Compounds labeled with stable or radioactive isotopes can accurately track and quantify individual atoms in metabolic pathways. Stable isotopes generally do not change molecular properties, but may slightly affect metabolic dynamics; radioactive isotopes may interfere with cells. Labeling can distinguish endogenous and exogenous metabolites, reduce false positives, and is beneficial for quantification and reconstruction of metabolic pathways [2]. In cell culture or enzyme reactions, the use of isotope labels can accurately control concentration and exposure time, making it easier to study metabolic reactions and enzyme activities. Through stable isotope-resolved metabolomics (SIRM), cellular metabolic networks can be studied, key metabolic nodes and regulatory mechanisms can be identified, and targets can be provided for compound development. Isotope-labeled compounds can be used in competition binding experiments to evaluate the affinity and binding kinetics of compounds with receptors, which helps optimize the design. Stable isotope labels are used as internal standards in mass spectrometry analysis to improve analytical accuracy and reproducibility and reduce matrix effect interference [3].

Isotope labels can non-invasively track the distribution, transformation, and clearance of compounds and their metabolites in the body through techniques such as mass spectrometry (MS) and nuclear magnetic resonance (NMR), which is beneficial for the study of drug metabolism dynamics (ADME). Isotope labeling can reveal specific steps in the metabolic pathway. Direct use of compounds with stable isotope labels at specific positions in human or animal models can also help verify drug mechanisms and evaluate unexpected side effects, improving the accuracy and efficiency of clinical research [3].

Absorption, Distribution and Excretion
When applied topically, chlorhexidine undergoes virtually no systemic absorption. Oral chlorhexidine, such as that used in dental mouthwash, is absorbed very poorly in the gastrointestinal tract—the peak plasma concentration (Cmax) after oral administration of 300 mg chlorhexidine is 0.206 μg/g, with a time to peak concentration (Tmax) of approximately 30 minutes after ingestion. No chlorhexidine levels were detected in plasma or urine after implantation of four Periodips in 18 adult patients. Chlorhexidine gluconate is almost entirely excreted in feces, with less than 1% of the ingested dose excreted in urine. One study investigated 34 newborns who underwent standard bathing with Hibiscrub to determine whether it was absorbed percutaneously. Low concentrations of chlorhexidine were detected in blood samples from 10 infants collected via heel prick, and also in 5 of the 24 infants who received intravenous blood samples. /Chlorhexidine Gluconate/
The transdermal absorption of the antibacterial agent chlorhexidine (carbon-14 labeled) was studied in rats. Within 5 days, the absorption rate of topically applied chlorhexidine was less than 5%. The absorbed radioactive material was primarily excreted in feces.
The transdermal absorption of chlorhexidine gluconate (chlorhexidine disodium; Hibitane) through the skin (with or without stratum corneum) of hairless rats was studied. In tests conducted on intact skin, drug storage in the skin tissue was more important than diffusion after 48 hours; the opposite was observed for skin with the stratum corneum removed. When the skin was peeled, the absorption increased approximately 100-fold, and the amount stored in the skin increased approximately 10-fold. The difference in chlorhexidine diffusion observed between intact and peeled skin was related to the physicochemical properties of chlorhexidine. /Chlorhexidine Gluconate/
This study aimed to evaluate the elimination kinetics of chlorhexidine in milk when used as an intramammary infusion to stop lactation in dairy cows. …The study was conducted in two phases. Three cows were studied in each phase. All cows received intramammary infusion of chlorhexidine suspension after two milkings 24 hours apart. Foremilk samples (100 mL) were collected from the udders of both the treated and untreated (control) cows. Chlorhexidine was extracted from the raw milk, and residual concentrations were quantitatively analyzed using high-performance liquid chromatography (HPLC). Foremilk samples were analyzed on days 2, 5, and 8 in the first phase; and on days 0, 3, 7, 14, 21, 28, 35, and 42 in the second phase. No quantitative transfer of chlorhexidine to milk was detected in the untreated mammary gland area in either phase. During the 42-day sampling period in the second phase, measurable chlorhexidine residues were detected in the milk from the treated mammary gland area of two cows. The mean elimination half-life of chlorhexidine in milk was estimated to be 11.5 days.
Metabolism/Metabolites
Due to the extremely low absorption rate of chlorhexidine in the gastrointestinal tract, significant metabolic transformation is unlikely.

