Fab fragments of ovine antibody to colchicine enhance its clearance in the rat.

CONTEXT: Colchicine is an anti-inflammatory alkaloid used for the treatment of acute gout, but has a narrow therapeutic index. Colchicine overdoses are relatively rare, but have high mortality requiring rapid treatment. OBJECTIVE: To evaluate the ability of a newly available ovine fragment antigen-binding (Fab) antibody to colchicine (ColchiFab(™)) to protect rats against renal and other injury 24 h after colchicine ingestion. MATERIALS AND METHODS: Rats were gavaged with colchicine (5 mg/kg), then 2 h later injected intraperitoneally with 5 ml of sterile saline, or Fab anti-colchicine, a newly available ovine antibody to colchicine. Samples of blood were taken at 1, 2, 5 and 24 h after gavage, and urine was collected from 5 to 24 h after gavage. Concentrations of colchicine in tissue, blood and urine were measured by liquid chromatography/mass spectrometry, concentrations of Fab anti-colchicine, urinary neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 or KIM-1 by enzyme-linked immunosorbent assay or ELISA, while concentrations of creatine kinase and creatinine (Cr) were measured enzymatically. RESULTS: Colchicine equilibrated rapidly throughout the body and increased serum creatine kinase. Fab anti-colchicine also rapidly redistributed to the blood and remained at high concentrations over 24 h. Fab anti-colchicine caused a rapid 7.1-fold increase in serum colchicine level, followed by excretion of both colchicine and Fab anti-colchicine through the urine. This was associated with the accumulation of colchicine in the kidney, a reversal of colchicine-induced diarrhoea, and increasing urinary NGAL level; from 168 ± 48 to 477 ± 255 ng/mmol Cr [mean ± standard deviation or SD]. DISCUSSION: Fab anti-colchicine greatly increased the clearance of colchicine, although increasing NGAL level suggested the presence of mild kidney damage. CONCLUSION: These data suggest clinical utility for Fab anti-colchicine in the treatment of colchicine overdose.

A fatal case of autumn crocus (Colchicum autumnale) poisoning in a heifer: confirmation by mass-spectrometric colchicine detection.

A heifer developed severe signs of acute gastrointestinal irritation 48 hr after ingesting fresh leaves of Colchicum autumnale growing on a damp meadow. Confirmation of the suspected toxicosis was obtained by detecting colchicine in serum and urine using liquid chromatography coupled with tandem mass spectrometry using atmospheric pressure chemical ionization. Although the serum colchicine concentration had declined to an apparently nontoxic level of 2.4 ng/ml, a more prominent concentration (640 ng/ml) indicative of colchicine poisoning was detected in the urine. This finding is consistent with the known toxicokinetic properties of colchicine, whereby a large volume of distribution results in low circulating blood concentrations and prolonged urinary excretion.

Simultaneous determination of five toxic alkaloids in body fluids by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

A novel analytical method was developed and validated for the rapid and simultaneous analysis of five toxic alkaloids: Brucine, Strychnine, Ephedrine, Aconitine and Colchicine, in blood and urine using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry in the multiple reaction monitoring (HPLC-ESI-MRM) mode. The linear range was 0.05-50.0 ng mL(-1) for Brucine, 0.1-50.0 ng mL(-1) for Strychnine and Ephedrine, 0.01-10.0 ng mL(-1) for Aconitine and Colchicine. The limits of quantification for Brucine, Strychnine, Ephedrine, Aconitine and Colchicine were found to be 0.03, 0.05, 0.20, 0.05, 0.01 ng mL(-1), respectively. The average extraction recoveries in urine ranged from 96.0 to 114.0% and in whole blood were 94.0 to 113.0%. The intra-day and inter-day RSDs were less than 8.3 and 10.6%, respectively. The five alkaloids could be well separated within 7 min in a single run. The established method should be suitable for the determination of trace alkaloids in body fluids.

Disposition and metabolism of the colchicine derivative [14C]-ZD6126 in rat and dog.

1. The tissue distribution, disposition and metabolism of ZD6126, a novel vascular targeting agent, were investigated in rat and dog. This paper comprises the findings of several investigations, including rat quantitative whole-body autoradiography (QWBA), rat and dog balance and metabolism (both in intact and bile-duct-cannulated animals), rat enterohepatic recirculation, and comparison of metabolism between young and mature rats. 2. Following intravenous administration of [(14)C]-ZD6126 to rats, quantitative whole-body autoradiography showed that radioactivity was widely distributed, then rapidly eliminated from the body. 3. ZD6126-related material was eliminated primarily in the faeces (approximately 86%) of both species, indicating the importance of biliary clearance. 4. Metabolite profiles from intact and bile-duct-cannulated animals suggest that ZD6126 is cleared primarily by metabolism. In rat, the major metabolites were ZD6126 phenol, its glucuronide and other metabolites consistent with O-demethylation and conjugation. ZD6126 was more extensively metabolized by male than female rats, and also in young compared with mature rats. In dog, metabolism occurred primarily via direct glucuronidation of the active species, ZD6126 phenol. 5. Following intraduodenal infusion of bile containing [(14)C]-ZD6126-related material to bile-duct-cannulated rats, 30% of the radioactivity was subsequently recovered in bile and urine, showing that one or more components in bile are reabsorbed and undergo enterohepatic recirculation.

