The Circadian PER2 Enhancer Nobiletin Reverses the Deleterious Effects of Midazolam in Myocardial Ischemia and Reperfusion Injury.

BACKGROUND: Recently, we identified the circadian rhythm protein Period 2 (PER2) in robust cardioprotection from myocardial ischemia (MI). Based on findings that perioperative MI is the most common major cardiovascular complication and that anesthetics can alter the expression of PER2, we hypothesized that an anesthesia mediated downregulation of PER2 could be detrimental if myocardial ischemia and reperfusion (IR) would occur. METHODS AND RESULTS: We exposed mice to pentobarbital, fentanyl, ketamine, propofol, midazolam or isoflurane and determined cardiac Per2 mRNA levels. Unexpectedly, only midazolam treatment resulted in an immediate and significant downregulation of Per2 transcript levels. Subsequent studies in mice pretreated with midazolam using an in-situ mouse model for myocardial (IR)-injury revealed a significant and dramatic increase in infarct sizes or Troponin-I serum levels in the midazolam treated group when compared to controls. Using the recently identified flavonoid, nobiletin, as a PER2 enhancer completely abolished the deleterious effects of midazolam during myocardial IR-injury. Moreover, nobiletin treatment alone significantly reduced infarct sizes or Troponin I levels in wildtype but not in Per2-/- mice. Pharmacological studies on nobiletin like flavonoids revealed that only nobiletin and tangeritin, both found to enhance PER2, were cardioprotective in our murine model for myocardial IR-injury. CONCLUSION: We identified midazolam mediated downregulation of cardiac PER2 as an underlying mechanism for a deleterious effect of midazolam pretreatment in myocardial IR-injury. These findings highlight PER2 as a cardioprotective mechanism and suggest the PER2 enhancers nobiletin or tangeritin as a preventative therapy for myocardial IR-injury in the perioperative setting where midazolam pretreatment occurs frequently.

Efficacy and safety of flumazenil injection for the reversal of midazolam sedation after elective outpatient endoscopy.

OBJECTIVE: Midazolam sedation during elective endoscopy is widely performed and flumazenil is frequently administered after endoscopy to reverse sedation in clinical practice. This study aimed to investigate the safety and efficacy of flumazenil injections after elective endoscopy under midazolam sedation. METHODS: Participants who underwent an upper endoscopy under midazolam sedation were randomly divided into two groups. In group I, flumazenil was administered i.v. 10 min after the patient's transfer to the recovery room, and no antidote was injected in group II. The time of stay in the recovery room and adverse events were reviewed through the nursing records. We asked the patients about their pain and degree of satisfaction according to a visual analogue scale (VAS), their memory of the procedure, mental status and the presence of uncomfortable symptoms on the day of the procedure and the day afterwards. RESULTS: The length of stay in recovery was significantly shorter in group I than in group II. No significant differences were found in the number of patients with pain (VAS ≥1), adverse events and discomfort between the two groups. Additionally, there were no differences in the patients' memory of the procedure, satisfaction with sedation, willingness to repeat the endoscopy and mental status. CONCLUSIONS: The time in the recovery room after flumazenil administration was significantly shortened, and the use of the drug did not increase the risk of adverse events or discomfort. The use of flumazenil for reversing midazolam sedation seems to be safe and effective.

A Review of the use of Flumazenil for the Reversal of Midazolam Conscious Sedation in Dentistry.

The practice of midazolam conscious sedation is well established in dentistry. The drug flumazenil is a specific benzodiazepine antagonist and is an essential requirement in settings where midazolam is used. A literature review has been carried out, examining the available information regarding flumazenil's safety, administration, potential complications and the regulatory documentation which governs its use. Flumazenil is a safe drug to use for the reversal of midazolam induced conscious sedation although the evidence surrounding its use is limited.

Injection anaesthesia with fentanyl-midazolam-medetomidine in adult female mice: importance of antagonization and perioperative care.

