Nivolumab plus ipilimumab in advanced melanoma.

BACKGROUND: In patients with melanoma, ipilimumab (an antibody against cytotoxic T-lymphocyte-associated antigen 4 [CTLA-4]) prolongs overall survival, and nivolumab (an antibody against the programmed death 1 [PD-1] receptor) produced durable tumor regression in a phase 1 trial. On the basis of their distinct immunologic mechanisms of action and supportive preclinical data, we conducted a phase 1 trial of nivolumab combined with ipilimumab in patients with advanced melanoma. METHODS: We administered intravenous doses of nivolumab and ipilimumab in patients every 3 weeks for 4 doses, followed by nivolumab alone every 3 weeks for 4 doses (concurrent regimen). The combined treatment was subsequently administered every 12 weeks for up to 8 doses. In a sequenced regimen, patients previously treated with ipilimumab received nivolumab every 2 weeks for up to 48 doses. RESULTS: A total of 53 patients received concurrent therapy with nivolumab and ipilimumab, and 33 received sequenced treatment. The objective-response rate (according to modified World Health Organization criteria) for all patients in the concurrent-regimen group was 40%. Evidence of clinical activity (conventional, unconfirmed, or immune-related response or stable disease for ≥24 weeks) was observed in 65% of patients. At the maximum doses that were associated with an acceptable level of adverse events (nivolumab at a dose of 1 mg per kilogram of body weight and ipilimumab at a dose of 3 mg per kilogram), 53% of patients had an objective response, all with tumor reduction of 80% or more. Grade 3 or 4 adverse events related to therapy occurred in 53% of patients in the concurrent-regimen group but were qualitatively similar to previous experience with monotherapy and were generally reversible. Among patients in the sequenced-regimen group, 18% had grade 3 or 4 adverse events related to therapy and the objective-response rate was 20%. CONCLUSIONS: Concurrent therapy with nivolumab and ipilimumab had a manageable safety profile and provided clinical activity that appears to be distinct from that in published data on monotherapy, with rapid and deep tumor regression in a substantial proportion of patients. (Funded by Bristol-Myers Squibb and Ono Pharmaceutical; ClinicalTrials.gov number, NCT01024231.).

Single-dose pharmacokinetics of boceprevir in subjects with impaired hepatic or renal function.

BACKGROUND AND OBJECTIVE: Boceprevir is a novel inhibitor of the hepatitis C virus NS3 protease and was recently approved for the treatment of patients with chronic hepatitis C infection. The objective of this study was to evaluate the impact of impaired hepatic or renal function on boceprevir pharmacokinetics and safety/tolerability. METHODS: We conducted two open-label, single-dose, parallel-group studies comparing the safety and pharmacokinetics of boceprevir in patients with varying degrees of hepatic impairment compared with healthy controls in one study and patients with end-stage renal disease (ESRD) on haemodialysis with healthy controls in the other. Patients with hepatic impairment (mild [n = 6], moderate [n = 6], severe [n = 6] and healthy controls [n = 6]) received a single dose of boceprevir (400 mg) on day 1, and whole blood was collected at selected timepoints up to 72 hours postdose to measure plasma drug concentrations. Patients with ESRD and healthy subjects received a single dose of boceprevir 800 mg orally on days 1 and 4, with samples for pharmacokinetic analyses collected at selected timepoints up to 48 hours postdose on both days. In addition, 4 hours after dosing on day 4, patients with ESRD underwent haemodialysis with arterial and venous blood samples collected up to 8 hours postdose. RESULTS: In the hepatic impairment study, there was a trend toward increased mean maximum (peak) plasma concentration (C(max)) and area under the plasma concentration-time curve (AUC) of boceprevir with increasing severity of liver impairment. Point estimates for the geometric mean ratio for C(max) ranged from 100% in patients with mild hepatic impairment to 161% in patients with severe hepatic impairment, with the geometric mean ratio for AUC ranging from 91% in patients with mild hepatic impairment to 149% for patients with severe hepatic impairment, relative to healthy subjects. The mean elimination half-life (t(1/2;)) and median time to C(max) (t(max)) values of boceprevir were similar in healthy subjects and patients with hepatic impairment. In the renal impairment study, mean boceprevir C(max) and AUC values were comparable in patients with ESRD and in healthy subjects, with point estimates for the geometric mean ratio of 81% and 90%, respectively, compared with healthy subjects. Mean t(1/2;), median t(max) and mean apparent oral total clearance (CL/F) values were similar in healthy subjects and patients with ESRD. Boceprevir exposure was also similar in patients with ESRD on day 1 (no dialysis) and day 4 (dialysis beginning 4 hours postdose), with point estimates for the geometric mean ratio of C(max) and AUC to the last measurable sampling time (AUC(last)) on day 1 compared with day 4 of 88% and 98%, respectively. Treatment-emergent adverse events were reported in one patient with severe hepatic impairment (mild vomiting) and two patients with ESRD (moderate ventricular extrasystoles, flatulence and catheter thrombosis). CONCLUSION: In the present studies, the pharmacokinetic properties of boceprevir were not altered to a clinically meaningful extent in patients with impaired liver or renal function. These data indicate that boceprevir dose adjustment is not warranted in patients with impaired hepatic function or ESRD, including those receiving dialysis. Boceprevir is not removed by haemodialysis.

