Interspecies variability in protein binding of antibiotics basis for translational PK/PD studies-a case study using cefazolin.

For antimicrobial agents in particular, plasma protein binding (PPB) plays a pivotal role in deciphering key properties of drug candidates. Animal models are generally used in the preclinical development of new drugs to predict their effects in humans using translational pharmacokinetics/pharmacodynamics (PK/PD). Thus, we compared the protein binding (PB) of cefazolin as well as bacterial growth under various conditions in vitro. The PB extent of cefazolin was studied in human, bovine, and rat plasmas at different antibiotic concentrations in buffer and media containing 20-70% plasma or pure plasma using ultrafiltration (UF) and equilibrium dialysis (ED). Moreover, bacterial growth and time-kill assays were performed in Mueller Hinton Broth (MHB) containing various plasma percentages. The pattern for cefazolin binding to plasma proteins was found to be similar for both UF and ED. There was a significant decrease in cefazolin binding to bovine plasma compared to human plasma, whereas the pattern in rat plasma was more consistent with that in human plasma. Our growth curve analysis revealed considerable growth inhibition of Escherichia coli at 70% bovine or rat plasma compared with 70% human plasma or pure MHB. As expected, our experiments with cefazolin at low concentrations showed that E. coli grew slightly better in 20% human and rat plasma compared to MHB, most probably due to cefazolin binding to proteins in the plasma. Based on the example of cefazolin, our study highlights the interspecies differences of PB with potential impact on PK/PD. These findings should be considered before preclinical PK/PD data can be extrapolated to human patients.

Antimicrobial Drug Penetration Is Enhanced by Lung Tissue Inflammation and Injury.

Rationale: Pneumonia is a frequent and feared complication in intubated critically ill patients. Tissue concentrations of antimicrobial drugs need to be sufficiently high to treat the infection and also prevent development of bacterial resistance. It is uncertain whether pulmonary inflammation and injury affect antimicrobial drug penetration into lung tissue.Objectives: To determine and compare tissue and BAL fluid concentrations of ceftaroline fosamil and linezolid in a model of unilateral acute lung injury in pigs and to evaluate whether dose adjustment is necessary to reach sufficient antimicrobial concentrations in injured lung tissue.Methods: After induction of unilateral acute lung injury, ceftaroline fosamil and linezolid were administered intravenously. Drug concentrations were measured in lung tissue through microdialysis and in blood and BAL fluid samples during the following 8 hours. The primary endpoint was the tissue concentration area under the concentration curve in the first 8 hours (AUC0-8 h) of the two antimicrobial drugs.Measurements and Main Results: In 10 pigs, antimicrobial drug concentrations were higher in inflamed and injured lung tissue compared with those in uninflamed and uninjured lung tissue (median ceftaroline fosamil AUC0-8 h [and interquartile range] = 26.7 mg ⋅ h ⋅ L-1 [19.7-39.0] vs. 16.0 mg ⋅ h ⋅ L-1 [13.6-19.9], P = 0.02; median linezolid AUC0-8 h 76.0 mg ⋅ h ⋅ L-1 [68.1-96.0] vs. 54.6 mg ⋅ h ⋅ L-1 [42.7-60.9], P = 0.01), resulting in a longer time above the minimal inhibitory concentration and in higher peak concentrations and dialysate/plasma ratios. Penetration into BAL fluid was excellent for both antimicrobials, but without left-to-right differences (ceftaroline fosamil, P = 0.78; linezolid, P = 1.00).Conclusions: Tissue penetration of two commonly used antimicrobial drugs for pneumonia is enhanced by early lung tissue inflammation and injury, resulting in longer times above the minimal inhibitory concentration. Thus, lung tissue inflammation ameliorates antimicrobial drug penetration during the acute phase.

