Analysis of therapeutic peptides in human urine by combination of capillary zone electrophoresis-electrospray mass spectrometry with preparative capillary isotachophoresis sample pretreatment.

The presented study deals with the off-line coupling of preparative isotachophoresis (pITP) with on-line combination of capillary zone electrophoresis with electrospray mass spectrometric detection (CZE-ESI-MS) used for the analysis of therapeutic peptides (anserine, carnosine, and buserelin) in complex matrix (urine). Preparative capillary isotachophoresis, operating in a discontinuous fractionation mode in column-coupling configuration, served as a sample pretreatment technique to separation, and fractionation of mixture of therapeutic peptides present in urine at low concentration level. The fractions isolated by pITP procedure were subsequently analyzed by capillary zone electrophoresis with electrospray mass spectrometric detection. Acetic acid at 200 mmol L(-1) concentration served as background electrolyte in CZE stage and it is compatible with MS detection in positive ionization mode. In pITP fractionation procedure, sodium cation (10 mmol L(-1) concentration) as leading ion and beta-alanine as terminating ion (20 mmol L(-1) concentration) were used. While using CZE-ESI-MS, the limits of detection were 0.18 µg mL(-1) for carnosine, 0.17 µg mL(-1) for anserine and 0.64 µg mL(-1) for buserelin in water and 0.19 µg mL(-1) for carnosine, 0.50 µg mL(-1) for anserine and 0.74 µg mL(-1) for buserelin in 10 times diluted urine, respectively. The cleaning power of pITP sample pretreatment was proved as the peptides provided the higher MS signals at lower concentration levels resulting from the minimized matrix effects. The quality of obtained MS/MS spectra was very good so that they can provide information about the structure of analytes, and they were used for verification of the analytes identities. The pITP pretreatment improved the detection limits of the analyzed therapeutic peptides at least 25 times compared to the CZE-ESI-MS itself.

Some possibilities of an analysis of complex samples by a mass spectrometry with a sample pretreatment by an offline coupled preparative capillary isotachophoresis.

This work deals with an analysis of biologically important compounds in complex matrices using preparative isotachophoresis (pITP) in column coupling configuration as a sample pretreatment technique followed by a direct infusion mass spectrometry with nano-electrospray ionization (DI-nESI-MS). Busereline was chosen as a model analyte, and urine was chosen as an example of complex matrix. In pITP experiments, sodium cation (10 mmol/L concentration) was used as a leading ion and ß-alanine as terminating ion (20 mmol/L concentration). The fractions, obtained by pITP pre-separation with the assistance of the mixture of discrete spacers, were finally analyzed by DI-nESI-MS. It was shown that pITP performed before DI-nESI-MS analysis can significantly simplify complex matrix, and, due to its concentration power, pITP can consequently decrease the concentration limit of detection. The concentration of buserelin in the urine samples analyzed by pITP-DI-nESI-MS was 10 µg/L (reflecting at a 8.10⁻9 mol/L concentration) in our work but from the ion intensities obtained in MS as well as MS/MS analyses, it is clear that this concentration level could be several orders of magnitude lower for reliable detection and identification of buserelin in urine analyzed using pITP with DI-nESI-MS detection.

Preparative isotachophoresis with surface enhanced Raman scattering as a promising tool for clinical samples analysis.

A surface enhanced Raman scattering (SERS) spectrometry is an interesting alternative for a rapid molecular recognition of analytes at very low concentration levels. The hyphenation of this technique with advanced separation methods enhances its potential as a detection technique. Until now, it has been hyphenated mainly with common chromatographic and electrophoretic techniques. This work demonstrates for a first time a power of preparative isotachophoresis-surface enhanced Raman scattering spectrometry (pITP-SERS) combination on the analysis of model analyte (buserelin) in a complex biological sample (urine). An off-line identification of target analyte was performed using a comparison of Raman spectra of buserelin standard with spectra obtained by the analyses of the fractions from preparative isotachophoretic runs. SERS determination of buserelin was based on the method of standard addition to minimize the matrix effects. The linearity of developed method was obtained in the concentration range from 0.2 to 1.5 nmol L(-1) with coefficient of determination 0.991. The calculated limit of detection is in tens of pico mols per liter.

Analysis of buserelin in urine by online combination of capillary zone electrophoresis with electrospray mass spectrometry.