Injection anaesthesia is commonly used in laboratory mice; however, a disadvantage is that post-anaesthesia recovery phases are long. Here, we investigated the potential for shortening the recovery phase after injection anaesthesia with fentanyl-midazolam-medetomidine by antagonization with naloxone-flumazenil-atipamezole. In order to monitor side-effects, the depth of anaesthesia, heart rate (HR), core body temperature (BT) and concentration of blood gases, as well as reflex responses, were assessed during a 50 min anaesthesia. Mice were allowed to recover from the anaesthesia in their home cages either with or without antagonization, while HR, core BT and spontaneous home cage behaviours were recorded for 24 h. Mice lost righting reflex at 330 ± 47 s after intraperitoneal injection of fentanyl-midazolam-medetomidine. During anaesthesia, HR averaged 225 ± 23 beats/min, respiratory rate and core BT reached steady state at 131 ± 15 breaths/min and 34.3 ± 0.25℃, respectively. Positive pedal withdrawal reflex, movement triggered by tail pinch and by toe pinch, still occurred in 25%, 31.2% and 100% of animals, respectively. Arterial blood gas analysis revealed acidosis, hypoxia, hypercapnia and a marked increase in glucose concentration. After anaesthesia reversal by injection with naloxone-flumazenil-atipamezole, animals regained consciousness after 110 ± 18 s and swiftly returned to physiological baseline values, yet they displayed diminished levels of locomotion and disrupted circadian rhythm. Without antagonization, mice showed marked hypothermia (22 ± 1.9℃) and bradycardia (119 ± 69 beats/min) for several hours. Fentanyl-midazolam-medetomidine provided reliable anaesthesia in mice with reasonable intra-anaesthetic side-effects. Post-anaesthetic period and related adverse effects were both reduced substantially by antagonization with naloxone-flumazenil-atipamezole.

Effects of an anesthetic mixture of medetomidine, midazolam, and butorphanol in rats-strain difference and antagonism by atipamezole.

An anesthetic mixture of medetomidine (MED), midazolam (MID), and butorphanol (BUT) has been used in laboratory animals. We previously reported that this anesthetic mixture produced closely similar anesthetic effects in BALB/c and C57BL/6J strains. We also demonstrated the efficacy of atipamezole (ATI), an antagonist of MED that produced quick recovery from anesthesia in mice. Anesthetics have various anesthetic effects among animal strains. However, the differences in the effects of anesthetic mixtures in rats are unclear. In the present study, we first examined effects of the abovementioned anesthetic mixture using three different rat strains: Wistar (WST), Sprague-Dawley (SD), and Fischer 344 (F344). Second, we examined how different dosages and optimum injection timing of ATI affected recovery from anesthesia in rats. We used the anesthetic score to measure anesthetic duration and a pulse oximeter to monitor vital signs. We found no significant differences in anesthetic duration among the three different strains. However, recovery from anesthesia in the SD strain took significantly longer than in the other strains. The antagonistic effects of ATI (0.15 mg/kg and 0.75 mg/kg) were equivalent when administered at 30 min after anesthetic mixture administration. The antagonistic effects of ATI 0.75 mg/kg were stronger than those of ATI 0.15 mg/kg at 10 min after anesthetic mixture administration. This anesthetic mixture is a useful drug that can induce similar anesthetic effects in three different strains and has an antagonist, ATI, that makes rats quickly recover from anesthesia. These results may contribute to the welfare of laboratory animals.

Risk Factors and Outcomes of Reversal Agent Use in Moderate Sedation During Endoscopy and Colonoscopy.

BACKGROUND: Moderate sedation has been standard for noninvasive gastrointestinal procedures for decades yet there are limited data on reversal agent use and outcomes associated with need for reversal of sedation. AIM: To determine prevalence and clinical significance of reversal agent use during endoscopies and colonoscopies. METHODS: Individuals with adverse events requiring naloxone and/or flumazenil during endoscopy or colonoscopy from 2008 to 2013 were identified. A control group was obtained by random selection of patients matched by procedure type and date. Prevalence of reversal agent use and statistical comparison of patient demographics and risk factors against controls were determined. RESULTS: Prevalence of reversal agent use was 0.03% [95% confidence interval (CI), 0.02-0.04]. Events triggering reversal use were oxygen desaturation (64.4%), respiration changes (24.4%), hypotension (8.9%), and bradycardia (6.7%). Two patients required escalation of care and the majority of patients were stabilized and discharged home. Compared with the control group, the reversal group was older (61±1.8 vs. 55±1.6, P=0.01), mostly female (82% vs. 50%, P<0.01), and had lower body mass index (24±0.8 vs. 27±0.7, P=0.03) but received similar dosages of sedation. When adjusted for age, race, sex, and body mass index, the odds of reversal agent patients having a higher ASA score than controls was 4.7 (95% CI, 1.7-13.1), and the odds of having a higher Mallampati score than controls was 5.0 (95% CI, 2.1-11.7) with P<0.01. CONCLUSIONS: Prevalence of reversal agent use during moderate sedation is low and outcomes are generally good. Several clinically relevant risk factors for reversal agent use were found suggesting that certain groups may benefit from closer monitoring.

