Tetracycline
Divalent cations, specifically Calcium (Ca²⁺). CTC forms a fluorescent chelate complex with Ca²⁺, and this complex binds to membranes. In mouse sperm, the CTC-Ca²⁺ complex localizes to the plasma membrane overlying the acrosomal region of the sperm head and the midpiece. [2]
Chlortetracycline hydrochloride inhibits bacterial protein synthesis by binding to the 16S portion of the 30S subunit of the bacterial ribosome. This binding prevents the aminoacyl-tRNA from binding to the A-site of the ribosome, thereby blocking the addition of new amino acids to the growing peptide chain. This mechanism effectively halts bacterial protein synthesis, leading to growth inhibition or bacterial death. The compound is bacteriostatic rather than bactericidal. Beyond its antibacterial mechanism, chlortetracycline hydrochloride functions as a cell-permeable fluorescent probe for calcium ions (Ca²⁺). It chelates divalent cations such as Ca²⁺ and Mg²⁺, enabling the visualization of calcium-dependent cellular processes. The primary mechanism of bacterial resistance involves loss of cell wall permeability, preventing the drug from reaching its ribosomal target.

Chlortetracycline is used as a fluorescent probe to detect the acrosome reaction in mouse sperm. Intact, acrosome-intact mouse sperm, when exposed to CTC in the presence of Ca²⁺, show bright, uniform fluorescence over the anterior portion of the sperm head (the acrosomal area). Sperm that have undergone the acrosome reaction do not show this fluorescence on the sperm head, which remains dark, while the midpiece retains its bright fluorescence as an internal control. This allows for reliable and quantifiable assessment of the percentage of sperm that have undergone the acrosome reaction in a motile population. [2]
Mouse sperm bound to the zonae pellucidae of eggs or to isolated zonae show the same CTC fluorescence pattern as intact sperm, with bright fluorescence on the anterior head, confirming that these sperm are acrosome-intact at the time of binding. Sperm located within the perivitelline space, which have penetrated the zona, show no CTC fluorescence on the sperm head, indicating they have completed the acrosome reaction. [2]
Incubation of mouse sperm in suspension with CTC reveals a predictable sequence of fluorescence patterns reflecting the stages of the acrosome reaction: from intact (bright, uniform acrosomal fluorescence), to an intermediate stage with intensely fluorescent spots on the acrosome, to the final reacted state (complete loss of fluorescence from the sperm head). [2]
In the absence of added Ca²⁺, exposure of mouse sperm to CTC results in considerably reduced fluorescence emission; the sperm head is virtually invisible, and the midpiece displays only weak fluorescence. The presence of Mg²⁺ does not produce the same fluorescence pattern as Ca²⁺. Addition of EGTA changes the fluorescence pattern observed with CTC plus Ca²⁺ to that observed with CTC in the absence of Ca²⁺. [2]
Tetracycline, when used under identical conditions (10 µM), produces no fluorescent image of mouse sperm. At a higher concentration (170 µM), it provides only a faint fluorescent image of the midpiece alone, with no regional fluorescence patterns on the sperm head. These patterns do not change with sperm incubation time, making tetracycline unsuitable as a probe for the acrosome reaction. [2]
In guinea pig sperm, which have larger acrosomes, CTC produces an analogous fluorescence pattern. The inner acrosomal membrane of reacted sperm is non-fluorescent. Approximately 50% of acrosome-reacted guinea pig sperm appear to acquire bright fluorescence in the post-acrosomal region. [2]

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In vitro studies demonstrate that chlortetracycline hydrochloride exhibits broad-spectrum antibacterial activity against both Gram-positive and Gram-negative bacteria. It is active against clinically important pathogens including Escherichia coli, Staphylococcus aureus, Salmonella, and Pseudomonas aeruginosa. Against Pseudomonas aeruginosa, chlortetracycline hydrochloride shows potent activity with a minimum inhibitory concentration (MIC) of 2 μg/mL. The compound also demonstrates activity against Mycoplasma putrefaciens isolates at concentrations ranging from 4 to 0.03 mg/mL. Studies on drug delivery formulations have shown that chlortetracycline hydrochloride can be successfully encapsulated in alginate beads with halloysite nanotubes (loading capacity of 59.85 ± 2.3 mg/g) or kaolin (loading capacity of 68.74 ± 2.1 mg/g), and these encapsulated formulations maintain antibacterial activity against E. coli and S. aureus after encapsulation. Solubility enhancement studies have demonstrated that complexation with hydroxypropyl-β-cyclodextrin (HP-β-CD) improves chlortetracycline hydrochloride solubility approximately 9-fold, from 4 mg/mL to 36 mg/mL, while maintaining or enhancing antibacterial activity.