A fast and precise analysis of the synthetic peptide buserelin in urine using CZE-ESI-MS method has been demonstrated. Formic acid at 50 mmol/L concentration served as background electrolyte in CZE stage and it is compatible with MS detection in positive ionization mode. Two injection modes were tested, i.e. pressure (50 mbar for 5 s) and electrokinetic injection (5 kV for 5 s), of which electrokinetic injection provided better calibration parameters. Buserelin LODs were 0.47 microg/mL in water and 0.63 microg/mL in ten times diluted urine samples using pressure injection, while they were 0.32 microg/mL in water and 0.34 microg/mL in ten times diluted urine samples using electrokinetic injection. Repeatability of buserelin migration times was below 6% (pressure injection mode) and 1% (electrokinetic injection mode). Repeatability of buserelin peak area in SIM mode (m/z=620.5+/-0.5) was less than 12% (pressure injection mode) and 5.8% (electrokinetic injection mode). In this work, no interferences were observed during the analyses of spiked urine samples.

Disposition of 3H-labelled buserelin continuously infused into rats.

The disposition of the gonadotropin-releasing hormone (GnRH) agonist buserelin was studied in male rats under conditions of long-term administration. Rats were continuously infused with about 30 pmole [3H]-buserelin/24 h subcutaneously by osmotic minipumps for 4-7 days. After killing the rats, the 3H-activity of the tissues was measured and was found to be highly concentrated (about 10-fold to plasma) only in the pituitary. The daily amounts of 3H-activity excreted in urine and faces were constant over the whole infusion period, suggesting steady state conditions. On a molar basis, of the infused dose of buserelin, 14.8% was found to be excreted into urine as intact peptide, and 16.5, 10.8 and 20.6% as the partial buserelin sequences 1-2, 1-3 and 5-9. It is concluded that the major elimination route of buserelin, constant with time, is glomerular filtration, followed by enzymatic degradation of part of the filtered peptide by kidney tubuli enzymes to the partial sequences 1-2, 1-3 and 5-9, which reflects the proteolytic breakdown of buserelin by kidney membrane peptidases in vitro. Based on the similarities in the pharmacokinetics, in vivo metabolities, and in vitro enzymatic degradabilities among the GnRH agonists that have the native GnRH sequence modified at position 6 with or without additional modification at the C-terminal, the elimination process as shown here for buserelin should also be valid for other GnRH agonists.

Disposition of the 3H-labeled gonadotropin-releasing hormone analog buserelin in rats.

The short-time disposition of 3H-labeled D-Ser(TBU)6-desGly10-GnRH-ethylamide ([3H]buserelin) was studied in rats after bolus intravenous and subcutaneous injections and killing the rats after 1 and 3 hr, respectively. When estimated as the percentage of the injected dose, 3H-activity within the whole blood rapidly declined from 25.5% at 2 min to 4.7% at 60 min after intravenous injection and remained nearly constant at 3.4% from 30 to 180 min after subcutaneous injection. More than 94% of the blood activity was confined to plasma. 3H-Activity was highly concentrated in the pituitary, as seen from the concentration ratio of activity tissue/plasma (ti/pl), being 12.6 and 8.0 at 60 and 180 min, respectively. A transient accumulation of activity was observed in kidney (ti/pl 9.5 and 2.2 at 60 and 180 min, respectively). All the other tissues studied (liver, spleen, adrenal, testis, epididymis, muscle, lung, fat, skin, heart, thyroid, stomach, and intestine) showed ratios ti/pl below 2.0, mostly below 1.0. The tissues within the blood-brain barrier cortex/thalamus and hypothalamus had the lowest ti/pl (0.08 at 60 min). Within 24 hr after intravenous injection of [3H]buserelin into rats, 58% of the administered 3H-dose was recovered in urine, 21.6% of the urinary radioactivity being identified as intact buserelin. Only 3.6% of the 3H-dose were found in the feces. It is concluded that buserelin is concentrated specifically only in its target organ pituitary, whereas kidney accumulates the peptide transiently due to glomerular filtration and presence of the peptide in the primary urine, part of the peptide being degraded to smaller peptides in the kidney tubuli before being excreted into urine.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics and endocrine effects of slow release formulations of LHRH analogues.