Anesthetic effects of a three-drugs mixture--comparison of administrative routes and antagonistic effects of atipamezole in mice.

The anesthetic mixture of medetomidine (MED), midazolam (MID) and butorphanol (BUT) produced anesthetic duration of around 40 minutes (min) in ICR mice. We reported that this anesthetic mixture produced almost the same anesthetic effects in both male and female BALB/c and C57BL/6J strains. Intraperitoneal (IP) administration of drugs has been widely used in mice. However, various injectable routes of the anesthetic mixture may cause different anesthetic effects. First, we examined effects of the anesthetic mixture by subcutaneous (SC) and intravenous (IV) injection compared to IP injection. After injection of the anesthetic mixture, administration of atipamezole (ATI) induced mice recovery from anesthesia. Secondly, we examined how different dosage and optimum injection timing of ATI affected mice recovery from anesthesia. We used an anesthetic score to measure anesthetic duration and a pulse oximeter to monitor vital signs under anesthesia. Usually, drugs from SC injection work more weakly than IP or IV injection. However, we found no significant differences of anesthetic duration among the three different injection routes. Antagonistic effects of ATI (0.3 mg/kg and 1.5 mg/kg) worked equally when administered at 30 min after injection of the anesthetic mixture. Antagonistic effects of ATI (1.5 mg/kg) were stronger than ATI (0.3 mg/kg) at 10 min after injection of the anesthetic mixture. The anesthetic mixture is a useful drug to induce nearly the same anesthetic effects by different injection routes and has an antagonist of ATI which helps mice quickly recover from anesthesia. These results may contribute to the welfare of laboratory animals.

A preliminary trial of the sedation induced by intranasal administration of midazolam alone or in combination with dexmedetomidine and reversal by atipamezole for a short-term immobilization in pigeons.

OBJECTIVE: To assess the sedative and immobilization effect of intranasal administration (INS) of midazolam (MID) without or with INS dexmedetomidine (DXM), and some physiological changes induced by the drugs. The ability of INS atipamezole to reverse the DXM component was also assessed. STUDY DESIGN: Prospective 'blinded' experimental study. ANIMALS: In total, 15 pigeons. METHODS: Pigeons were sedated by INS MID alone at a dose of 5 mg kg(-1) (group MID, n = 6) or in combination with INS DXM at a dose 80 µg kg(-1) (group MID-DXM, n = 6). Measurements were made of heart rate (HR), respiratory rate (fR ) and cloacal temperature (CT). The degree of sedation was assessed at 15 minutes prior to, immediately after, and at intervals until 100 minutes after drug administrations. Following MID-DXM, INS atipamezole (250 µg kg(-1) ) was administered and the same indices measured 5 and 10 minutes later. RESULTS: MID had no effect on HR and fR , and although CT decreased, it remained within physiological range. MID-DXM caused significant falls in HR, fR and CT that persisted until the end of sedation. Atipamezole antagonized sedation and cardiorespiratory side effects of MID-DXM within 10 minutes of application. In addition, for MID compared to MID-DXM, the lowest sedation scores [10 (7-14) and 10.5 (5-14) versus 2 (1-4) and 2 (1-5)] were achieved in the 10th and 20th minute versus the 20th and 30th minute of the sedation, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: MID alone, given INS had minimal side effects on vital functions but caused inadequate immobilization of pigeons for restraint in dorsal recumbency. MID-DXM caused an effective degree of immobilization from 20 to 30 minutes after administration, at which time birds tolerated postural changes without resistance. Atipamezole antagonized both side effects and sedation, but complete recovery had not occurred within 10 minutes after its application.

Significant and safe shortening of the recovery time after flumazenil-reversed midazolam sedation.