In vivo studies demonstrate the therapeutic efficacy of chlortetracycline hydrochloride in animal models of bacterial infection. Research in naturally infected chickens showed that chlortetracycline hydrochloride injection (20 mg/kg) exhibited good antibacterial activity and therapeutic effects, with pathological tissue images confirming treatment efficacy. Notably, chlortetracycline hydrochloride/HP-β-CD inclusion complex injection demonstrated superior therapeutic effects compared to florfenicol injection at the same dose. Pharmacokinetic studies in ducks revealed that chlortetracycline hydrochloride soluble powder (15 mg/kg body weight) reached peak plasma concentration at approximately 1.5 hours post-administration. In veterinary medicine, chlortetracycline hydrochloride is commonly used to treat conjunctivitis in cats, as well as infections in cattle (0.1-10 mg per lb body weight per day), swine (10 mg per lb body weight per day), and turkeys (25 mg per lb body weight per day), administered via oral route.

Methodology for Ribosome Binding Assay (Cell-Free System): To assess the direct binding of chlortetracycline hydrochloride to bacterial ribosomes, a cell-free translation system can be employed. Purified 70S ribosomes from E. coli are isolated and suspended in binding buffer containing appropriate concentrations of Mg²⁺ and monovalent cations. Chlortetracycline hydrochloride (radiolabeled with ³H or ¹⁴C, or fluorescently labeled) is incubated with the ribosome preparation at 37°C for a specified duration (typically 15-30 minutes). The reaction mixture is then filtered through nitrocellulose membranes to separate ribosome-bound antibiotic from unbound drug. The retained radioactivity or fluorescence on the filters is quantified by liquid scintillation counting or fluorometry. Competition binding assays can be performed using unlabeled tetracycline to determine specific binding. Alternatively, surface plasmon resonance (SPR) can be employed with ribosomes immobilized on a sensor chip to measure real-time binding kinetics and calculate association (ka) and dissociation (kd) rate constants, from which the dissociation constant (Kd) is derived.

Mouse sperm suspensions are prepared by mincing the excised caudae epididymides of a mature Swiss mouse in 0.4 ml of TN medium (Tris-NaCl). Particulate tissue is removed after 10-15 min of sperm dispersion. Aliquots of the epididymal suspension are diluted 1:5 in the indicated test medium. For CTC assay, sperm are exposed to a final concentration of 10 µM chlortetracycline in the presence of 1.7 mM Ca²⁺. The sperm suspension is then placed on a slide and observed immediately under a microscope equipped with epifluorescence illumination. The fluorescence pattern on the sperm head is used to classify sperm as acrosome-intact (bright, uniform anterior head fluorescence) or acrosome-reacted (no head fluorescence). The midpiece fluorescence serves as a positive internal control. To assess the acrosome reaction in sperm bound to zonae, gametes are recovered 15 min post-insemination and exposed to 10 µM CTC. For sperm within the perivitelline space, gametes are inseminated in the presence of 3 µM A23187 to increase the number of penetrating sperm, recovered 2h later, washed, and exposed to 20 µM CTC. [2]