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Biological Half-Life
This study aimed to evaluate the elimination kinetics of chlorhexidine in milk when used as an intramammary infusion to stop lactation in dairy cows. …The study was conducted in two phases. Three cows were studied in each phase. All cows received chlorhexidine suspension infusion after two milkings 24 hours apart, with the suspension injected into the mammary gland area affected by mastitis. Foremilk samples (100 mL) were collected from the treated and untreated (control) areas of each cow. Chlorhexidine was extracted from the raw milk, and residual concentrations were quantitatively analyzed using high-performance liquid chromatography. In the first phase, foremilk samples were analyzed on days 2, 5, and 8; in the second phase, samples were analyzed on day 0 and on days 3, 7, 14, 21, 28, 35, and 42. In both phases, no quantitative transfer of chlorhexidine to milk was detected in the untreated mammary gland area. During the 42-day sampling period in the second phase, measurable chlorhexidine residues were detected in the milk from the treated mammary gland areas of two cows. The estimated mean elimination half-life of chlorhexidine in breast milk is 11.5 days.

Toxicity Summary
Identification and Uses: Chlorhexidine forms solid crystals. Chlorhexidine diacetate is currently registered for use in the United States, but approved pesticide uses may change periodically, so it is essential to consult federal, state, and local authorities for currently approved uses. Currently, two end products containing 2% chlorhexidine diacetate are registered for use as hard surface treatment disinfectants/viricides. Chlorhexidine is primarily used in its salt forms, such as dihydrochloride, diacetate, and digluconate, for use in disinfectants (skin and hand disinfectants), cosmetics (additives in creams, toothpastes, deodorants, and antiperspirants), and pharmaceuticals (preservatives in eye drops, active ingredients in wound dressings, and mouthwashes). Human Contact and Toxicity: Chlorhexidine diacetate is highly acutely toxic upon contact with the eyes. Skin reaction tests for chlorhexidine acetate and chlorhexidine gluconate were conducted on patients with eczema. In the initial test, 52 out of 1063 subjects (5.4%) showed a positive reaction. Of the 29 subjects who underwent retesting, 21 remained positive. Chlorhexidine-specific IgE was detected only in Japanese individuals who had previously experienced anaphylactic shock, and not in Japanese nurses and patients or a group of British nurses and hospital staff frequently exposed to chlorhexidine. All chromogenic agents, when used in combination with chlorhexidine, rather than chlorhexidine alone, caused some degree of discoloration in hydroxyapatite and human teeth. A 67-year-old male undergoing colectomy for colon cancer accidentally received an intravenous injection of 0.8 mg chlorhexidine gluconate and subsequently developed acute respiratory distress syndrome. There have been two case reports of healthcare workers developing occupational asthma due to exposure to chlorhexidine and alcohol aerosols. Another case report describes six patients experiencing urticaria, dyspnea, and anaphylactic shock after topical application of chlorhexidine gluconate solution. Even highly diluted chlorhexidine solutions can cause significant articular cartilage resorption, leading to severe permanent knee joint damage. Animal studies: Rabbits experienced severe eye irritation after treatment with chlorhexidine acetate. No skin irritation was reported in rabbits within 72 hours of treatment with the investigational drug. In developmental studies, no observable malformations or developmental toxicities were observed at any tested dose. Mutagenicity of chlorhexidine was observed in both positive and negative results in bacterial studies. However, no mutagenic activity was observed in in vivo micronucleus assays or mammalian cytogenetics assays using Chinese hamster ovary cells. No carcinogenic effects were observed in long-term animal studies.

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Interactions
Chlorhexidine enhances the activity of itraconazole against Candida isolates; the itraconazole-chlorhexidine combination showed synergistic activity in culture media.

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Non-human toxicity values
Oral LD50 in rats: 5,000 mg/kg
Oral LD50 in male rats: 1710 mg/kg / chlorhexidine diacetate/
Oral LD50 in female rats: 1180 mg/kg / chlorhexidine diacetate/
Dermal LD50 in rabbits: >2000 mg/kg / chlorhexidine diacetate/
For more complete non-human toxicity data for chlorhexidine (6 types), please visit the HSDB record page.

[1]. Impact of Deuterium Substitution on the Pharmacokinetics of Pharmaceuticals. Ann Pharmacother. 2019;53(2):211-216.

[2]. Soil and environmental analysis[M]. Marcel Dekker Incorporated, 2000.