BACKGROUND: Endoscopy under midazolam sedation requires a 2-h recovery facility. AIM: To study the potential of shortening patients' stay without jeopardizing patients' safety by the use of the benzodiazepine-antagonist flumazenil in the everyday practice and to investigate the feasibility of a study comparing midazolam with recovery with midazolam-flumazenil and immediate discharge. METHODS: Consecutive ambulatory patients referred for endoscopy under midazolam sedation with ASA I or II, escorted by a person, were eligible. Flumazenil was given on arrival in the recovery room. Patients were discharged when adequate Aldrete scores and physical mobility were present. The next day, they were contacted by telephone. RESULTS: A total of 1,506 patients participated. They received 5 mg midazolam, while 887 patients also received 50 mcg fentanyl. The median dose of flumazenil was 0.2 mg. Oxygen desaturation (sO2 <92%) occurred in 15% during the procedure without an effect on recovery and discharge times. Patients left the department 65 min after the last midazolam administration. The majority (82.7%) were fully alert during their journey home. At home, 2.7% went to bed, 45.2% took a nap, and 40% undertook activities. Almost every patient (98.8%) liked the shortened recovery time. Three patients had an incident (fainting, fall, and near-fall) without consequences. Based on this low incidence, a non-inferiority comparison of midazolam-flumazenil with midazolam-recovery would require a total of 32,650 patients. CONCLUSIONS: Administration of flumazenil resulted in a safe shortening of the recovery period and offers the possibility for substantial savings in time, space, and nurse resources. A non-inferiority comparison will not be practicable.

Quantitative pharmacological analyses of the interaction between flumazenil and midazolam in monkeys discriminating midazolam: Determination of the functional half life of flumazenil.

The duration of action of a drug is commonly estimated using plasma concentration, which is not always practical to obtain or an accurate estimate of functional half life. For example, flumazenil is used clinically to reverse the effects of benzodiazepines like midazolam; however, its elimination can be altered by other drugs, including some benzodiazepines, thereby altering its half life. This study used Schild analyses to characterize antagonism of midazolam by flumazenil and determine the functional half life of flumazenil. Four monkeys discriminated 0.178mg/kg midazolam while responding under a fixed-ratio 10 schedule of stimulus-shock termination; flumazenil was given at various times before determination of a midazolam dose-effect curve. There was a time-related decrease in the magnitude of shift of the midazolam dose-effect curve as the interval between flumazenil and midazolam increased. The potency of flumazenil, estimated by apparent pA2 values (95% CI), was 7.30 (7.12, 7.49), 7.17 (7.03, 7.31), 6.91 (6.72, 7.10) and 6.80 (6.67, 6.92) at 15, 30, 60 and 120min after flumazenil administration, respectively. The functional half life of flumazenil, derived from potency estimates, was 57±13min. Thus, increasing the interval between flumazenil and midazolam causes orderly decreases in flumazenil potency; however, across a broad range of conditions, the qualitative nature of the interaction does not change, as indicated by slopes of Schild plots at all time points that are not different from unity. Differences in potency of flumazenil are therefore due to elimination of flumazenil and not due to pharmacodynamic changes over time.

Antagonistic effect of flumazenil after midazolam sedation on arterial-cardiac baroreflex.

BACKGROUND: Flumazenil is generally administered to antagonise the sedative effect of midazolam. However, although flumazenil completely antagonises the sedative effect of midazolam, a few effects remain unantagonised. Hence, it is unclear whether flumazenil restores the attenuation of the arterial-cardiac baroreflex (i.e. arterial-heart rate reflex) induced by midazolam. We investigated the antagonistic effect of flumazenil administered after midazolam on cardiac baroreflex, to reveal whether complete recovery from midazolam-induced sedation by flumazenil administration is accompanied by restoration of midazolam's attenuating effects on the cardiac baroreflex. METHOD: Twelve healthy male subjects received midazolam followed by flumazenil until complete recovery from midazolam sedation. Before and during midazolam sedation, and after flumazenil administration, cardiac baroreflex function was assessed by sequence analysis and transfer function analysis between spontaneous oscillations in systolic arterial pressure and R-R interval. RESULTS: During midazolam sedation, defined by an Observer's Assessment of Alertness/Sedation scale score of 3, BIS value decreased significantly. Simultaneously, the baroreflex indices of the two analyses decreased significantly compared with baseline, suggesting attenuated cardiac baroreflex function. With complete recovery from midazolam sedation by flumazenil, indicated by an Observer's Assessment of Alertness/Sedation scale score of 5, BIS values returned to the baseline level. Simultaneously, cardiac baroreflex indices also returned to baseline levels. CONCLUSION: The present results suggest that complete recovery from midazolam sedation by flumazenil is accompanied by restoration of the attenuated cardiac baroreflex function induced by midazolam.