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Methodology for Antibacterial Susceptibility Testing (MIC Determination): The minimum inhibitory concentration (MIC) of chlortetracycline hydrochloride against bacterial strains is determined using the broth microdilution method as recommended by CLSI standards. Bacterial isolates (e.g., E. coli, S. aureus, P. aeruginosa) are cultured overnight in appropriate growth medium (Mueller-Hinton broth). The bacterial suspension is adjusted to 0.5 McFarland standard (~1.5 × 10⁸ CFU/mL) and further diluted 1:100 to achieve a final inoculum of ~5 × 10⁵ CFU/mL. Serial two-fold dilutions of chlortetracycline hydrochloride are prepared in 96-well plates, ranging from 64 μg/mL to 0.03 μg/mL. Each well receives 100 μL of bacterial suspension and 100 μL of antibiotic dilution. Positive controls (bacteria without antibiotic) and negative controls (medium only) are included. Plates are incubated at 37°C for 18-24 hours. The MIC is defined as the lowest concentration of antibiotic that completely inhibits visible bacterial growth, as evidenced by a lack of turbidity or pH color change compared to the drug-free growth control. For fluorescence-based assays (calcium probe application), cells (e.g., spermatozoa or neuronal cells) are loaded with chlortetracycline hydrochloride (50-100 μM) in appropriate buffer containing Ca²⁺, incubated for 30-60 minutes, washed, and examined under a fluorescence microscope (excitation ~400 nm, emission ~520 nm).

Methodology for Pharmacokinetic Study in Ducks: The pharmacokinetics of chlortetracycline hydrochloride soluble powder in ducks is conducted as follows. Male or female ducks (approximately 90 days old) are randomly assigned to treatment groups (n=10 per group). Chlortetracycline hydrochloride is administered as a single oral gavage dose of 15 mg/kg body weight (calculated as chlortetracycline hydrochloride). Blood samples (approximately 1-2 mL) are collected from the wing vein at predetermined time points: pre-dose (0), and at 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 24 hours post-administration. Plasma is separated by centrifugation at 3000 rpm for 10 minutes and stored at -20°C until analysis. Chlortetracycline hydrochloride concentrations in plasma are quantified using high-performance liquid chromatography with UV detection or LC-MS/MS. Pharmacokinetic parameters including Tmax (time to maximum concentration), Cmax (maximum plasma concentration), AUC₀₋ₜ (area under the concentration-time curve from zero to last measurable time point), t₁/₂ (elimination half-life), and MRT (mean residence time) are calculated using non-compartmental or compartmental analysis. For infection model studies, chickens naturally infected with bacterial pathogens (e.g., E. coli, Salmonella) are treated with chlortetracycline hydrochloride injection at 20 mg/kg, and therapeutic effects are assessed by clinical observation and pathological tissue examination.

The pharmacokinetic profile of chlortetracycline hydrochloride has been characterized in several animal species. In ducks, following oral administration of chlortetracycline hydrochloride soluble powder at 15 mg/kg body weight, the time to maximum concentration (Tmax) was approximately 1.5 hours post-administration, with enhanced absorption and effective blood concentration maintenance observed when using ultra-fine powder formulations (800 mesh) compared to conventional particle sizes. The elimination half-life (t₁/₂) in ducks was significantly longer for the ultra-fine powder formulation compared to standard formulations. Comparative pharmacokinetic studies across tetracyclines reveal that chlortetracycline achieves a maximum plasma concentration (Cmax) of 1,560.8 ± 450.04 ng/mL and an area under the curve (AUC₀₋ₜ) of 12,780.81 ± 2,923.61 μg·hour/mL, with an elimination half-life (t₁/₂β) of 30.59 ± 18.01 hours and absolute bioavailability (F) of 30.54 ± 6.99%. The compound is significantly bound to plasma proteins. Incomplete gastrointestinal absorption is observed in many species, and the drug distributes widely into tissues, with particular affinity for bone and teeth due to chelation with calcium. Excretion occurs primarily via urine and bile. The USP specifies that chlortetracycline hydrochloride tablets must contain not less than 90.0% and not more than 120.0% of the labeled amount, and must disintegrate within 1 hour in simulated gastric fluid.

