CRISPR-Cas9: A New Addition to the Drug Metabolism and Disposition Tool Box.

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9), i.e., CRISPR-Cas9, has been extensively used as a gene-editing technology during recent years. Unlike earlier technologies for gene editing or gene knockdown, such as zinc finger nucleases and RNA interference, CRISPR-Cas9 is comparably easy to use, affordable, and versatile. Recently, CRISPR-Cas9 has been applied in studies of drug absorption, distribution, metabolism, and excretion (ADME) and for ADME model generation. To date, about 50 papers have been published describing in vitro or in vivo CRISPR-Cas9 gene editing of ADME and ADME-related genes. Twenty of these papers describe gene editing of clinically relevant genes, such as ATP-binding cassette drug transporters and cytochrome P450 drug-metabolizing enzymes. With CRISPR-Cas9, the ADME tool box has been substantially expanded. This new technology allows us to develop better and more predictive in vitro and in vivo ADME models and map previously underexplored ADME genes and gene families. In this mini-review, we give an overview of the CRISPR-Cas9 technology and summarize recent applications of CRISPR-Cas9 within the ADME field. We also speculate about future applications of CRISPR-Cas9 in ADME research.

Pharmacological indices and pulmonary distribution of rifampicin after repeated oral administration in healthy foals.

BACKGROUND: The treatment of equine lung infections by Rhodococcus equi with rifampicin is empirically based because pharmacokinetic/pharmacodynamic (PK/PD) indices and pivotal clinical outcome data are not available. OBJECTIVES: To evaluate the pharmacokinetics and pulmonary distribution of rifampicin into epithelial lining fluid (ELF) and bronchoalveolar lavage cells (BALC) to predict antimicrobial activity in the lung using PK/PD indices. STUDY DESIGN: Controlled, randomised, two-period, crossover, repeated-dose study with an initial arm to measure disposition after i.v. administration of rifampicin. METHODS: Pharmacokinetics and lung distribution were evaluated in six healthy foals treated with 10 mg/kg bwt rifampicin i.v. (initial arm) and with repeated oral doses of rifampicin at 10 mg/kg bwt and 20 mg/kg bwt once per day for 10 days (crossover arms). ELF and BALC were sampled by bronchoalveolar lavage 24 h after the last oral dosing. Rifampicin and 25-O-desacetyl rifampicin were quantified using liquid chromatography tandem-mass spectrometry. Enzyme induction by rifampicin was confirmed by evaluation of plasma 4ß-OH-cholesterol:cholesterol ratios. RESULTS: The distribution volume of rifampicin administered i.v. was ~0.85 L/kg. Terminal elimination half-life was ~11 h. Orally given rifampicin was slowly absorbed (Tmax , range: 2.5-8.0 h) and eliminated with apparent half-lives of ~6-8 h. Trough concentrations in ELF and BALC were 1.01 ± 0.20 µg/mL and 1.25 ± 0.29 µg/mL, respectively, after 10 mg/kg bwt rifampicin and 2.71 ± 1.25 µg/mL and 3.09 ± 1.63 µg/mL, respectively, after 20 mg/kg bwt rifampicin. The average ratios of area under the plasma concentration time curve during an administration interval of 24 h (AUC0-24 h ) to minimum inhibitory concentration (MIC) were 145 and 322 h, respectively, for less susceptible strains of R. equi (MIC90 : 0.5 µg/mL). MAIN LIMITATIONS: The clearance and bioavailability of rifampicin after repeated oral dosing were not evaluated. CONCLUSIONS: Treatment with rifampicin at 10 mg/kg bwt administered once per day is suitable to generate drug concentrations above the MIC90 in the ELF and BALC of foals. Future clinical studies with rifampicin in combination with macrolide antibiotics with low drug interaction potential are required to translate the PK/PD indices into protocols for the treatment of R. equi lung infections.

Systems pharmacology augments drug safety surveillance.

Small molecule drugs are the foundation of modern medical practice, yet their use is limited by the onset of unexpected and severe adverse events (AEs). Regulatory agencies rely on postmarketing surveillance to monitor safety once drugs are approved for clinical use. Despite advances in pharmacovigilance methods that address issues of confounding bias, clinical data of AEs are inherently noisy. Systems pharmacology-the integration of systems biology and chemical genomics-can illuminate drug mechanisms of action. We hypothesize that these data can improve drug safety surveillance by highlighting drugs with a mechanistic connection to the target phenotype (enriching true positives) and filtering those that do not (depleting false positives). We present an algorithm, the modular assembly of drug safety subnetworks (MADSS), to combine systems pharmacology and pharmacovigilance data and significantly improve drug safety monitoring for four clinically relevant adverse drug reactions.

Impact of efavirenz on intestinal metabolism and transport: insights from an interaction study with ezetimibe in healthy volunteers.