Midazolam increases bite force during intravenous sedation.

PURPOSE: Although there have been many reports on the effects of midazolam on vital function and the recovery profile, little is known about muscle power during sedation. The purpose of this study was to investigate the effects of midazolam on muscle power during moderate sedation. MATERIALS AND METHODS: The subjects were 20 male volunteers classified as American Society of Anesthesiologists physical status I. Each subject underwent 2 experiments in a randomized crossover manner (midazolam and control groups). After baseline data were obtained, midazolam (0.05 mg/kg) was administered. Thirty minutes after midazolam administration, flumazenil (0.5 mg) was administered to antagonize the sedative effects of midazolam in the midazolam group. Heart rate, noninvasive blood pressure, arterial oxygen saturation, respiratory rate, and the bispectral index value were monitored. The Observer's Assessment of Alertness/Sedation scale and the correct-answer rate of the Stroop color word test were assessed. To evaluate muscle power, grip strength and bite force were measured. After baseline measurement, all variables were measured 2, 5, 10, 20, and 30 minutes after midazolam administration and 5, 10, and 20 minutes after flumazenil administration. For statistical comparisons, repeated measures analysis of variance, the Friedman χ(2) test, and the Student t test for paired samples were used. RESULTS: No significant changes were observed for any variable in the control group. In the midazolam group, the bispectral index value and the Observer's Assessment of Alertness/Sedation scale decreased during midazolam sedation. The correct-answer rate of the Stroop color word test decreased 5 and 10 minutes after midazolam administration. Grip strength decreased during midazolam sedation. Bite force increased immediately after midazolam administration and remained increased even after flumazenil administration. CONCLUSIONS: Although the detailed mechanisms are unknown, bite force increases despite the muscle-relaxant action of midazolam during sedation and persists even with flumazenil reversal.

Quantitative analyses of antagonism: combinations of midazolam and either flunitrazepam or pregnanolone in rhesus monkeys discriminating midazolam.

Adverse effects of benzodiazepines limit their clinical use; these effects might be reduced without altering therapeutic effects by administering other positive GABA(A) modulators (i.e., neuroactive steroids) with benzodiazepines. One concern with this strategy involves reversing these combined effects in case of overdose. The current study examined whether flumazenil can attenuate the combined effects of two benzodiazepines, midazolam and flunitrazepam, and the combined effects of midazolam and the neuroactive steroid pregnanolone, in four monkeys discriminating midazolam. Each positive modulator produced ≥80% midazolam-lever responding. Interactions between midazolam and either flunitrazepam or pregnanolone were additive. Flumazenil antagonized the benzodiazepines when they were administered alone or in combination. Schild analyses yielded slopes that did not deviate from unity, regardless of whether benzodiazepines were administered alone or together; the pA(2) value for flumazenil was 7.58. In contrast, flumazenil enhanced the effects of pregnanolone with 0.32 mg/kg flumazenil shifting the pregnanolone dose-effect curve 2-fold leftward. Flumazenil attenuated the combined effects of midazolam and pregnanolone, although antagonism was not dose-dependent. Thus, the interaction between two benzodiazepines was similar to that of a benzodiazepine and a neuroactive steroid; however, flumazenil more efficiently attenuated a combination of two benzodiazepines compared with a combination of a benzodiazepine and a neuroactive steroid. Although the magnitude of antagonism of a benzodiazepine combined with a neuroactive steroid was reduced, these results support continued exploration of the use of combinations of positive modulators to enhance therapeutic effects while reducing adverse effects.

Propofol increases the rate of albumin-unbound free midazolam in serum albumin solution.

Propofol and midazolam have a synergistic anesthetic action. One of the reasons for this is thought to be the inhibitory effect of propofol on midazolam metabolism. However, because both drugs bind strongly to serum protein, their interaction may not only involve the effects of propofol on midazolam metabolism, but may also involve propofol's effects on serum protein-binding. Against this background, we investigated the characteristics of midazolam binding to serum albumin, and evaluated the effects of both propofol and ketamine on this binding. Midazolam was added to a serum albumin solution with propofol or ketamine, and, after incubation for 1 h, albumin-free solution was separated from the sample and the midazolam concentration was measured using a high-performance liquid chromatography system. The albumin-unbound rate of midazolam was evaluated and compared with the rate in the control solution (only midazolam). Propofol significantly raised the rate of albumin-unbound free midazolam, while ketamine had no effect on the binding of midazolam to serum albumin. These findings suggest that the increase in albumin-unbound free midazolam brought about by propofol is involved in the synergistic effect of these two agents.