Single- and repeated-dose pharmacokinetics of intramuscular thiocolchicoside in healthy volunteers.

OBJECTIVE: The aim of this study was to investigate the pharmacokinetics and the accumulation and stationarity of thiocolchicoside after repeated intramuscular administration. METHOD: The pharmacokinetics of thiocolchicoside were studied in 6 healthy male volunteers after one single dose and repeated intramuscular doses of 4 mg twice a day for seven days. Plasma and urine samples were assayed for thiocolchicoside levels by a radioimmunoassay (RIA) using a cross-reacting colchicine-specific polyclonal antibody. The pharmacokinetic parameters between the first and the last days were compared using Student's t-test. RESULTS: Thiocolchicoside pharmacokinetic parameters, calculated after the single dose using non-compartmental analysis, were in good agreement with those obtained in previous studies. Following the repeated-dose regimen, the terminal half-life was not significantly different (2.7 (0.3) h) from that predicted from a single-dose (2.8 (0.2) h). The accumulation ratio, based on the repeated-dose/single-dose ratio of AUCtau was approximately 1.25. A decrease of CLT/f was found between day 1 (24.1 (5.2) l/h) and day 7 (19.9 (3.4) l/h), suggesting that moderate time-related alterations occur in the pharmacokinetics of thiocolchicoside, which may be due to a change in its CL(NR) (CL(R) was constant) or to the extent of bioavailability, explained by enterohepatic recirculation. CONCLUSION: Serum thiocolchicoside concentrations accumulated to steady-state when the drug was given twice a day for seven days and the pharmacokinetics were modified. But no adjustments of dose or dosing interval were necessary because the accumulation did not lead to marked change in the plasma levels.

Markedly altered colchicine kinetics in a fatal intoxication: examination of contributing factors.

1. Colchicine poisoning, which is relatively rare, is associated with significant morbidity and mortality. Whilst a new treatment modality, in the form of colchicine-specific Fab fragments is on the horizon, currently available therapy is largely supportive. 2. The elimination of colchicine occurs primarily by hepatic metabolism, following a first-order process, with significant enterohepatic circulation. Renal extraction is responsible for approximately 20% of colchicine elimination. 3. We report a case of colchicine intoxication, complicated by the presence of co-ingestants, in which serum colchicine concentrations remained quasi-constant over the 3 days of the patient's survival, consistent with marked alterations both in metabolism and excretion. The initial presentation was relatively benign but the subsequent course was one of severe colchicine poisoning, resulting in death. 4. Severe colchicine toxicity appears to have resulted in a vicious cycle of progressive organ dysfunction and impaired elimination. 5. Josamycin, one of the co-ingestants and an inhibitor of P-glycoprotein, the membrane pump responsible for multidrug resistance, may have played a significant role in impeding the cellular and biliary elimination of colchicine. Co-ingested opioid and anticholinergic compounds may have altered colchicine absorption and gastrointestinal transit. 6. This case serves as a reminder of the need for attention to co-ingested drugs, to early aggressive therapy, and if available, to consideration of immunotherapy.

High-performance liquid chromatography coupled to ion spray mass spectrometry for the determination of colchicine at ppb levels in human biofluids.

An original method based upon high-performance liquid chromatography coupled to ion spray mass spectrometry (HPLC-ISP-MS) has been developed for the identification and quantification of colchicine (COL) in human blood, plasma or urine. After single-step liquid-liquid extraction by dichloromethane at pH 8.0 using tofisopam (TOF) as an internal standard, solutes are separated on a 5-microns C18 Microbore (Alltech) column (250 x 1.0 mm, I.D.), using acetonitrile-2 mM NH4COOH, pH 3 buffer (75: 25, v/v) as the mobile phase (flow-rate 50 microliters/min). Detection is done by a Perkin-Elmer Sciex API-100 mass analyzer equipped with a ISP interface (nebulizing and curtain gas: N2, quality U; main settings: ISP, +4.0 kV; OR, +50 V; Q0, -10 V; Q1, -13 V; electron multiplier, +2.2 kV); MS data are collected as either total ion current (TIC, m/z 100-500 or 380-405), or selected ion monitoring (SIM) at m/z 400 and 383 for COL and TOF, respectively. COL mass spectrum shows a prominent molecular ion [M + H]+ at m/z 400. Increasing OR potential fails to provide a significant fragmentation. Retention times are 2.70 and 4.53 min for COL and TOF, respectively. The quantification method shows a good linearity (r = 0.998) over a concentration range from 5 to 200 ng/ml. The lower limit of detection in SIM mode is 0.6 ng/ml COL, making the method convenient for both clinical and forensic purposes.

