.5 µg/ml) [2].

Antifungal spectrum of 1-hydroxy-2(1H)pyridinethione (sodium salt, MC 3277) tested on malt-yeast extract dextrose agar (streakplate method) gave MICs: Trichophyton violaceum 2 µg/ml (most resistant), Microsporum audouini (Kligman strain) <0.06 µg/ml, Candida albicans 0.08 µg/ml, Cryptococcus neoformans 0.08 µg/ml, Saccharomyces cerevisiae 0.08 µg/ml, Trichophyton mentagrophytes 0.15 µg/ml [2].

Inactivation studies: Saliva (30% substitution in medium) increased MIC of 1-hydroxy-2(1H)pyridinethione (sodium salt, MC 3277) against Micrococcus pyogenes var. aureus from 0.15 µg/ml to 3.5 µg/ml; against Streptococcus pyogenes from 0.3 to 0.6 µg/ml; against Klebsiella pneumoniae from 2 to 3 µg/ml; against Pseudomonas aeruginosa from 5 to 6 µg/ml. Mucin (0.1%) caused about 25% inactivation against Saccharomyces cerevisiae. Human cerebrospinal fluid (30%) did not change MIC (0.15 µg/ml for M. pyogenes, 0.10 µg/ml for C. neoformans). In 50% beef serum at 5 µg/ml concentration, some inactivation was observed after 24 hours at 37°C; at higher concentrations (100-1000 µg/ml) no apparent inactivation occurred [2].

1-hydroxy-2(1H)pyridinethione (sodium salt, MC 3277) showed no activity in mice in preliminary tests against Streptococcus pyogenes C-203 [2]

For aconitase assay: S. cerevisiae BY4741 cultures were treated with Zinc Pyrithione (ZPT) overnight at 30°C, collected, washed, and concentrated 20-fold in 100 mM NaCl–20 mM Tris (pH 7.4). Cells were lysed with glass beads (10 cycles of 1 min vortexing and 1 min ice chilling). Supernatant (50 µl) was assayed in 200 µl volume in a 96-well UV-transparent plate using a kit method (increase in OD at 340 nm from 1 to 5 min). Background was subtracted from samples without NADP⁺. Aconitase specific activity expressed as µmol product/min/mg protein [1].

For isopropylmalate isomerase (Leu1): DY150 strain harboring a LEU2-containing plasmid was grown in SD media with various ZPT concentrations for 5 h at 30°C. Cells harvested at OD 0.5-1.0, lysed, and assayed by measuring absorbance increase at 235 nm (double bond of intermediate). Specific activity calculated using molar extinction coefficient of 4.530 mmol⁻¹ l for 1.0 cm path length [1].

For glutamate synthase: Extracts prepared from 10-20 U of cells in pH 7.5 buffer, assayed as described previously [1].

For malate dehydrogenase: Extracts prepared by washing 8 U of cells, suspending in 0.1 M potassium phosphate (pH 7.4) with 1 mM phenylmethylsulfonyl fluoride, vortexing with glass beads (5 times 1 min at 4°C), centrifuging at 3,000 × g for 5 min, and assaying supernatant [1].

For alcohol dehydrogenase (ADH): Extracted and assayed as described previously [1].

For 1-hydroxy-2(1H)pyridinethione (sodium salt, MC 3277), a turbidimetric tube assay was developed using Saccharomyces cerevisiae Squibb strain in modified Difco penassay broth containing 1% dextrose. The assay procedure followed Donovick et al. Statistical analysis showed that for a single assay on any one day, the assay will be within 8.5% of the true value 95% of the time [2].

Growth inhibition of Zinc Pyrithione (ZPT) against S. cerevisiae: Overnight culture (5 µl) added to 190 µl YPD medium with up to 5 µl test material in 96-well plates (Costar 3596). Plates were incubated without shaking overnight at 30°C in humidified chambers. After growth, plates were shaken for 30 s and OD600 measured using a plate reader. OD measured in plate reader corresponded to about 60% of value in a cuvette spectrophotometer [1].

Deletion library screening: Stock plates of ∼4,700 haploid deletion mutants were thawed, mixed, and 5 µl transferred to 200 µl YPD for 2 days. Then 5 µl of that culture was used to inoculate 190 µl YPD containing test compound (5 µl). Plates incubated overnight, OD measured. Each mutant tested with three ZPT concentrations (2.5, 5, 10 µM), two doses of ZnCl₂ (1.1 and 2.2 mM), and DMSO control (2.5%). Sensitivity ranking based on Jonckheere-Terpstra test [1].