Identification of a Synthetic Polyhydroxyphenolic Resveratrol Analogue, 3,3',4,4',5,5'-Hexahydroxy-trans-Stilbene with Anti-SARS-CoV-2 Activity.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus has been causing the COVID-19 pandemic since December 2019, with over 600 million infected persons worldwide and over six million deaths. We investigated the anti-viral effects of polyphenolic green tea ingredients and the synthetic resveratrol analogue 3,3',4,4',5,5'-hexahydroxy-trans-stilbene (HHS), a compound with antioxidant, antitumor and anti-HIV properties. In the TCID50 assay, four out of nine green tea constituents showed minor to modest cell protective effects, whereas HHS demonstrated the highest reduction (1103-fold) of the TCID50, indicating pronounced inhibition of virus replication. HHS was also a highly effective inhibitor of SARS-CoV-2 proliferation in VeroE6 cells with an IC50 value of 31.1 µM. HSS also inhibited the binding of the receptor-binding domain (RBD) of the spike protein to the human angiotensin-converting enzyme 2 (ACE2) receptor (RBD-ACE2) binding with 29% at 100 µM and with 9.2% at 50 µM indicating that the SARS-CoV-2 inhibitory effect might at least in part be attributed to the inhibition of virus binding to ACE2. Based on the chemical similarity to other polyphenols, the oral bioavailability of HHS is likely also very low, resulting in blood levels far below the inhibitory concentration of EGCG against SARS-CoV-2 observed in vitro. However, administration of HHS topically as a nose or throat spray would increase concentrations several-fold above the minimal inhibitory concentration (MIC) in the mucosa and might reduce virus load when administered soon after infection. Due to these promising tissue culture results, further preclinical and clinical studies are warranted to develop HHS as an additional treatment option for SARS-CoV-2 infection to complement vaccines, which is and will be the main pillar to combat the COVID-19 pandemic.

Involvement of UDP-Glucuronosyltransferases and Sulfotransferases in the Excretion and Tissue Distribution of Resveratrol in Mice.

Resveratrol is a naturally occurring polyphenolic compound with various pharmacological activities. It is unknown whether the expression of metabolizing enzymes correlates with resveratrol levels in organs and tissues. Therefore, we investigated the metabolism and tissue distribution of resveratrol in mice and assessed its association with the expression of UDP-glucuronosyltransferase (Ugt) and sulfotransferase (Sult) genes. Plasma, urine, feces, and various organs were analyzed using high-performance liquid chromatography at up to 8 h after intragastric resveratrol administration. The metabolism of resveratrol was pronounced, leading to the formation of resveratrol glucuronides and sulfates. Concentrations of resveratrol and its metabolites were high in the gastrointestinal organs, urine, and feces, but low in the liver and kidneys. In lung, heart, thymus, and brain tissues, parent resveratrol levels exceeded the sulfate and glucuronide concentrations. The formation of resveratrol conjugates correlated with the expression of certain Ugt and Sult genes. Reverse transcription quantitative PCR (RT-qPCR) analysis revealed high mRNA expression of Ugt1a1 and Ugt1a6a in the liver, duodenum, jejunum, ileum, and colon, leading to high concentrations of resveratrol-3-O-glucuronide in these organs. Strong correlations of resveratrol-3-O-sulfate and resveratrol-3-O-4'-O-disulfate formation with Sult1a1 mRNA expression were also observed, particularly in the liver and colon. In summary, our data revealed organ-specific expression of Sults and Ugts in mice that strongly affects resveratrol concentrations; this may also be predictive in humans following oral uptake of dietary resveratrol.

Interplay of drug metabolizing enzymes with cellular transporters.

Many endogenous and xenobiotic substances and their metabolites are substrates for drug metabolizing enzymes and cellular transporters. These proteins may not only contribute to bioavailability of molecules but also to uptake into organs and, consequently, to overall elimination. The coordinated action of uptake transporters, metabolizing enzymes, and efflux pumps, therefore, is a precondition for detoxification and elimination of drugs. As the understanding of the underlying mechanisms is important to predict alterations in drug disposal, adverse drug reactions and, finally, drug-drug interactions, this review illustrates the interplay between selected uptake/efflux transporters and phase I/II metabolizing enzymes.

Resveratrol and its major sulfated conjugates are substrates of organic anion transporting polypeptides (OATPs): impact on growth of ZR-75-1 breast cancer cells.