Pharmacokinetics of thiocolchicoside in humans using a specific radioimmunoassay.

The myorelaxant thiocolchicoside (TC), an analogue of colchicine (COL), was assayed in plasma and urine by a radioimmunoassay (RIA) using a cross-reacting COL-specific polyclonal antibody. Cross-reactivity was 56% for TC, giving a limit of quantification of 0.5 ng/ml and a linear response from 0.5 to 100 ng/ml. Specificity was checked by cross-reactivity studies with COL analogues and by using liquid chromatography and RIA in tandem on urine samples. Two immunoreactive peaks were detected, but the nonspecific peak represented < 2% of the total urine concentration of TC. Pharmacokinetics of TC following infusion of 4 mg in two subjects revealed a moderate distribution (Vss from 31 to 35 L) and mainly extrarenal elimination (75% of the total body clearance). Terminal half-lives ranged from 2.4 to 2.7 h in plasma and from 3.2 to 3.7 h in urine.

Colchicine poisoning: report of a fatal case with body fluid analysis by GC/MS and histopathologic examination of postmortem tissues.

A suicide caused by oral ingestion of colchicine is reported. Postmortem examination revealed circulating Pelger-Huet polymorphonuclear leukocytes and numerous mitotic and chromatin bodies in tissues with rapid cell turnover. Colchicine was identified by gas chromatography/mass spectrometry (GC/MS) of an organic extract of a urine specimen obtained about 36 h after the patient's original hospitalization. The clinical history and pathology of this rarely encountered intoxication are correlated with previous reports, and the rapid detection of colchicine by GC/MS is discussed.

Determination of the antimitotic agents N-desacetylcolchicine, demecolcine and colchicine in serum and urine.

In an effort to characterize the pharmacokinetic behavior of the antimitotic agent N-desacetylcolchicine a selective, sensitive high-performance liquid chromatographic method was developed for the determination of N-desacetylcolchicine, demecolcine and colchicine in serum or urine. To 0.5 ml of serum or 0.1 ml of urine diluted to 0.5 ml were added 50 microliters demecolcine (2 micrograms/ml) which serves as the internal standard. The sample was extracted using a C2 reversed-phase solid extraction column. N-Desacetyl-colchicine, colchicine and the internal standard were eluted from the column with methanol. The combined eluates were evaporated to dryness and the residue was reconstituted with water. The reconstituted sample was injected into a C18 reversed-phase column and eluted using a mobile phase consisting of 0.1 M potassium dihydrogenphosphate, 5 mM 1-pentanesulfonic acid in methanol and acetonitrile with a final pH of 6.0, at a flow rate of 1.5 ml/min. N-Desacetylcolchicine, colchicine and the internal standard were detected using a variable-wavelength ultraviolet detector at 254 nm. The limit of detection was 0.4 ng/ml for desacetylcolchicine and 4.0 ng/ml for colchicine. The method is linear over a concentration range of 1.0-200 ng/ml. The method has been shown to be a rapid, reliable method to monitor N-desacetylcolchicine levels in clinical trials in cancer patients.

Pharmacokinetics/bioavailability of colchicine in healthy male volunteers.

In a randomized 2-way cross-over study with twelve healthy male volunteers, two colchicine preparations (tablets, A vs. oral solution, B) were tested. The preparations were administered as single doses of 1 mg; prior to and up to 72 h after medication blood samples were collected and the plasma colchicine concentrations determined. Additionally urine samples were collected at 0-2, 2-4, 4-6, 6-8, 8-10, 10-24, 24-48, 48-72 and 72-96 h intervals. The colchicine plasma and urine concentrations were determined by a newly developed and validated RIA method. The mean area under the plasma concentration-time curve (AUC-1, AUC-3) was calculated as 23.95 +/- 12.10 (AUC-1) and 26.73 +/- 12.75 (AUC-3) h.ng/ml after application of A and 28.01 +/- 14.74 (AUC-1) and 31.57 +/- 16.58 (AUC-3) h.ng/ml after application of B, respectively. Mean peak plasma concentrations of 4.15 +/- 2.35 (A) and 4.88 +/- 3.90 (B) ng/ml were reached at 1.15 +/- 0.38 (A) and 1.13 +/- 0.42 (B) h after application. The mean terminal half-lives accounted for 9.31 +/- 3.98 (A) and 10.57 +/- 5.53 (B) h. The mean total clearance (Cl/F) and volume of distribution (V/F) were found to be 40.12 +/- 20.87 (A) and 46.58 +/- 24.65 (B) l/h and 472.59 +/- 196.46 (A) and 624.89 +/- 304.09 (B) l, respectively. The mean total amount excreted in urine (Ae) was 172.66 +/- 91.51 (A) and 174.85 +/- 63.53 (B) micrograms.(ABSTRACT TRUNCATED AT 250 WORDS)

