Colchicine antibodies - Principles of treatment and future

B. Mégarbane, F.J. BaudB. Mégarbane, F.J. Baud

Réanimation Médicale et Toxicologique, Réanimation Médicale et Toxicologique,

INSERM U705, Université Paris-Diderot, INSERM U705, Université Paris-Diderot,

Hôpital Lariboisière, Paris, FranceHôpital Lariboisière, Paris, France

Colchicine is an alkaloid derived from Colchicum autumnale (Liliaceae family) - Native to temperate areas of Europe, Asia, and America - All poisonous plant, high concentrations in the corm or bulb

History:- Ephemeron (Theophrastlus of Eresus, 370 BC )- Destructive fire of Colchican Medea (Nicander, 150 BC)- Treatment of gout (Alexander of Tralles, 550 AD)- Chemical synthesis (Laborde & Houdé, 1884)

Introduction (1)Introduction (1)

Introduction (2)Introduction (2)

Indications: Gouty arthritis

Familial Mediterranean fever

Scleroderma, amylmoidosis, Behcet’s disease, Paget’s disease

Dermatitis herpetiformis, Acute neutrophilic dermatosis

Erythema nodosum leprosum, Condyloma acuminata

Resistant idiopathic thrombocytopenic purpura

Biliary and liver cirrhosis

Acute colchicine poisoningAcute colchicine poisoning

• Rare in Western countries

• Resulting from both accidental and intentional overdoses

• Responsible of a high-rate mortality

Colchicine toxicity is predictable and dose-dependent with multiorgan involvement and delayed onset.

323 case mentions208 single exposures

51 None31 Minor19 Moderate3 Major4 Deaths

AAPCC-TESS, Clin Tox 2006

355 exposures in 33 countries in Europe during 1999-2003 with 4% fatal rate

Kupferschmidt H, EAPCCT 2005

Mechanisms of colchicine actionMechanisms of colchicine action

• Selective and reversible binding to the 65 subunits of microtubules - In vitro : high affinity for dividing cells, mitotic apparatus, cilia, sperm tails and brain - Forensic studies: cell blockage in metaphase and abnormal nuclear morphology

• Alteration in multiple cellular functions: - Cell shape, mobility, ability to phagocytosis - Cell division

• Affection of organs exhibiting the highest cell turnover (GI mucosa, bone marrow)

• Other effects: - collagenase activity - production of PG-B and PG-F2 in synovial cells - transport of nucleosides

Toxicokinetics of colchicine

Conflicting data ...

• A narrow therapeutic index

• Rapid absorption with variable bioavailability (25 - 50%)

• Hepatic metabolism (deacetylation), biliary secretion, enterohepatic cycle

• Renal elimination (20%, unchanged form)

• Role of co-ingested drugs (CYP3A4 inhibitors, macrolides)

Bi-exponential curve

Elimination half-life : 10 - 37 hrs

Distribution volume : 21 l/kg

Rochdi M. Hum Exp Toxicol 1992

Colchicine poisoningColchicine poisoning

Stage I (0-12 h) GI symptoms

Volume depletion

Peripheral leukocytosis

Stage II (2-7 d) Respiratory distress, ARDS, hypoxemia

Cardiovascular shock

Thromboyctopenia, DIC

Myelosuppression, neutropenia

Hyponatremia, hypocalcemia, hypophosphatemia

Metabolic acidosis

Rhabdomyolysis, myoglobinuria, oliguric renal failure

Stage III (1-2 wk) Rebound leukocytosis

Alopecia.

Signs are well documented, follow a typical pattern, and involve multiorgan systems

Experimental assessment of cardiotoxicityExperimental assessment of cardiotoxicity

• Impairment of myocardial contractility: maximum shortening velocity (-32% and -61%) active isometric force (-47% and -65%) peak power output (-57% and -69%)

• Impairment of isotonic relaxation and load dependence of relaxation, suggesting a decrease in sarcoplasmic reticulum function

• Acceleration of isometric relaxation, suggesting a decrease in Ca++ myofilament sensitivity

• Marked negative inotropic effects if impairment of myothermal economy

Mery P. Intensive Care Med 1994

Impairment of the intrinsec contractility of rat left ventricular papillary muscle

(2 and 4mg/kg colchicine versus saline)

Colchicine cardiac toxicityColchicine cardiac toxicity

2 series with very elevated mortality rate:

• Bismuth C. Presse Med 1977:

11 cases ≥ 0.8mg/kg GI troubles + DIC + aplasia + cardiac shock = 100% death

• Sauder P. Hum Toxicol 1983:

8 cases 9-160 mg, 4 cases with cardiac failure ( cardiac index, systemic resistance) = 100%

death

Few cases report with survive despite cardiogenic shock:

• Baron DA. Ouest Med 1972 F 38 yrs, 30 mg, WBC 27,000 /mm3, FV 22%

• Bismuth C. Nouv Presse Méd 1981 M 37 yrs, 0.8 mg/kg, WBC 12,000 /mm3, PT

19%

• De Villota E. Crit Care Med 1979 M 50 yrs, 30 mg, WBC 9,100 /mm3

Cardiovascular and direct negative inotropic effects were largely assessed.

