Crossreactivity of isolated everolimus metabolites with the Innofluor Certican immunoassay for therapeutic drug monitoring of everolimus.

Everolimus is an immunosuppressant used as rejection prophylaxis in patients undergoing transplants. It requires blood concentration-guided dosing and is extensively metabolized. It was the goal to assess the crossreactivity of the major everolimus metabolites in the blood of patients undergoing kidney graft with the Innofluor Certican Assay (Seradyn, Inc., Indianapolis, IN), a clinical assay used to quantify the concentrations of everolimus in patients' blood samples. The three main hydroxy metabolites of everolimus (46-, 24-, and 25-hydroxy everolimus) and all other minor hydroxylated and demethylated metabolites were generated using pooled human liver microsomes and purified using semipreparative high-performance liquid chromatography with ultraviolet detection. Structures were confirmed using liquid chromatography-mass spectrometry/ion trap mass spectrometry and analysis of the fragmentation patterns. Blank blood samples were spiked with the isolated metabolites to determine the specific crossreactivity with the immunoassay. Crossreactivity testing with the immunoassay showed 1% or less for 46-hydroxy and 24-hydroxy everolimus and 6% or less crossreactivity for 25-hydroxy everolimus at therapeutically relevant concentrations. Crossreactivity testing of the minor metabolites showed crossreactivities of 16.3% for 45-hydroxy, 33.0% for 12-hydroxy, 18.3% for 11-hydroxy, 15.3% for 14-hydroxy, 38.7% for OH-piperidine I, 46.3% for OH-piperidine II, 43% for 39-O-desmethyl, 142% for 27-O-desmethyl, and 68% for 40-O-desethylhydroxy everolimus (sirolimus).

Identification of everolimus metabolite patterns in trough blood samples of kidney transplant patients.

Everolimus is used as an immunosuppressant after organ transplantation. It is extensively metabolized, mainly by cytochrome P4503A enzymes, resulting in several hydroxylated and demethylated metabolites. The structures of these metabolites after in vitro metabolism of everolimus by human liver microsomes have recently been identified. It was the goal to elucidate the everolimus metabolite patterns in 128 trough blood samples from kidney graft patients using high-performance liquid chromatography (LC)-ion trap mass spectrometry (MS) in combination with analysis of the fragmentation patterns of the metabolites isolated from patient blood and comparison with the metabolites generated in vitro. After identification, concentrations of the metabolites were estimated using LC-MS. Relative to the everolimus concentrations in trough blood samples, metabolite concentrations were [median (range), n = 128] 46-hydroxy 44.1% (0-784%), 24-hydroxy 7.7% (0-85.6%), and 25-hydroxy 14.4% (0-155.4%); 11-Hydroxy, 12-hydroxy, 14-hydroxy, 49-hydroxy, two hydroxy-piperidine everolimus metabolites, 16-O-desmethyl, 16,39-O-didesmethyl, 16,27-O-didesmethyl, and 27,39-O-didesmethyl everolimus were also detected. However, when detectable, concentrations were consistently between the lower limit of detection (0.1 microg/L) and the lower limit of quantification (0.25 microg/L) of our LC-MS assay. In most trough blood samples, the total metabolite concentrations were between 50% and 100% of the everolimus concentrations. The clinical importance of everolimus metabolites in blood of patients including pharmacodynamics, toxicodynamics, and cross-reactivity with the antibodies of immunoassays used for therapeutic drug monitoring remains to be evaluated.

Evaluation of a fluorescent polarization immunoassay for whole blood everolimus determination using samples from renal transplant recipients.

OBJECTIVES: This study compared the performance of a fluorescent polarization immunoassay (FPIA) against HPLC-tandem mass spectrometry (HPLC-MS) for the measurement of everolimus in renal transplant recipients. DESIGN AND METHODS: A total of 333 pre-dose samples from 45 renal transplant patients were analyzed by FPIA and HPLC-MS. RESULTS: The inter-batch inaccuracy and precision of the FPIA for control samples were

Long-acting octreotide and prolonged-release lanreotide formulations have different pharmacokinetic profiles.