[3]. Stable isotope-resolved metabolomics and applications for drug development[J]. Pharmacology & therapeutics, 2012, 133(3): 366-391.

Chlorhexidine is a biguanide compound whose structure consists of two (p-chlorophenyl)guanidine units linked by a hexamethylene bridge. It possesses anti-infective and antibacterial activity. It belongs to the biguanide and monochlorobenzene classes and is functionally related to biguanide compounds. Chlorhexidine is a broad-spectrum antibacterial biguanide compound used as a topical disinfectant and in dental treatment for inflammatory dental diseases caused by microorganisms. It is one of the most commonly used skin and mucous membrane disinfectants. The molecule itself is a cationic biguanide, consisting of two 4-chlorophenyl rings and two biguanide groups linked by a central hexamethylene chain. Topical chlorhexidine used for disinfection and mouthwashes used in dentistry are active against a variety of pathogens, including bacteria, yeasts, and viruses. Chlorhexidine was developed by Imperial Chemical Industries in the early 1950s and introduced to the United States in the 1970s. Due to numerous reports indicating that the use of 0.5% chlorhexidine gluconate tincture could cause chemical and thermal burns, the U.S. Food and Drug Administration (FDA) revoked the product's marketing authorization. Other formulations of chlorhexidine continue to be sold. Chlorhexidine's physiological mechanism of action is through disruption of cell wall integrity. Chlorhexidine is a biguanide compound used as a disinfectant with local antibacterial activity. Chlorhexidine carries a positive charge and reacts with the negatively charged surface of microbial cells, thereby disrupting cell membrane integrity. Subsequently, chlorhexidine penetrates the cell, causing leakage of intracellular components and ultimately cell death. Gram-positive bacteria are more sensitive to this disinfectant because they carry a higher negative charge. Chlorhexidine is a disinfectant and a topical anti-infective agent, and can also be used as a mouthwash to prevent dental plaque. Drug Indications Chlorhexidine can be used as a topical disinfectant for sterilization before surgery and/or medical procedures. It is available without a prescription and in various formulations (e.g., solution, sponge, cloth, swabs). Dental preparations require a prescription and include mouthwash for treating gingivitis and slow-release "chips" that can be implanted in periodontal pockets to reduce pocket depth in adults with periodontitis, serving as an adjunct to scaling and root planing.
FDA Label

Mechanism of Action

Chlorhexidine's broad-spectrum antibacterial action stems from its ability to disrupt microbial cell membranes. Positively charged chlorhexidine molecules react with negatively charged phosphate groups on the surface of microbial cells—this reaction both disrupts cell integrity, leading to intracellular leakage, and allows chlorhexidine to enter the cell, causing precipitation of cytoplasmic components, ultimately leading to cell death. The specific mechanism of cell death depends on the concentration of chlorhexidine—low concentrations have an antibacterial effect, leading to leakage of intracellular substances such as potassium and phosphorus; while high concentrations have a bactericidal effect, leading to cytoplasmic precipitation.
Therapeutic Use

Disinfectant; bactericide. (Veterinary): Disinfectant; bactericide.
Cleansing Agents: Used as surgical hand sanitizers, skin wound cleaners, general skin cleansers, healthcare worker hand sanitizers, and preoperative skin preparation agents. Chlorhexidine gluconate significantly reduces the number of microorganisms on the hands and forearms before surgery or patient care. /Chlorhexidine gluconate - Topical/
Exploratory Treatment: Designed to determine whether chlorhexidine can be used as an intervention to prolong the recurrence time of oral candidiasis. Subjects and Methods: This study was a double-blind, randomized clinical trial in 75 HIV/AIDS patients with oral candidiasis. All subjects received clotrimazole lozenges and were followed up every 2 weeks until the lesions were completely cleared. Subsequently, subjects were randomized to two groups: the 0.12% chlorhexidine group (n = 37, age 22–52 years, mean age 34 years) and the 0.9% saline group (n = 38, age 22–55 years, mean age 38 years). Follow-up was conducted every 2 weeks until the next recurrence was observed. Results: There was no statistically significant difference in the recurrence time of oral candidiasis between the chlorhexidine group and the saline group (P > 0.05). The following variables were significantly associated with recurrence time: frequency of antifungal treatment (P = 0.011), total lymphocyte count (P = 0.017), alcohol consumption (P = 0.043), and gingival candidiasis (P = 0.048). Subjects with lower lymphocyte counts had shorter recurrence-free periods of oral candidiasis (P = 0.034). Conclusion: Chlorhexidine showed a small but not statistically significant effect in maintaining recurrence-free periods of oral candidiasis. This lack of significant effect may be due to the small sample size. Further research should be conducted to better assess the magnitude of the effect or confirm our findings.
/Experimental Therapy:/ Rats were subcutaneously injected weekly with 10 mg/kg azomethane for 12 weeks to induce colorectal cancer. At week 20, rats with colorectal tumors but without peritoneal implantation or liver metastasis underwent subtotal colectomy. During surgery, the excised tumor portion was placed in the peritoneal cavity for 30 minutes; then, rats were randomly assigned to receive peritoneal lavage with either chlorhexidine or sterile water (control group). Autopsy was performed 8 weeks post-surgery. At this time, histological evaluation was performed on obvious and suspected recurrent lesions and the anastomotic area. Chlorhexidine showed significant differences compared to water in both grossly visible tumors (P=0.05) and microscopically visible tumors (P<0.05).