For Malassezia globosa: Cells inoculated at OD600 0.1 into minimal medium (composition detailed) containing test materials, incubated 4 days at 30°C with shaking. Cells harvested by centrifugation, washed four times with 0.9% NaCl–0.4% detergent to remove residual lipid, then twice with 0.9% NaCl. Lyophilized pellets used for atomic emission [1].

For 1-hydroxy-2(1H)pyridinethione (sodium salt, MC 3277), MIC determinations: Two-fold dilution procedure with incubation for 17 hours at 37°C for most bacteria in Difco penassay broth. Cultures in logarithmic phase with approximately 1,000 organisms per ml were used. Complete inhibition of growth (absence of turbidity) defined as MIC. For fungi, streakplate method on malt-yeast extract dextrose agar was used [2].

Inactivation assays: Media were prepared with 30% of water substituted by saliva, human cerebrospinal fluid, or 50% Bacto beef serum. MICs determined by two-fold procedure. For serum inactivation, 1-hydroxy-2(1H)pyridinethione (sodium salt, MC 3277) at 1000, 500, 100, and 5 µg/ml in 50% sterile Bacto beef serum or distilled water were incubated at 37°C. Aliquots were drawn at 0, 1, 3, and 24 hours and assayed against Saccharomyces cerevisiae [2].

Absorption, Distribution and Excretion
Following oral administration, only a portion of pyrithione is absorbed. Less than 1% of zinc pyrithione is absorbed through the skin. In rats, rabbits, and monkeys, the absorption rate of radiolabeled zinc pyrithione in the bloodstream reaches 80-90% after oral or intraperitoneal injection. In rats, the primary route of excretion after oral administration is urine, with the major metabolite being S-glucuronide of 2-mercaptopyridine-N-oxide and the minor metabolite being 2-mercaptopyridine-N-oxide. Most zinc is excreted in feces after oral administration. After transdermal administration, the recovery rate from the administration site flushing fluid in pigs exceeds 90%. The urinary excretion rate in animals with intact skin is 3%. Metabolites/Metabolites In rabbits, rats, monkeys, and dogs, zinc pyrithione is bioconverted to 2-pyridinethiol 1-oxide S-glucuronide and 2-pyridinethiol S-glucuronide after oral administration.

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1-hydroxy-2(1H)pyridinethione (sodium salt, MC 3277) is partially inactivated by saliva (30% substitution caused up to 23-fold increase in MIC against M. pyogenes), by mucin (0.1% caused ~25% inactivation against S. cerevisiae), and by serum at low concentration (5 µg/ml in 50% beef serum showed some inactivation after 24 h at 37°C). No inactivation was observed with human cerebrospinal fluid (30%) [2].

[1]. Zinc pyrithione inhibits yeast growth through copper influx and inactivation of iron-sulfur proteins. Antimicrob Agents Chemother. 2011 Dec;55(12):5753-60.

[2]. In vitro studies with 1-hydroxy-2(1H) pyridinethione. Proc Soc Exp Biol Med. 1953 Jan;82(1):122-4.

