Chemistry

The tetracyclines are polyketides with the core structure consisting of a hydronaphthacene nucleus. Different functional groups and moieties determine the solubility and biological activity of the tetracycline (Figure 14.2).

The tetracyclines are amphoteric drugs that are ionized at all pH values. The second‐generation tetracyclines are 3–5 times more lipophilic than the first‐generation drugs; OTC the least lipophilic and MIN the most lipophilic. The tetracyclines are poorly stable in aqueous solutions but relatively stable at acidic pH values. As the tetracyclines are sparingly water soluble, they are formulated as acid or basic salts that are administered orally or parenterally. In solution, they form a mixture of cations, anions, and zwitterions, for which the respective proportions are pH dependent. At pH values ranging between 4 and 7, the zwitterionic form predominates; its null net charge favors drug passage across cell membranes. The keto‐enol systems lead to different pH‐dependent tautomers and are chelation sites for multivalent cations (e.g., Ca²⁺, Mg²⁺, Zn²⁺, Cu²⁺, Fe²⁺, Fe³⁺, Al³⁺). The tetracyclines form insoluble complexes with calcium, magnesium, iron or aluminum. They act as ionophores, forming lipid‐soluble complexes with metal cations. Tetracyclines are photosensitive and can form colored complexes with multivalent metal cations.

Mechanism of Action

The tetracyclines are bacteriostatic, concentration‐time (or co‐dependent) protein synthesis inhibitors. Upon binding to the 16S RNA (rRNA) and S7 protein of the 30S bacterial ribosome, they allosterically inhibit the binding of aminoacylated transfer RNA (AA‐tRNA) to their docking site (A‐site) on the ribosome. This halts the process of peptide synthesis. To interact with their targets, the tetracyclines traverse one or more membrane systems depending on whether the susceptible organism is Gram‐positive or Gram‐negative. Tetracyclines cross the outer membrane of Gram‐negative bacteria through the OmpF and OmpC porin channels, as positively charged cation–tetracycline coordination complexes. The cationic metal ion–tetracycline complex accumulates in the periplasm, where the complex dissociates to liberate uncharged tetracycline, which diffuses through the lipid bilayer of the cytoplasmic membrane. Similarly, the electroneutral, lipophilic form diffuses across the cytoplasmic membrane of Gram‐positive bacteria. Uptake of tetracyclines across the cytoplasmic membrane is energy dependent. Within the cytoplasm, tetracycline molecules become chelated since the internal pH and divalent metal ion concentrations are higher than those outside the cell and it is a metal–tetracycline complex that binds to the ribosome. Recently, it has been discovered that TTC also directly disturbs bacterial cell membrane organization and localization of membrane proteins (Wenzel et al., 2021).

The tetracyclines exert antiparasitic activity by inhibiting protein synthesis in endosymbionts or organelles that possess a genome and prokaryote‐like ribosomal components. For instance, they alter the apicoplasts of Plasmodium falciparum, coccidia, and Babesia.

Tetracyclines also have a wide variety of other activities, often at subantimicrobial concentrations. Tetracyclines or CMT analogs can have antifungal, antitumoral, antiinflammatory, and immunomodulating activities. The use of tetracyclines with antimicrobial activity for nonantimicrobial purposes is not consistent antimicrobial stewardship.

Antimicrobial Activity

The tetracyclines exhibit broad‐spectrum antimicrobial activity.

• Good to moderate activity (MIC ≤4 μg/ml): they exhibit good to moderate activity against the following Gram‐positive aerobes: Bacillus spp., Corynebacterium spp., Erysipelothrix rhusiopathiae, Listeria monocytogenes and some streptococci, and against the following Gram‐negative bacteria: Actinobacillus spp., Bordetella spp., Borrelia spp., Brucella spp., Campylobacter fetus, Francisella tularensis, Haemophilus spp., Lawsonia intracellularis, Leptospira spp., Mannheimia spp., Pasteurella spp. (including P. multocida), and Yersinia spp. They are also active against Anaplasma spp., Chlamydia and Chlamydophila spp., Coxiella burnetii, Ehrlichia spp., Mycoplasma spp., Rickettsia and Neorickettsia, and some anaerobes including Actinomyces spp. and Fusobacterium spp.
  
• Variable susceptibility: because of acquired resistance, among Gram‐positive bacteria, many isolates of enterococci, staphylococci and streptococci are resistant. Among Gram‐negative bacteria many Enterobacterales including Enterobacter spp., E. coli, Klebsiella spp., Proteus spp. and Salmonella spp. are resistant. Many isolates of Mannheimia haemolytica are also resistant. Anaerobes such as Bacteroides spp. and Clostridium spp. show variable susceptibility.
  