Determination of flunixin in edible bovine tissues using liquid chromatography coupled with tandem mass spectrometry.

An accurate, reliable, and reproducible assay was developed and validated to determine flunixin in bovine liver, kidney, muscle, and fat. The overall recovery and percent coefficient of variation (%CV) of twenty-eight determinations in each tissue for flunixin free acid were 85.9% (5.9% CV) for liver, 94.6% (9.9% CV) for kidney, 87.4% (4.7% CV) for muscle, and 87.6% (4.4% CV) for fat. The theoretical limit of detection was 0.1 microg/kg (ppb, ng/g) for liver and kidney, and 0.2 ppb for muscle and fat. The theoretical limit of quantitation was 0.3, 0.2, 0.6, and 0.4 ppb for liver, kidney, muscle, and fat, respectively. The validated lower limit of quantitation was 1 ppb for edible tissues with the upper limit of 400 ppb for liver and kidney, 100 ppb for fat, and 40 ppb for muscle. Accuracy, precision, linearity, specificity, ruggedness, and storage stability were demonstrated. Briefly, the method involves an initial acid hydrolysis, followed by pH adjustment ( approximately 9.5) and partitioning with ethyl acetate. A portion of the ethyl acetate extract was purified by solid-phase extraction using a strong cation exchange cartridge. The eluate was then evaporated to dryness, reconstituted, and analyzed using LC/MS/MS. The validated method is sensitive and specific for flunixin in edible bovine tissue.

Determination and confirmation of 5-hydroxyflunixin in raw bovine milk using liquid chromatography tandem mass spectrometry.

A method was developed and validated to determine 5-hydroxyflunixin in raw bovine milk using liquid chromatography tandem mass spectrometry (LC/MS/MS). The mean recovery and percentage coefficient of variation (%CV) of 35 determinations for 5-hydroxyflunixin was 101% (5% CV). The theoretical limit of detection was 0.2 ppb with a validated lower limit of quantitation of 1 ppb and an upper limit of 150 ppb. Accuracy, precision, linearity, specificity, ruggedness, and storage stability were demonstrated. A LC/MS/MS confirmatory method using the extraction steps of the determinative method was developed and validated for 5-hydroxyflunixin in milk from cattle. Briefly, the determinative and confirmatory methods were based on an initial solvent (acetone/ethyl acetate) precipitation/extraction of acidified whole milk. The solvent precipitation/extraction effectively removed incurred ((14)C) residues from milk samples. The organic extract was then purified by solid phase extraction (SPE) using a strong cation exchange cartridge (sulfonic acid). The final SPE-purified sample was analyzed using LC/MS/MS. The methods are rapid, sensitive, and selective and provide for the determination and confirmation of 5-hydroxyflunixin at the 1 and 2 ppb levels, respectively.

Flunixin residues in milk after intravenous treatment of dairy cattle with (14)C-flunixin.

Flunixin meglumine is used in veterinary medicine as an alternative to narcotic analgesics and as an antiinflammatory agent. Eight Holstein dairy cows were dosed intravenously once daily on three consecutive days with (14)C-flunixin meglumine at approximately 2.2 mg of flunixin free acid/kg of body weight. Milk was collected twice daily to determine the decline of the total radioactive residues (TRR) in milk and to identify or characterize residue components. TRR in milk declined rapidly and averaged 66, 20, and 14 ppb, respectively, for the first, second, and third milkings after administration of the last dose. Milk was extracted, and the extracts were examined for radioactive residues. Mean extractability of milk TRR was always greater than 80%. Flunixin and 5-hydroxyflunixin were identified by coelution with analytical standards using reverse phase HPLC. These two residues were the main radioactive residues found in milk and together accounted for 64, 37, and 44% of the extractable residues, for the first, second, and third milkings, respectively, after administration of the last dose. The presence of 5-OH flunixin in milk was confirmed by HPLC/MS/MS.