Single-dose pharmacokinetic (PK) profiles and multiple-dose PK modeling were compared for long-acting octreotide (20 or 60 mg) and prolonged-release lanreotide (90 or 120 mg) over 91 days; steady-state profiles were simulated. All treatments were well tolerated. Octreotide 20-mg profile showed increased concentration on day 1, lag from days 2 to 6, then prolonged plateau phase (days 11-41); 60-mg PK was dose proportional. Lanreotide 90-mg profile showed C(max) on day 1 then elimination (apparent t1/2 25.5 days); 120-mg profile was underproportional. Steady-state PK of octreotide 20 mg/28 d suggested a C(mean) of 1216 rhog/mL (range, 1065-1585) with low fluctuation index (43%). Steady-state PK of lanreotide 90 mg/28 d suggested a C(mean) of 4455 rhog/mL (range, 2499-9279) with high fluctuation index (152%). Long-acting octreotide had more predictable PK than prolonged-release lanreotide. Simulated steady-state profiles suggest long-acting octreotide could be optimized to meet individual patient needs. In contrast, prolonged-release lanreotide requires exposure constantly above the therapeutic target to enable monthly long-term therapy.

Pulsatile and nocturnal growth hormone secretions in men do not require periodic declines of somatostatin.

Using a continuous subcutaneous octreotide infusion to create constant supraphysiological somatostatinergic tone, we have previously shown that growth hormone (GH) pulse generation in women is independent of endogenous somatostatin (SRIH) declines. Generalization of these results to men is problematic, because GH regulation is sexually dimorphic. We have therefore studied nine healthy young men (age 26 +/- 6 yr, body mass index 23.3 +/- 1.2 kg/m2) during normal saline and octreotide infusion (8.4 microg/h) that provided stable plasma octreotide levels (764.5 +/- 11.6 pg/ml). GH was measured in blood samples obtained every 10 min for 24 h. Octreotide suppressed 24-h mean GH by 52 +/- 13% (P = 0.016), GH pulse amplitude by 47 +/- 12% (P = 0.012), and trough GH by 39 +/- 12% (P = 0.030), whereas GH pulse frequency and the diurnal rhythm of GH secretion remained essentially unchanged. The response of GH to GH-releasing hormone (GHRH) was suppressed by 38 +/- 15% (P = 0.012), but the GH response to GH-releasing peptide-2 was unaffected. We conclude that, in men as in women, declines in hypothalamic SRIH secretion are not required for pulse generation and are not the cause of the nocturnal augmentation of GH secretion. We propose that GH pulses are driven primarily by GHRH, whereas ghrelin might be responsible for the diurnal rhythm of GH.

Distribution of STI-571 to the brain is limited by P-glycoprotein-mediated efflux.

The adequate distribution of STI-571 (Gleevec) to the central nervous system (CNS) is critical for its effective use in CNS tumors. P-glycoprotein-mediated efflux in the blood-brain barrier may play a role in the CNS delivery of this drug. Whether STI-571 is a substrate of P-glycoprotein was determined by examining the directional flux of [(14)C]STI-571 in parental and MDR1-transfected Madin-Darby canine kidney (MDCK) II epithelial cell monolayers. The basolateral-to-apical flux of STI-571 was 39-fold greater than the apical-to-basolateral flux in the MDR1-transfected cells and 8-fold greater in the parental cell monolayers. This difference in directional flux was significantly reduced by a specific P-glycoprotein inhibitor (2R)-anti-5-[3-[4-(10,11-difluoromethanodibenzo-suber-5-yl)piperazin-1-yl]-2-hydroxypropoxy]quinoline trihydrochloride (LY335979). The role of P-glycoprotein in the CNS distribution of STI-571 was examined in vivo, using wild-type and mdr1a/b (-/-) knockout mice that were orally administered 25 mg/kg [(14)C]STI-571. In the wild-type mice, the brain-to-plasma STI-571 concentration ratio at all time points was low (1-3%); however, there was an 11-fold greater brain partitioning of STI-571 at 1 h postdose in the mdr1a/b (-/-) mice compared with the wild-type mice. When 12.5 mg/kg STI-571 was given intravenously, the brain-to-plasma ratio of STI-571 in the mdr1a/b (-/-) mice was approximately 7-fold greater than that of wild-type mice up to 120 min postdose. These data indicate that STI-571 is a substrate of P-glycoprotein, and that the inhibition of P-glycoprotein affects the transport of STI-571 across MDCKII monolayers. Moreover, P-glycoprotein plays an important role in limiting the distribution of STI-571 to the CNS.