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Drug Warnings
For external use only: For external use only. Avoid contact with eyes, ears, and mouth. Chlorhexidine gluconate should not be used for preoperative skin disinfection of the face or head. Misuse of products containing chlorhexidine gluconate, if it enters the eyes during surgery and remains there, can cause serious permanent eye damage. If chlorhexidine gluconate comes into contact with these areas, rinse thoroughly with cold water immediately. Avoid contact with nerves. Do not use on the genital area.
/Chlorhexidine gluconate - External Use/
Allergy: Contraindicated in patients with known hypersensitivity to chlorhexidine gluconate or any of its components.
Hypersensitivity: Products containing chlorhexidine have been reported to cause irritation, sensitization, and systemic allergic reactions, particularly in the genital area. If an adverse reaction occurs and lasts for more than 72 hours, discontinue use immediately; if the reaction is severe, contact a healthcare professional.
Deafness: There have been reports of deafness caused by chlorhexidine gluconate instilled into the middle ear through a tympanic membrane perforation. /Chlorhexidine Gluconate - Topical Use/
For more complete data on chlorhexidine (8 of 8), please visit the HSDB records page.

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Pharmacodynamics
Chlorhexidine is a broad-spectrum antibacterial agent that has been shown to be active against Gram-positive and Gram-negative bacteria, yeasts, and viruses. Antibacterial activity is dose-dependent—chlorhexidine has bacteriostatic activity at low concentrations (0.02%-0.06%) and bactericidal activity at high concentrations (>0.12%). Pharmacokinetic studies of chlorhexidine mouthwash have shown that approximately 30% of the active ingredient remains in the oral cavity after rinsing and is subsequently released slowly into the oral fluid. Like tetracycline antibiotics (such as doxycycline), chlorhexidine has the ability to adhere to dentin; this ability, known as "persistence," is a result of chlorhexidine's positive charge. This persistence likely plays a role in chlorhexidine's antibacterial activity, as it allows it to adhere persistently to surfaces such as dentin, thereby preventing microbial colonization. Use of chlorhexidine mouthwash may cause staining of oral surfaces, such as teeth. This effect is not universal and appears to be more pronounced with long-term treatment (e.g., up to 6 months). However, chlorhexidine mouthwash should be used with caution in patients who cannot tolerate oral staining, and the duration of use should be minimized. Allergic reactions to chlorhexidine may be associated with the occurrence of anaphylactic shock.

C22H22D8CL2N10

513.50

504.203

1246816-96-5

9552079

Crystals from methanol
Solid

257-259°C

0.1

6

2

13

34

649

0

C1=CC(=CC=C1N/C(=N/C(=NCCCCCCN=C(/N=C(/NC2=CC=C(C=C2)Cl)\N)N)N)/N)Cl

GHXZTYHSJHQHIJ-UHFFFAOYSA-N

InChI=1S/C22H30Cl2N10/c23-15-5-9-17(10-6-15)31-21(27)33-19(25)29-13-3-1-2-4-14-30-20(26)34-22(28)32-18-11-7-16(24)8-12-18/h5-12H,1-4,13-14H2,(H5,25,27,29,31,33)(H5,26,28,30,32,34)

(1E)-2-[6-[[amino-[(E)-[amino-(4-chloroanilino)methylidene]amino]methylidene]amino]hexyl]-1-[amino-(4-chloroanilino)methylidene]guanidine

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.)