The LHRH agonists are antigonadotropic agents for reversible ovarian suppression in gynaecology and in oncology. In oncology, pituitary inhibition is maintained with high release rates preferably by implant or microcapsule injection. The pharmacokinetics of buserelin after injection, infusion, and during implant treatment (controlled release) are described. The release rate is monitored by urinary buserelin excretion (fractional excretion of 30% of the daily dose). During therapy, LHRH agonists in serum are measured by specific radioimmunoassays, with or without extraction. A more convenient non-invasive procedure is to measure the amount of buserelin in 24-h urine samples (during injections or nasal spray), or the urinary buserelin/creatinine ratio in morning urine samples (during infusions or implants). After high dose injection, buserelin has a half-life of 80 min, therapeutic plasma concentrations are maintained for 8-12 h. In long-term maintenance with buserelin implants (polylactide-glycolide, 75:25), serum concentrations and urinary excretion showed an extended plateau phase indicating a suitable dose interval of 2-3 months. In endometriosis and leiomyoma, the minimum release rate (urinary buserelin) required for maintenance of steroid suppression was established (buserelin excretion of about 0.5 microgram/g creatinine). Buserelin implants in prostate carcinoma are effective for 2 or 3 months, after a single dose of 6.6 or 10 mg buserelin, respectively. A consistent suppression of serum testosterone secretion was confirmed for more than 2 yr. Buserelin microparticles are effective in rhesus monkeys to completely suppress follicular maturation and oestrogen secretion during 4-6 weeks after a single dose of 3.6 mg buserelin. Recent results on the controlled release of an LHRH antagonist (Hoe 013) from biodegradable microparticles in rats with DMBA-induced mammary tumours indicate that tumour suppression by LHRH antagonists is well tolerated and highly effective. The local tolerance at the injection site of antagonist microparticles is excellent as in the case of LHRH agonists like buserelin.

Urinary excretion of [D-Ser(t-Bu)6,Des-Gly10]GnRH ethylamide (buserelin) during therapy of central precocious puberty: a multicentre study.

In order to compare the clinical effects of buserelin on central precocious puberty to its excretion in urine, as a parameter of metabolism or compliance, we have studied 52 patients treated either sc or intranasally. In girls with good control, urinary buserelin excretion represented 0.5 +/- 0.1% of the daily intranasal dose vs 12.5 +/- 2.3% of the daily sc dose. In boys, it represented 0.8 +/- 0.2 and 9.9 +/- 2.7%, respectively. In the sc treated group, 3 patients (2 girls and 1 boy) with poor control who exhibited excretion levels similar to those with good control were classified as resistant to therapy. Clinical control was poor in 4 intranasally treated girls: 2 had low excretion values suggesting poor compliance or failure of absorption by the nasal mucosa, and 2 appeared resistant to therapy, as urinary excretion levels of buserelin were similar to those of well-controlled patients. In addition, these data suggest that the small amount of buserelin absorbed by the nasal mucosa, as expressed by urinary excretion, is sufficient to desensitize the pituitary gonadotropes without any significant first-pass effect in the systemic circulation. This may explain the clinical effectiveness of the intranasal route for administration of small hormonal peptides acting on the pituitary gland.

Controlled release of a GnRH agonist from a polyhydroxybutyric acid implant: reversible suppression of the menstrual cycle in the macaque.

The use of polyhydroxybutyric acid as a matrix for delivery of the GnRH agonist buserelin was studied in adult stumptailed macaques with regular menstrual cycles. This material contained 5.0 mg buserelin in a disk of 0.6 cm diameter and 0.15 cm thickness. The effects on pituitary-ovarian function were examined and buserelin release profiles from 30,000 and 150,000 molecular weight polyhydroxybutyric acid matrices were compared. In an attempt to reduce the high initial release rate of buserelin from these disks, and to prolong the effective period of release, implants of 30,000 molecular weight were coated with cyanoacrylate. The implants were removed after 28, 35 or 42 days for the 30,000, the 150,000 and the coated implants, respectively. The urinary excretion profile for the agonist revealed a high initial release of buserelin from these implants followed by a rapid decline. The coated disks released significantly less (p less than 0.05) buserelin over the first 3 days than did either of the uncoated implants, but maintained release of the agonist over a longer period. Inhibition of ovulation and suppression of estradiol secretion lasted for the duration of treatment in 3 of 5 macaques receiving the 30,000 implant, and in 5 of 5 receiving the 150,000 or the coated implant. In 3 macaques in whom the coated implant was left in situ, ovulation was suppressed for 104 +/- 3.7 days. These results demonstrate that a polyhydroxybutyric acid matrix can be used as a depot formulation for a GnRH agonist and that improved control of release of the agonist can be achieved by coating such implants with cyanoacrylate. This approach may be useful in experimental and clinical situations and may be applicable for delivery of other peptide hormones.