Population pharmacodynamic analysis of octreotide in acromegalic patients.

INTRODUCTION: Octreotide is an octapeptide analog of somatostatin used to normalize growth hormone levels in acromegaly. This article presents a population analysis of the relationship between octreotide and growth hormone concentrations in 94 patients with acromegaly, including 10 patients responding incompletely to subcutaneous treatment (poor responders). METHODS: Growth hormone and octreotide concentrations were recorded hourly over 12-hour time periods during long-term subcutaneous treatment. Twelve-hour profiles were also collected on different days up to 2 months after intramuscular injection of the long-acting formulation. We modeled the inhibition of growth hormone secretion by octreotide with a direct maximum inhibition model. A joint analysis of both formulations was performed with NONMEM (GloboMax, LLC, Hanover, Md). During model building, we examined the relationships between parameters and demographic covariates or formulations with the use of likelihood ratio tests. RESULTS: The baseline growth hormone level was higher in poor responders and was best described by a bimodal distribution. The maximum inhibition was common to both formulations and had a mean of 90%, with low interindividual variability. Sensitivity to octreotide (50% inhibitory concentration) was found to be slightly lower on average with intramuscular administration than with subcutaneous administration. CONCLUSION: Given adequate doses of octreotide, in 72% of 94 patients, growth hormone would decrease to levels below 2.5 ng. mL(-1), considered to be a desirable target concentration in acromegaly. This study provides a way to identify poor responders during subcutaneous treatment, allowing an early clinical decision to be made to switch nonresponders to alternative therapies.

Partial tachyphylaxis to somatostatin (SST) analogues in a patient with acromegaly: the role of SST receptor desensitisation and circulating antibodies to SST analogues.

OBJECTIVE: Somatostatin (SST) analogues are a key option in the management of a variety of conditions, including acromegaly. Tachyphylaxis to SST analogues is not documented in acromegaly. We describe such a phenomenon. DESIGN AND METHODS: A 74-year-old female with acromegaly previously treated with (90)Y implant, external radiotherapy and thrice daily s.c. octreotide had stable GH levels of 19 mU/l. GH progressively rose following switches to lanreotide and depot octreotide as Sandostatin LAR: from 29 to 126 mU/l. Magnetic resonance imaging and (111)In-pentetreotide scanning revealed no tumour growth or alteration in SST receptor (SSTR) status. Tachyphylaxis to SST analogues was considered. Therapy was discontinued and re-introduced in daily 200 microg/24 h increments by continuous s.c. infusion, to a maximum of 1000 microg/24 h, and maintained over 3 weeks with daily, followed by weekly, GH profiles. Competitive (125)I-octreotide radioligand binding assays measured in vitro bio-activity of anti-SST analogue antibodies. In vitro SSTR binding studies utilised SSTR-expressing rat cortex membrane. RESULTS: Median GH fell by 93% from 504 to 39.5 mU/l and rose reproducibly on continued infusion to 120 mU/l. Octreotide withdrawal for 16 h produced a 64% increase in sensitivity. High-affinity IgG anti-lanreotide (IC(50)=187 pmol/l) and anti-octreotide (IC(50)=82 nmol/l) antibody, with no crossreactivity with natural SST, was demonstrated. In vitro inhibition of (125)I-octreotide SSTR binding by anti-SST analogue crossreacting antibody was observed at 1:1 serum dilution. CONCLUSIONS: This is the first report of tachyphylaxis to SST analogues in acromegaly. We believe that the short time course of resensitisation following acute octreotide withdrawal is suggestive of an effect(s) on receptor function or on the receptor signal transduction cascade at sites further downstream, rather than an immune-mediated phenomenon.