SCOPE: Resveratrol is a naturally occurring polyphenolic compound with various pharmacological activities. These effects are observed despite its low bioavailability, which is particularly caused by extensive phase II metabolism. It is unknown whether resveratrol and its metabolites can accumulate to bioactive levels in organs and tissues through protein-mediated transport mechanisms. Because organic anion transporting polypeptides (OATPs) mediate the uptake of many clinically important drugs, we investigated their role in the cellular transport of resveratrol and its major glucuronides and sulfates. METHODS AND RESULTS: Uptake experiments were performed with resveratrol and its glucuronides and sulfates in OATP-expressing Chinese hamster ovary (CHO) and breast cancer (ZR-75-1) cells. The uptake rates for resveratrol in OATP1B1-, OATP1B3-, and OATP2B1-transfected Chinese hamster ovary cells were four- to sixfold higher compared to wild-type cells. Resveratrol-3-O-4'-O-disulfate was transported by OATP1B1 and OATP1B3, while resveratrol-3-O-sulfate was exclusively transported by OATP1B3. However, resveratrol-4'-O-sulfate, resveratrol-3-O-glucuronide, and resveratrol-4'-O-glucuronide did not show any affinity for these OATPs. OATP-dependent uptake of resveratrol was also confirmed in ZR-75-1 cells. CONCLUSION: Our data revealed that OATPs act as cellular uptake transporters for resveratrol and its major sulfates, which must be considered in humans following oral uptake of dietary resveratrol.

Cytochrome P450 3A-mediated metabolism of the topoisomerase I inhibitor 9-aminocamptothecin: impact on cancer therapy.

The metabolism of 9-aminocamptothecin (9-AC) was investigated in human and rat liver microsomes. In both species 9-AC was almost exclusively biotransformed to dihydroxy-9-AC (M1) and monohydroxy-9-AC (M2). The enzymatic efficiencies of the formation of M1 and M2 (V(max)/K(m)) were 1.7- and 2.7­fold higher in rat than in human liver microsomes indicating species-related differences in 9-AC hydroxylation. Incubation in the presence of human recombinant cytochrome P450 (CYP) enzymes demonstrated that the formation of M1 and M2 is mainly catalyzed by CYP3A4 and only to a minor extent by extrahepatic CYP1A1. The predominant role of CYP3A4 was further supported by a dramatic inhibition of metabolite formation in the presence of the CYP3A4 substrates troleandomycin and ketoconazole. Experiments conducted in isolated perfused rat livers further demonstrated that biliary excretion of 9-AC, M1 and M2 during 60 min of perfusion was pronounced and accounted for 17.7±2.59, 0.05±0.01 and 2.75±0.14% of total 9-AC applied to the liver, respectively. In summary, this study established that CYP3A-dependent hydroxylation is the main metabolic pathway for 9-AC in rat and human liver, which have to be taken into consideration during cancer therapy of patients.

Pharmacokinetics of ganciclovir during continuous venovenous hemodiafiltration in critically ill patients.

Ganciclovir is an antiviral agent that is frequently used in critically ill patients with cytomegalovirus (CMV) infections. Continuous venovenous hemodiafiltration (CVVHDF) is a common extracorporeal renal replacement therapy in intensive care unit patients. The aim of this study was to investigate the pharmacokinetics of ganciclovir in anuric patients undergoing CVVHDF. Population pharmacokinetic analysis was performed for nine critically ill patients with proven or suspected CMV infection who were undergoing CVVHDF. All patients received a single dose of ganciclovir at 5 mg/kg of body weight intravenously. Serum and ultradiafiltrate concentrations were assessed by high-performance liquid chromatography, and these data were used for pharmacokinetic analysis. Mean peak and trough prefilter ganciclovir concentrations were 11.8 ± 3.5 mg/liter and 2.4 ± 0.7 mg/liter, respectively. The pharmacokinetic parameters elimination half-life (24.2 ± 7.6 h), volume of distribution (81.2 ± 38.3 liters), sieving coefficient (0.76 ± 0.1), total clearance (2.7 ± 1.2 liters/h), and clearance of CVVHDF (1.5 ± 0.2 liters/h) were determined. Based on population pharmacokinetic simulations with respect to a target area under the curve (AUC) of 50 mg · h/liter and a trough level of 2 mg/liter, a ganciclovir dose of 2.5 mg/kg once daily seems to be adequate for anuric critically ill patients during CVVHDF.