Zero-order absorption and linear disposition of oral colchicine in healthy volunteers.

The pharmacokinetics of colchicine has been studied in nine healthy male volunteers after oral doses of 0.5, 1, and 1.5 mg as tablets. Plasma and urine samples were collected over 48 h and analysed for colchicine by radioimmunoassay. Individual colchicine concentration profiles in plasma and urine were well described by a two-compartment open model with zero-order input. Considering the absorption variables as specific to each experiment, the lag time (0-0.35 h) and duration (0.39-2.38 h) of absorption were found to be independent of dose, while the zero-order rate constant of absorption (k0) increased linearly with dose. Disposition variables were taken as common to the three experiments, except in six subjects in whom renal excretion varied significantly across experiments in a dose-independent manner. For seven subjects the terminal half-life was 19.4 h, the oral apparent volume of distribution at steady-state (Vss/f) was 691 l, and the oral systemic clearance (CL/f) was 33.1 l.h-1. In the two other subjects, the values were unreliable, but the estimated terminal half-life was greater than 48 h, Vss/f ranged from 1690 to 3480 l, and CL/f was in the range of the other subjects in 1 subject, and it was about 15 l.h-1 in the other. In the latter subject, these estimates, together with the observation that plasma concentration reached a plateau at 2 to 5 h after ingestion, suggest enterohepatic cycling of colchicine. Overall, the disposition of colchicine was linear in the dose range 0.5-1.5 mg, with a long terminal half-life, and absorption obeyed zero-order kinetics, with k0 proportional to dose.

Radioimmunoassay for colchicine in plasma and urine.

A radioimmunoassay for the measurement of colchicine (in quantities as small as 0.05 nanogram) in plasma and urine was developed with the use of an antibody from immunized rabbits. After the intravenous injection of 2 milligrams of colchicine in seven subjects, the calculated zero-time concentration in the plasma was 2.9 +/- 1.5 micrograms per deciliter, and the mean half-time in the plasma was 58 +/- 20 minutes. Declining, but measurable, amounts of colchicine could be detected in urine up to day 9 after the drug was administered.

Massive colchicine overdose: a report on the toxicity.

A case of an overdose of colchicine for suicidal purposes is presented. The main complications observed involved the hematopoietic, the gastrointestinal, the respiratory and the central nervous system. Heinz bodies were noticed in the red blood cells, a finding which had not been previously documented in patients with colchicine overdose. Despite intensive management the patient died on the thirteenth hospital day of central nervous system complications.

[Determination of colchicine in biological fluids]. / Dosage de la colchicine dans les liquides biologiques

The authors described a fluorimetric method of estimation of colchicine applicable to biological fluids, during treatment and, especially, during acute poisoning. Blood and urinary concentrations confirm the data in the literature.

Biliary excretion of colchicine.

After intravenous administration of 3H-colchicine (0.2 mg/kg) to rats, 68 percent was excreted in the feces in 48 hours suggesting bile might be the major route of excretion for colchicine. Rats with cannulated bile ducts excreted 50 percent of a 2 mg/kg dose into the bile within 2 hours; half of this was colchicine, and the rest was desmethylcolchicine and more polar metabolites. Colchicine was excreted into the bile of the rat against a bile/plasma concentration gradient of 800 which resulted from a liver/plasma ratio of 15 and a bile/liver ratio of 60. The biliary excretion of colchicine varied widely among the hamster, dog and rabbit. Of the administered colchicine, 32, 20 and 16 percent were excreted by the hamster, dog and rabbit, respectively, within 2 hours. There was also a species difference in the percentage of the radioactivity present in bile as the parent drug. In the hamster, dog and rabbit, the percentage of radioactivity excreted into the bile as colchicine was 45, 34 and 72 percent, respectively. These species excreted colchicine into the bile against a bile/plasma gradient ranging from 19 to 870. Partition of these gradients between liver/plasma and bile/liver ratios demonstrated that both ratios were greater than one, suggesting that colchicine is excreted by an active process. The liver/bile gradient was always the larger of the two ratios.