Hypercholesterolemia frequently occurs in patients treated with efavirenz who cannot be treated adequately with statins because of drug interactions. These patients may benefit from cholesterol-lowering therapy with ezetimibe. This study determined the influence of single-dose and multiple-dose efavirenz (400 mg/day for 9 days) on the pharmacokinetics and sterol-lowering of ezetimibe (10 mg) in 12 healthy subjects. In addition, the influence of efavirenz on genome-wide intestinal expression and in vitro function of ABCB1, ABCC2, UGT1A1, and OATP1B1 was studied. Efavirenz (multiple dose) had no influence on the pharmacokinetics and lipid-lowering functions of ezetimibe. Intestinal expression of enzymes and transporters (e.g., ABCB1, ABCC2, and UGT1A1) was not affected by chronic efavirenz. Efavirenz (single dose) slightly increased ezetimibe absorption and markedly decreased exposure to ezetimibe-glucuronide (single dose and multiple dose), which may be explained by inhibition of UGT1A1 and ABCB1 (in vitro data). Ezetimibe had no effect on the disposition of efavirenz. Consequently, ezetimibe may be a safe and efficient therapeutic option in patients with HIV infection.

Drug interactions between the immunosuppressant tacrolimus and the cholesterol absorption inhibitor ezetimibe in healthy volunteers.

Immunosuppressive therapy is frequently associated with hypercholesterolemia, calling for lipid-lowering treatment without adverse drug interactions. One option is treatment with the cholesterol absorption inhibitor ezetimibe. We have shown in vitro that ezetimibe and tacrolimus may interact in competition for intestinal UGT1A1 and ABCB1 at concentrations reached in gut lumen after oral administration. However, this clinical study in healthy volunteers showed that the expected pharmacokinetic interaction between ezetimibe and tacrolimus is not of clinical relevance.

Pharmacokinetic and pharmacodynamic interactions between the immunosuppressant sirolimus and the lipid-lowering drug ezetimibe in healthy volunteers.

Organ transplant recipients who have dyslipidemia related to immunosuppression may benefit from cholesterol-lowering therapy with ezetimibe, a substrate of ABCB1, ABCC2, and OATP1B1. Adverse pharmacokinetic interactions are hypothesized with sirolimus, which is a substrate of OATP1B1 and OATP1B3 and an inhibitor of ABCB1, OATP1B1, and OATP1B3 but not of ABCC2. However, competition between sirolimus and ezetimibe for ABCB1 and OATP1B1 is not of major clinical relevance, as confirmed in our randomized, controlled, single-dose study in healthy subjects.

Effect of anticholinergics (atropine, glycopyrrolate) and prokinetics (metoclopramide, cisapride) on gastric motility in beagles and labrador retrievers.

The effect of atropine, glycopyrrolate, metoclopramide and cisapride on the antral motility was investigated in eight dogs (four Beagles and four Labradors) using passive telemetry. Both anticholinergics induced a pronounced and lasting reduction of the intensity and frequency of the contractions. A definite dose-related inhibition of the antral motility was seen in Beagles, similar for both active substances. Low doses of atropine (0.02 mg/kg BW i.m.) and glycopyrrolate (0.005 mg/kg BW i.m.) completely inhibited the gastric motility for at least 30 min, whereas higher doses (0.04 or 0.01 mg/kg BW) caused a cessation of activity for more than 3 h. In Labradors, the effects of both active substances were not so dose related and the effect of glycopyrrolate lasted at least 6 h, whereas the effect of atropine gradually decreased after 3 h. A distinct breed difference regarding the effect of the two prokinetics on the antral motility was also observed. In Beagles, the prokinetics, at a low dose (metoclopramide 0.3 mg/kg BW, cisapride 0.2 mg/kg BW), resulted in a significant increase in the amplitude integral. Higher doses (metoclopramide 0.6 mg/kg BW, cisapride 0.5 mg/kg BW) also increased the integrals of the pressure profiles, but significantly less than with the lower doses. In Labradors, both medications, mainly at higher doses, resulted in an increase of the contraction amplitudes. The low dose had no (cisapride) or only a transient effect (metoclopramide). The frequency of the antral contractions was not at all influenced by cisapride, and only in Beagles metoclopramide resulted in a dose-related increase. It is not clear if the different results in Labradors and Beagles are because of breed or body weight.

[Mitotane treatment in a dog with a recurring adrenocortical carcinoma--a case report]. / Mitotane-Behandlung bei einem Hund mit einem rezidivierenden Nebennierenrindenkarzinom--ein Fallbericht.

In a 7 year old female poodle an adrenocortical tumor was diagnosed on basis of laboratory and ultrasonographic examinations. One year after adrenalectomy, a relapse was diagnosed, at that time the suspicion of metastases in the liver arose for the first time. By treatment with Mitotane in a dose aiming at completely destroying the adrenal cortex, a complete disappearance of the tumor as well as a dramatic reduction of the size of the metastases could be achieved. 12 months after the begin of the chemotherapy, the dog is in good general condition.

Why not the eighth nerve? Neurovascular compression--probable cause for pulsatile tinnitus.

Carotid arteriograms on three patients with unilateral pulsatile tinnitus demonstrated an ipsilateral atypical trigeminal artery extending from the cavernous portion of the internal carotid artery to form the posterior inferior cerebellar artery. Illustrations and a dissection of a human fetus with a similar finding show this artery crossing the cochlear nerve near its insertion in the pons. Evidence is presented suggesting that neurovascular compression of the eighth nerve is the source of pulsatile tinnitus in these patients.