The toxicological profile of chlortetracycline hydrochloride indicates a low degree of acute toxicity in mammals. In humans, no toxic effects were reported from treatment of premature infants, children, adults, or elderly with chlortetracycline. Effects after short- and long-term exposure included increased body-weight gain and prophylactic activity against infections. A 52-week study in rats fed a diet containing 1% chlortetracycline hydrochloride (equivalent to 1000 mg/kg body weight per day) showed no significant difference in body weight and no evidence of toxicity. According to GHS classification, chlortetracycline hydrochloride is classified as: Skin Irritation Category 2 (H315: Causes skin irritation), Eye Irritation Category 2 (H319: Causes serious eye irritation), Specific Target Organ Toxicity - Single Exposure Category 3 (H335: May cause respiratory irritation), and Aquatic Chronic Category 2 (H411: Toxic to aquatic life with long-lasting effects). The compound has an EC50 of 3.1 mg/L in the green alga Selenastrum capricornutum and is not considered bioaccumulative. Bound residues in bone are microbiologically inactive but can be released under acidic conditions. The drug is contraindicated in patients with known hypersensitivity to chlortetracycline or other tetracycline-class antibiotics.

[1]. Role of excreted chlortetracycline in modifying the decomposition process in feedlot waste. Bull Environ Contam Toxicol. 1971 Mar-Apr;6(2):129-32.

[2]. Mouse gamete interactions during fertilization in vitro. Chlortetracycline as a fluorescent probe for the mouse sperm acrosome reaction. J Cell Biol. 1979 Dec;83(3):544-55.

Chlortetracycline hydrochloride is a tetracycline drug with broad-spectrum antibacterial and antiprotozoal activity. It is a bacteriostatic agent that inhibits bacterial protein synthesis by binding to the 30S ribosomal subunit, thereby preventing the addition of amino acids to the growing peptide chain. This tetracycline is effective against a variety of Gram-positive and Gram-negative bacteria, spirochetes, rickettsiae, certain protozoa, and mycoplasma and chlamydia.
A tetracycline drug with a chlorine substitution at the 7-position.
See also: Chlortetracycline hydrochloride (note moved to).

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In this study, chlortetracycline is not used for its antibiotic properties but as a vital fluorescent probe to monitor the status of the sperm acrosome. Its utility is based on its ability to chelate Ca²⁺ and form a fluorescent complex that partitions into the lipid environment of membranes. The loss of CTC fluorescence from the sperm head correlates with the loss of the plasma membrane and outer acrosomal membrane during the acrosome reaction. This assay was crucial for demonstrating that only acrosome-intact mouse sperm bind to the zona pellucida, defining the sequence of events during fertilization: sperm binding via its plasma membrane, followed by the acrosome reaction at the zona surface. The CTC assay was also used to develop a method for inducing high rates of acrosome reaction in mouse sperm in vitro using hyperosmolar medium and the ionophore A23187. [2]

C22H24CL2N2O8

515.3406

514.09

C, 68.11; H, 5.42; N, 10.18; O, 16.28

64-72-2

Chlortetracycline;57-62-5;Chlortetracycline-d6 hydrochloride; 27823-62-7 (bisulfate); 64-72-2 (HCl)

54682468

Light yellow to yellow solid powder

694.1ºC at 760 mmHg

210-215 °C (dec.)(lit.)

1.941

7

9

2

34

1010

5

ClC1C([H])=C([H])C(=C2C(=C3C([C@@]4(C(=C(C(N([H])[H])=O)C([C@]([H])([C@]4([H])C([H])([H])[C@]3([H])[C@@](C([H])([H])[H])(C2=1)O[H])N(C([H])([H])[H])C([H])([H])[H])=O)O[H])O[H])=O)O[H])O[H].Cl[H]

CBHYYLPALVVVEY-MRFRVZCGSA-N

InChI=1S/C22H23ClN2O8.ClH/c1-21(32)7-6-8-15(25(2)3)17(28)13(20(24)31)19(30)22(8,33)18(29)11(7)16(27)12-10(26)5-4-9(23)14(12)21/h4-5,7-8,15,26,28-29,32-33H,6H2,1-3H3,(H2,24,31)1H/t7-,8-,15-,21-,22-/m0./s1

(4S,4aS,5aS,6S,12aS)-7-chloro-4-(dimethylamino)-3,6,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide hydrochloride

Chlortetracycline HCl; Chlortetracycline; Aureomycin; Chlortetracycline hydrochloride; Chlorotetracycline hydrochloride; 64-72-2; Isphamycin; Aureociclina; U-6780; U 6780; U6780; Clorocipan; Isphamycin

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : ~10 mg/mL ( ~19.4 mM )
Water : 2~6.66 mg/mL(~12.92 mM)

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.)