Interplay between metabolism and transport of resveratrol.

Resveratrol exhibits a variety of biological and pharmacological activities despite its extensive metabolism to sulfates and glucuronides in the intestine and liver. The metabolism of resveratrol is cell specific and strongly correlates with enzyme expression levels. However, a high rate of biotransformation, in concert with the action of the efflux transporters MRP2, MRP3, and ABCG2, reduces intracellular resveratrol concentrations, and may thereby decrease its pharmacological activity. Interestingly, biotransformation is also dependent on disease status. For example, significantly greater sulfation of resveratrol occurs in human breast tumor tissue than in adjacent nonmalignant tissue. The observed differences, however, do not correlate with the expression of sulfotransferases responsible for catalyzing resveratrol sulfation, but rather with significantly higher steroid sulfatase mRNA levels. The in vitro activity of resveratrol sulfates may not necessarily reflect their in vivo function, given the fact that ubiquitously existing human sulfatases can convert the metabolites back to active resveratrol in humans.

Good penetration of moxifloxacin into human abscesses.

Abscesses are often treated with antibiotics in addition to incision or when incision is unfeasible, but accurate information about antibiotic abscess penetration in humans is missing. This study aimed at evaluating the penetration of moxifloxacin into human abscesses. After administration of a single dose of 400 mg moxifloxacin, drug concentrations were measured in 10 differently located abscesses at incision, and in plasma over 8 h. At incision performed 0.9-4.8 h after administration, moxifloxacin concentrations in abscesses ranged from ≤0.01 to 9.2 mg/l (1.9 ± 3.4 mg/l), indicating pronounced drug accumulation in some abscesses. The degree of abscess penetration could not be explained by covariates like the ratio of surface area to volume or pH of abscesses, or by moxifloxacin plasma concentrations. Concluding, moxifloxacin was detectable in most abscesses and may be a useful antibiotic for this indication. However, antibiotic abscess penetration was highly variable and unpredictable, suggesting surgical abscess incision whenever possible.

Abscess penetration of cefpirome: concentrations and simulated pharmacokinetic profiles in pus.

PURPOSE: Abscess patients frequently receive antibiotic therapy when incision cannot be performed or in addition to incision. However, antibiotic concentrations in human abscesses are widely unknown. METHODS: Pharmacokinetics of cefpirome in 12 human abscesses located in different body regions was studied. Cefpirome (2 g) was administered as an intravenous short infusion, and concentrations were measured in plasma over an 8-h period and in abscesses at incision. A pharmacokinetic two-stage model was applied. RESULTS: At abscess incision performed 158 ± 112 min after the start of the infusion, the cefpirome concentrations in the abscess fluid varied markedly, ranging from ≤0.1 (limit of quantification) to 47 (mean 8.4 ± 14.1 ) mg/L. Cefpirome was detectable in nine of 12 abscesses. Maximum concentrations were calculated to be 183 ± 106 mg/L in plasma and 12 ± 16 mg/L in the abscess. A cefpirome concentration of 2 mg/L, which is the minimum concentration inhibiting growth of 90% of the most relevant bacterial pathogens, was exceeded spontaneously in six of 12 abscesses after a single dose. Cefpirome concentrations in the abscess did not correlate with either the pH or the ratio of surface area to volume of the abscesses, nor with plasma pharmacokinetics. CONCLUSIONS: Cefpirome may be useful to treat abscess patients because it was detectable in most abscesses after a single dose. However, the penetration of cefpirome into abscesses is extremely variable and cannot be predicted by measuring other available covariates.

Hepatic glucuronidation of resveratrol: interspecies comparison of enzyme kinetic profiles in human, mouse, rat, and dog.