Serum concentration and urinary excretion of the luteinizing hormone-releasing hormone agonist buserelin in patients with endometriosis.

We studied the pharmacokinetics of iv and intranasally administered buserelin, a LHRH agonist peptide, in 14 women with endometriosis. Serum and urinary buserelin concentrations were determined by specific RIA (buserelin antiserum AS-639). Intact buserelin and the metabolites in urine were separated by reverse phase high performance liquid chromatography and measured by RIA. The mean serum buserelin concentrations were 101 +/- 33 (+/- SD) ng/mL 20 min and 1.12 +/- 0.12 ng/mL 360 min after its iv injection in 6 women, and the mean elimination half-life between 20 and 360 min was 51 min. In serum, intact buserelin was the main constituent (10 min, 90%; 120 min, 74%; 360 min, 52%), and the major metabolite was the buserelin-(5-9) pentapeptide (10 min, 0.6%; 120 min, 19%; 360 min, 12%). In the urine collected 0-1 h after buserelin administration, intact buserelin was 66% and the 5-9 pentapeptide was 28% of the total excretion. In the urine collected between 6-24 h after buserelin administration, intact buserelin accounted for 67% and the 5-9 pentapeptide for 32% of the total excretion. The urinary buserelin concentration was 1345 +/- 156 micrograms/g creatinine 1 h and 25 +/- 5 micrograms/g creatinine 6-24 h after buserelin administration. Serum LH, FSH, and estradiol concentrations increased acutely up to 10-fold above basal values; the mean peak LH, FSH, and estradiol values occurred at 180-240 min, 240 min, and 24 h, respectively. In therapeutic studies with buserelin nasal spray in 5 women, serum concentrations of 0.9-1.4 ng/mL were found 15 min after a single dose of 300 micrograms, intranasally, and the urinary excretion was 2.52-3.68 micrograms/24 h during daily administration of 3 doses of 300 micrograms at intervals of 8 h. These results confirm that buserelin is slowly inactivated and remains available to pituitary receptors for a prolonged period after its iv or intranasal administration.

A pharmacological evaluation of a new 3-month depot preparation of buserelin for prostatic cancer.

A new slow-release formulation of buserelin given as a 3-month depot injection was evaluated in four patients with advanced prostatic cancer. Treatment was maintained for a mean period of 15 months. Steady-state urinary buserelin concentrations were reached by the beginning of the 3rd week of treatment and maintained until the end of the 3rd month. Serum testosterone remained suppressed in the castrate range for the duration of the study. This preparation offers an advantage for the patient and clinician over existing methods of gonadotrophin-releasing hormone (GnRH) analogue administration and will enter further clinical trial.

The adjuvant effect of bacitracin on nasal absorption of gonadorelin and buserelin in rats.

Nasal absorption of gonadorelin (luteinizing hormone-releasing hormone; LH-RH) and buserelin, an LH-RH agonist, was studied in anesthetized rats. Administration of peptides was by nasal instillation of aqueous peptide/buffer solutions. Peptide absorption was monitored using different techniques: (a) by specific radioimmunoassays for serum levels of lutropin (LH), (b) by the cumulative urinary excretion of buserelin, and (c) by the ovulatory activity after nasal LH-RH and buserelin, respectively. Without adjuvant the nasal absorption of LH-RH and buserelin was relatively poor compared to subcutaneous or intravenous injection. Using absorption adjuvants of different types, e.g., sodium taurodihydrofusidate (STDHF) and bacitracin, marked increases in nasal absorption and, therefore, significant nasal adjuvant activity were found, as demonstrated by an increase in the biological response after nasal administration of the peptides. The mucosal compatibility of bacitracin at the concentrations used for enhancement of absorption was confirmed by an in vitro investigation using isolated gastric mucosa of guinea pigs as a test model.