The use of flumazenil after midazolam-induced conscious sedation.

OBJECTIVE: To investigate the use of flumazenil after midazolam-induced conscious sedation. DESIGN AND SETTING: A prospective audit was carried out in the Department of Sedation and Special Care Dentistry at Guy's Hospital, King's College, London, 2009.Subjects Patients sedated with midazolam for dental treatment. METHOD: All clinical staff completed the data capture proforma when flumazenil was administered to a patient after sedation with midazolam. RESULTS: Four hundred and fifty-three patients were sedated with midazolam. Flumazenil was used in 32 cases. No cases required flumazenil for the emergency treatment of respiratory depression. CONCLUSIONS: The results of the audit confirmed the safe and appropriate use of midazolam for conscious sedation within the Department of Sedation and Special Care Dentistry at Guy's Hospital and demonstrated that flumazenil use was low and in accordance with current best practice. The audit has highlighted distinct indications for the post-operative use of flumazenil in specifically selected cases. Each case should be individually considered, justified and documented within the patient's clinical record.

Midazolam inhibits hippocampal long-term potentiation and learning through dual central and peripheral benzodiazepine receptor activation and neurosteroidogenesis.

Benzodiazepines (BDZs) enhance GABA(A) receptor inhibition by direct actions on central BDZ receptors (CBRs). Although some BDZs also bind mitochondrial receptors [translocator protein (18 kDa) (TSPO)] and promote the synthesis of GABA-enhancing neurosteroids, the role of neurosteroids in the clinical effects of BDZs is unknown. In rat hippocampal slices, we compared midazolam, an anesthetic BDZ, with clonazepam, an anticonvulsant/anxiolytic BDZ that activates CBRs selectively. Midazolam, but not clonazepam, increased neurosteroid levels in CA1 pyramidal neurons without changing TSPO immunostaining. Midazolam, but not clonazepam, also augmented a form of spike inhibition after stimulation adjacent to the pyramidal cell layer and inhibited induction of long-term potentiation. These effects were prevented by finasteride, an inhibitor of neurosteroid synthesis, or 17PA [17-phenyl-(3α,5α)-androst-16-en-3-ol], a blocker of neurosteroid effects on GABA(A) receptors. Moreover, the synaptic effects were mimicked by a combination of clonazepam with FGIN (2-[2-(4-fluorophenyl)-1H-indol-3-yl]-N,N-dihexylacetamide), a selective TSPO agonist, or a combination of clonazepam with exogenous allopregnanolone. Consistent with these in vitro results, finasteride abolished the effects of midazolam on contextual fear learning when administrated 1 d before midazolam injection. Thus, dual activation of CBRs and TSPO appears to result in unique actions of clinically important BDZs. Furthermore, endogenous neurosteroids are shown to be important regulators of pyramidal neuron function and synaptic plasticity.

Pharmacokinetics and pharmacodynamics of GS-9350: a novel pharmacokinetic enhancer without anti-HIV activity.

GS-9350 is a new chemical entity under development as a potent, mechanism-based inhibitor of human cytochrome P450 3A (CYP3A) isoforms. Its intended use is to increase the systemic exposure of coadministered agents that are metabolized by CYP3A enzymes. Unlike ritonavir, which is in current clinical use for this purpose, GS-9350 is devoid of anti-HIV activity. The pharmacokinetics of GS-9350 and its efficacy in increasing systemic exposure of the probe CYP3A substrate midazolam were examined in a study involving single- and multiple-dose escalations of GS-9350 from 50 to 400 mg. Single-dose escalation from 50 to 400 mg resulted in a 164-fold increase in GS-9350 exposure, whereas multiple-dose escalation in the dosage range of 50-300 mg resulted in a 47-fold increase in exposure. GS-9350 potently inhibited midazolam apparent clearance (95% reduction), similar in effect to ritonavir 100 mg. GS-9350 was generally well tolerated at all doses, and there was no evidence of dose-limiting toxicity. Establishing proof-of-concept, GS-9350 is currently under phase II development as a potential alternative to ritonavir for use with antiretroviral agents (including the HIV integrase inhibitor elvitegravir) that are often prescribed along with a "booster" drug.