Pyrithione is a pyridinethione, namely pyridine-2(1H)-thione, in which the hydrogen atom bonded to the nitrogen atom is replaced by a hydroxyl group. It is a zinc ion carrier; its zinc salt can be used as an antifungal and antimicrobial agent. It has the function of an ion carrier. It is a pyridinethione and a monohydroxypyridine. It is a tautomer of pyridine-2-thiol N-oxide. Zinc pyrithione, or zinc pyrithione, is a coordination compound formed by the chelation of zinc ions with pyrithione ligands through oxygen and sulfur centers. In the crystalline state, it exists as a centrosymmetric dimer. Due to its dynamic antibacterial and antifungal properties, zinc pyrithione can be used to treat dandruff and seborrheic dermatitis. Dandruff is a common scalp condition affecting more than 40% of the adult population worldwide, and its pathogens may be fungi such as Malassezia globosa and Malassezia restricta. Zinc pyrithione is a common active ingredient in over-the-counter anti-dandruff topical products such as shampoos. Its mechanism of action involves increasing intracellular copper levels and disrupting iron-sulfur clusters in proteins essential for fungal metabolism and growth. Due to its low solubility, zinc pyrithione released from topical formulations can deposit relatively effectively and remain on the target skin surface. Other uses of zinc pyrithione include as an additive in antifouling coatings and algaecides. Although approved for use by the U.S. Food and Drug Administration (FDA) as early as the early 1960s, the safety and efficacy of zinc pyrithione have been established over decades. Pyrithione has not shown any significant estrogenic activity according to in vivo and in vitro studies. Pyrithione has been found in Sargassum (Marsypopetalum modestum), and relevant data exist. Pyrithione is an antibacterial and antimicrobial derivative of aspergillus acid. Although its exact mechanism of action is not fully elucidated, pyrithione appears to interfere with membrane transport, ultimately leading to a loss of metabolic control. See also: Zinc pyrithione (active ingredient); Sodium pyrithione (salt form). Indications For the treatment of dandruff and seborrheic dermatitis. Mechanism of Action Zinc pyrithione inhibits fungal growth and is associated with increased copper uptake and elevated intracellular copper levels, manifested as decreased CTR1-lacZ expression and slightly increased CUP1-lacZ expression in infected microorganisms. Upon dissociation of the zinc pyrithione coordination complex, the pyrithione ligand forms a CuPT complex with available extracellular copper in the target organism. Pyrithione acts as an ion carrier, interacting nonspecifically with the plasma membrane to transport copper into the cell and promoting copper transmembrane transport. Copper may be transported to mitochondria. Copper inactivates iron-sulfur cluster (Fe-S) proteins through a mechanism similar to copper-induced bacterial growth inhibition. Reduced Fe-S protein activity leads to inhibition of fungal metabolism and growth. Studies have shown that zinc pyrithione can slightly increase zinc levels.

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Pharmacodynamics
Zinc pyrithione has broad-spectrum antibacterial activity, including against fungi, Gram-positive bacteria, and Gram-negative bacteria. Zinc pyrithione is effective against Malassezia and all other fungi, especially Malassezia species on the scalp. For patients with dandruff, zinc pyrithione treatment can reduce the number of fungi on the scalp, thereby reducing the content of free fatty acids, and thus reducing dandruff and itching.

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Zinc Pyrithione (ZPT) mechanism of action model: Pyrithione exchanges zinc for copper from extracellular copper, forming CuPT. Pyrithione acts as an ionophore, shuttling copper across the plasma membrane and intracellular membranes (including mitochondria). Increased copper inactivates Fe-S cluster assembly, leading to loss of Fe-S enzyme activity (aconitase, Leu1, glutamate synthase). This results in auxotrophy for glutamate and lysine, which can be rescued by adding L-lysine (and to a lesser extent L-glutamate) to the medium. In S. cerevisiae, L-lysine provided nearly as much protection as both amino acids [1].

ZPT treatment increases transcription of the iron regulon (FET3, etc.) due to Fe-S protein defect, not due to low iron per se. However, ZPT did not increase accumulated iron levels; decreased cellular iron may result from export of damaged Fe-S clusters [1].

For Malassezia globosa, ZPT increased cellular copper levels and downregulated CTR1 homolog, similar to S. cerevisiae. The antifungal activity of ZPT on scalp may be attributed to copper supplied by the immune system (e.g., phagosomal copper) or copper released during skin renewal [1].

1-hydroxy-2(1H)pyridinethione (sodium salt, MC 3277) was reported as a broad-spectrum antimicrobial in 1953, with striking activity against Candida albicans and Cryptococcus neoformans. Its lack of in vivo activity in mice was noted [2].

C5H5NOS

127.1643

127.009

1121-30-8

15922-78-8 (hydrochloride salt)

1570

Light yellow to yellow solid powder

1.43g/cm3

253.8ºC at 760mmHg

107.3ºC

0.00275mmHg at 25°C

1.732

1.454

1

2

0

8

162

0

YBBJKCMMCRQZMA-UHFFFAOYSA-N

InChI=1S/C5H5NOS/c7-6-4-2-1-3-5(6)8/h1-4,7H

1-hydroxypyridine-2-thione

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product is not stable in solution, please use freshly prepared working solution for optimal results.

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

DMSO : ~125 mg/mL (~983.01 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (16.36 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 (16.36 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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Solubility in Formulation 3: ≥ 2.08 mg/mL (16.36 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 20.8 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.)