• Resistant (MIC ≥16 mg/ml): most Mycobacterium spp., some Enterobacterales (e.g., Proteus mirabilis, Serratia spp.), and P. aeruginosa are resistant. Some Mycoplasma spp., Chlamydia suis, and bovine Anaplasma spp. may be resistant. Some pathogens intermediate or resistant to tetracycline may be susceptible to the second‐generation tetracyclines, and pathogens resistant or intermediate to DXC may be susceptible to MIN.

The tetracyclines are considered as concentration‐time antimicrobials because both %fT>MIC and fAUC/MIC can be predictive of efficacy in veterinary species, where f describes the free (protein‐unbound) drug concentration (Toutain et al., 2021). The Clinical and Laboratory Standards Institute (CLSI) considers only fAUC/MIC values of ≥25 to be predictive for the clinical efficacy of the tetracyclines. However, AUC/MIC values ranging from 12 to 819 are reported depending on the species treated, the bacterial pathogens targeted, and the tetracycline used. Values also vary depending on the bacteriological effect and the body site targeted. Some studies suggest T>MIC is a more predictive index than AUC/MIC (Li et al., 2021; Zhang et al., 2019). The fAUC/MIC is the preferred PK/PD index for long‐acting oxytetracycline injectables.

The tetracyclines tend to have postantibiotic effects for 2–3 hours for both Gram‐positive and Gram‐negative pathogens.

Resistance to Tetracyclines

The widespread use of tetracyclines in food animal production contributes to the worldwide spread of resistance in Gram‐positive and Gram‐negative bacteria and Mycoplasma spp. Resistance to tetracyclines is due to the acquisition of mobile genetic elements carrying tetracycline‐specific resistance genes, mutations within the ribosomal binding site, and/or chromosomal mutations leading to increased expression of intrinsic resistance mechanisms. Currently, over 1000 resistance genes are reported, including mosaic genes. Mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins (RPPs). They are formed when two or more RPP‐encoding genes recombine in a bacterium, resulting in a functional chimera.

The following mechanisms of resistance are described, with the first two mechanisms being the most common: (1) energy‐dependent efflux pumps, most of which are antiporters that exchange an extracellular H⁺ for a cytoplasmic tetracycline‐Mg²⁺ complex; (2) synthesis of ribosomal protection proteins that dissociate the tetracycline from the binding site near the ribosomal AA‐tRNA docking site; (3) synthesis of antibiotic‐inactivating enzymes; (4) ribosomal 16S RNA mutation at the primary binding site of tetracyclines; (5) stress‐induced downregulation of the porins through which the drug crosses the outer Gram‐negative wall; and (6) multidrug transporters with similar mechanisms to efflux pumps.

Because of the absence of the dimethylamine group on carbon 4, the CMTs do not select for antimicrobial resistance (Figure 14.2).

The oral administration of tetracyclines results in the fecal excretion of resistant bacteria as well as unchanged drug. The presence of tetracyclines in the environment selects for resistance in bacterial populations and alters microbial activities such as the decay process. While the tetracyclines are usually degraded with half‐lives ranging from less than 1 hour to 22 days, their persistence for 180 days in soil to six months in marine sediment has been reported. Composting contaminated manure may efficiently accelerate the degradation of the tetracyclines but the degradation products of some tetracyclines remain biologically active (Granados‐Chinchilla and Rodríguez, 2017).

Pharmacokinetic Properties

In addition to the usual PK factors (e.g., molecular size, lipid solubility, degree of protein binding), the pharmacokinetics of tetracyclines can be greatly affected by exposure to multivalent cations (e.g., Ca²⁺, Mg²⁺, Zn²⁺, Cu²⁺, Fe²⁺, Fe³⁺, Al³⁺) and interactions with P‐glycoprotein (P‐gp) transporters. There are also wide species variations in the PK parameters. The oral bioavailability of the first‐generation tetracyclines is relatively low. If calcium, magnesium, iron or aluminum ions are present within the gastrointestinal tract, oral bioavailability is markedly reduced. Oral bioavailability is improved in fasted animals, but this is not a clinically feasible adjustment for sick animals.

Large animal formulations are often administered by IM or SC injection. The type of salt, solvents or vehicles present in the formulation and the pH of the products influence the bioavailability.

The extent of protein binding of the tetracyclines is highly variable and dependent on the tetracycline, the species treated, and the type of proteins present in the biological media. Several tetracyclines display an atypical nonlinear binding to serum protein, where the free fraction of the tetracycline decreases at higher total plasma concentrations, in contrast with what occurs with drugs for which binding is saturable (Toutain et al., 2021). Free tetracycline molecules circulate mostly as Ca²⁺ and Mg²⁺ chelates, which may facilitate the bacterial penetration.