The enzyme kinetic profiles of the formation of resveratrol-3-O-glucuronide (R3G) and resveratrol-4'-O-glucuronide (R4'G) by liver microsomes from humans, dogs, and rodents were investigated. Glucuronidation by human and dog liver microsomes to R3G and R4'G occurred for about 65% of applied resveratrol, and was significantly reduced to 10% when substrate concentration was increased 10-fold. In contrast, rodent microsomes glucuronidated about 90% of applied resveratrol independently of substrate concentration. Furthermore, in mouse and rat liver microsomes, resveratrol was almost exclusively conjugated at position 3, whereas human and dog livers also glucuronidated resveratrol at position 4' (ratio R3G:R4'G = 5:1). Interspecies differences were also found when calculating the enzyme kinetic profiles of both conjugates. Formation of R4'G in human and dog microsomes followed Michaelis-Menten kinetics, while R3G showed substrate inhibition at higher resveratrol concentrations. In mouse and rat microsomes, however, both R3G and R4'G formation exhibited auto-activation kinetics. Formation of R3G and R4'G by recombinant UGT1A1 also showed substrate inhibition kinetics that led to decreased intrinsic clearance values, while UGT1A9-catalyzed glucuronidation demonstrated substrate inhibition kinetics at position 3 and Hill kinetics for the formation of R4'G. In conclusion, resveratrol glucuronidation exhibited species-dependent differences, with the dog as the animal model that most closely represents humans in terms of this process.

In vitro metabolism and disposition of honokiol in rat and human livers.

The biotransformation of honokiol, a major constituent of the bark of Magnolia officinalis, was investigated in rat and human livers. When isolated, rat livers were perfused with 10 µM honokiol and two metabolites, namely hydroxylated honokiol conjugated with glucuronic and sulfuric acid (M1) and honokiol monoglucuronide (M2), were quantified in bile and perfusate by high-performance liquid chromatography. The hepatic extraction ratio and clearance of honokiol was very high in rat liver (E: 0.99 ± 0.01 and 35.8 ± 0.04 mL/min, respectively) leading to very low bioavailability (F = 0.007 ± 0.001). M2 formation was also highly efficient in human liver microsomes [V(max) /K(m) = 78.1 ± 6.73 µL/(min mg)], which appeared to be catalyzed mainly by UDP-glucuronosyltransferases 1A1, A3, 1A8, and 1A10, indicating hepatic and extrahepatic glucuronidation. Monosulfation of honokiol to the minor metabolite honokiol monosulfate [V(max) /K(m) = 27.9 ± 4.33 µL/(min mg)] by human liver cytosol was less pronounced and is mediated by sulfotransferases 1A1* 1, 1A1* 2, 1A2, 1A3, 1B1, and 1E1. P450-mediated oxidation of honokiol by liver microsomes, however, was below detection limit. In summary, this study established that glucuronidation and sulfation are the main metabolic pathways for honokiol in rat and human liver, suggesting their major contribution to clearance in vivo.

Multiple-dose pharmacokinetics of anidulafungin during continuous venovenous haemofiltration.

BACKGROUND: Clinical studies support a role for anidulafungin as first-line treatment of invasive candidiasis in critically ill patients and postulate no need for dose adjustments in mild to severe renal failure. Although intensive care patients requiring renal replacement therapy are at particular risk of invasive fungal infection, no pharmacokinetic data on anidulafungin during continuous venovenous haemofiltration (CVVHF) are available. PATIENTS AND METHODS: Ten patients with CVVHF due to acute renal failure were included. Anidulafungin was infused on 3 consecutive days starting with a loading dose of 200 mg on day 1, followed by doses of 100 mg on each of days 2 and 3. During the 72 h study phase of CVVHF, blood and ultrafiltrate samples were collected at corresponding times. Anidulafungin concentrations were determined by HPLC. RESULTS: Peak plasma concentrations were reached 3 h after the start of infusion and were 8.5±3.6 mg/L at the pre-filter port. The mean arterial area under the curve (AUC0-24) of the study population was 109.9±49.82 mg·h/L, the total clearance was 1.08±0.41 L/h, the volume of distribution was 41.97±22.64 L and the elimination half-life was 28.78±10.40 h. Anidulafungin was not filtered, but CVVHF resulted in a substance loss of ∼20%, due to adherence to synthetic surfaces. CONCLUSIONS: Pharmacokinetics of anidulafungin during CVVHF resembled findings in healthy adults and adults with fungal infections. Therefore we recommend a loading dose of 200 mg intravenous anidulafungin on the first day and 100 mg on consecutive treatment days in patients during CVVHF.