The ability to cross biological membranes varies between the tetracyclines. While a high degree of protein binding tends to limit tissue distribution, lipophilicity favors passage across cell membranes. Therefore, despite extensive binding to plasma proteins, the second‐generation tetracyclines have more extensive tissue penetration than the first‐generation tetracyclines.

The tetracyclines have generally good distribution in highly vascularized tissues without anatomical barriers (e.g., liver, gallbladder, salivary gland, seminal vesicles, kidney, heart, lungs, endometrium), reaching higher concentrations than in plasma. Diffusion into interstitial fluid such as pulmonary epithelial fluid, peritoneal and pleural fluids can be variable. The distribution in intermediately vascularized tissues, such as muscle and skin, is variable but can reach higher concentrations than in plasma. The concentrations reached in poorly perfused tissues and/or those with anatomical barriers (e.g., adipose tissue, joints, tendons, brain, CSF, prostate, ocular tissues) is generally low; however, moderate to high concentrations are achieved in milk. The passage of tetracyclines across the blood–brain barrier and placenta may be influenced by their affinity to P‐gp transporters. In infected tissues, the concentrations of tetracyclines can be higher than in healthy tissues, such as in infected lungs or in mastitic milk. The tetracyclines are among a limited number of osteotropic drugs and deposit in teeth or in sites of new bone formation.

Tetracyclines are eliminated by glomerular filtration, biliary secretion, and intestinal excretion. As efflux transporters, P‐gp has a role in the elimination of some tetracyclines. The elimination of the second‐generation tetracyclines is mainly by nonrenal routes, but the concentrations reached in urine and bile are higher than plasma concentrations for all tetracyclines.

Enterohepatic recirculation contributes to the unusually long elimination half‐life for drugs mainly eliminated by renal route, and can cause a second plasma peak that has been detected in sheep, horses, and donkeys following parenteral administration (Chapuis et al., 2021a, b).

Pharmacokinetic parameters of the tetracyclines can vary between species of a same genus, such as reported between adult horses, donkeys, and ponies. Young animals or neonates can have different PK parameters from adults, but the variation is species dependent. Comparing results of studies in ruminants, the stage of maturity of rumen function affects the PK of some tetracyclines even when administered parenterally.

Drug Interactions

The oral absorption of the tetracyclines is impaired by antacids containing multivalent cations, such as magnesium and aluminum, by iron‐containing preparations such as ferrous sulfate, and by bismuth subsalicylate. Manipulation of the P‐gp transporters by tetracyclines can be done deliberately to change drug PK. For example, DXC was effective at inhibiting the efflux of ivermectin and doramectin in alpaca cells, which may improve CNS concentrations for more effective treatment of parelaphostrongylosis (Agbedanu et al., 2015).

Evidence of synergism of tetracyclines with other antimicrobials is scant. Studies of synergistic effects are mainly performed in vitro, using models, or evaluating the correlation of mass administration of medicated feed and animal performance. DXC and MIN showed in vitro synergism when combined with clarithromycin against R. equi (Erol et al., 2022).

As tetracyclines are typically bacteriostatic, they are considered antagonistic to bactericidal drugs that target actively growing cells (Ocampo et al., 2014).

Formulations

Oral formulations of CTC, TTC, and OTC are available as premixes for medicated feeds for food animals and for milk replacer for calves. Water additives of TTC and OTC for food animals are also available. TTC is available as human and small animal tablets or capsules and as boluses and intramammary products for large animals. OTC is available in a number of formulations for parenteral use in large animals; the composition of the carrier solution determines the absorption and elimination from IM or SC injection sites (propylene glycol – “short‐acting”, polyethylene glycol and 2‐pyrrolidone – “long‐acting”).

Pharmacokinetics

The oral absorption of CTC, TTC, and OTC varies widely between species and is greatly affected by complexation with multivalent cations that precipitate as pH increases (e.g., calcium in dairy products) and by food particles (Decundo et al., 2019). In pigs, feed did not affect the bioavailability of OTC (3% in both fasted and fed pigs); for TTC there was a significantly higher bioavailability in fasted (18%) than in fed (5%) pigs while for CTC the bioavailability was not significantly different between fasted (11%) and fed pigs (6%) (Nielsen and Gyrd‐Hansen, 1996). Therefore, therapeutic concentrations in plasma or tissues are not achieved after oral administration of any of the three tetracyclines to fed or fasted pigs and any benefit of oral administration is due to impact on gastrointestinal flora. Poor oral bioavailability also occurs in poultry and ruminants fed tetracyclines. Exposing the microbiota to high concentrations of the tetracyclines enhances development of drug‐resistant strains and the lack of absorption also results in tetracycline dissemination in the environment (Ricker et al., 2020).

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