Sauder P. Hum Toxicol, 1983

Assessment of colchicine mechanism of cardiac Assessment of colchicine mechanism of cardiac toxicitytoxicity

Profound EKG changes :Profound EKG changes :

Murray. Mayo Clin Proc 1983Mendis. Postgraduate J 1989Wells. Vet Hum Toxicol 1989Mullins. Am J Emerg Med 2000Weakleyy-Jones. Am J Forensic Med Pathol 2001Brvar. Crit Care 2003Miller. J Emerg Med 2005Van Heyningen C. Emerg Med J 2005

T-waves negativation, ST elevation in leads I, II V2-V6

Stahl. Am J Med Sci 1979Stapczynski. Ann Emer Med 1982Hobson. Anaesth Intensive Care 1986Wells. Vet Hum Toxicol 1989Stemmermann. Hum Pathol 1972McIntyre. J Forensic Sci 1994Weakley-Jones. Am J Forensic Med Pathol 2001Wells. Vet Hum Toxicol 2000Brvar. Wien Klin Wochenschr 2004

Cardiac dysrhythmias (sinus tachycardia, sinus bradycardia, VF,

sinus arrest, complete AV blockade)

Colchicine may impair impulse generation and cardiac conduction, in addition to the electrolyte and acid-base

disturbances

Direct colchicine-related cardiac injuriesDirect colchicine-related cardiac injuries

• Biology alterations: Delayed elevation of myocardial enzymes (troponine, CPK-MB)Mullins ME. Am J Emerg Med 2000

Sussman JS. Ther Drug Monit 2004

•  Pathology: a series of 12 fatal cases Interstitial edema without cell necrosis in all cases. Interstitial myocarditis in 2 cases.

Hoang C. Ann Pathol 1982

Van Heyningen C. Emerg Med J 2005

Prognostic factorsPrognostic factors

• The supposed ingested dose

Mortality< 0.5 mg/kg < 5 %0.5 à 0.8 mg/kg 10-50 %> 0.8 mg/kg 90 %

• Prothrombin index ≤ 20%• WBC ≥ 18x109 /l• Onset of cardiogenic shock• Onset of ARDS

in the 24th hours

in the 72th hours

Bismuth C. Presse Med 1977

Colchicine poisoning managementColchicine poisoning management

• Management includes early GI decontamination, careful monitoring of physical examination and laboratory tests (electrolytes, blood gases, appropriate cultures) and supportive treatments:

• To date, there is no successful commercially available specific therapy, although several experimental studies and one human case report assessed

the efficiency of colchicine-specific Fab fragments.

Fluids (diarrhea) Vasopressors (shock) Oxygen supplementation, mechanical ventilation (ALI, ARDS) Antibiotics (fever) GCSF (neutropenia) Transfusions (thrombocytopenia, anemia)

Principles of immunotoxicotherapyPrinciples of immunotoxicotherapy

• A procedure able to simultaneously sequester, extract or redistribute, and eliminate the toxin by using specific active binding sites derived from different antibody molecular entities. • Currently used in humans for treating cardiac glycoside and venom poisonings. Since the initial report in 1976 by Smith et al., it has become the first-line treatment of life-threatening intoxication by cardiac glycosides, including digoxin, digitoxin, and other structurally related cardiotoxins from Nerium, Thevetia sp. (oleander), and Bufo sp. (toads).

Scherrmann JM. Clin Toxicol 1989

Specific active biding sites derived from Specific active biding sites derived from antibodyantibody

• Industrial manufacturing

• Polyclonal antibodies of ovine origin

• Fragmentation of antibodies: - useful for haptens (< 1000 d) - increased safety - limited instability

• Future trends: monoclonal, humanized, semi-synthetic, further fragmentation, ...

Toxin sequestrationToxin sequestration

Pharmacokinetic characteristics:

•  Vdtoxin/Vdantitoxin ratio 1 to probability of interaction

For toxins with vascular distribution: IgG, IgM (5 l)

For toxin with extravascular distribution: Fab (30 l)

• Equilibration time in the distribution space: 2-4h (Fab) versus 12-24 h (IgG,

Fab’2)

Affinity issues:• Affinity ≥ 109 M-1 (critical minimal value) to form stable complexes

• Prefer polyclonal to monoclonal antibodies, based on manufacturing capacity

+ to enlarge specificity to epitopes.• Prefer ovine to equine antibodies, to reduce serum sickness + to produce a higher proportion of specific IgG

Toxin extraction or redistributionToxin extraction or redistribution

Limiting factors:- Reversibility of binding (reversible intracellular binding)- Kinetics of the toxin release from the receptor

t1/2 dissociation of colchicine / tubulin : 20 h

t1/2 dissociation of digoxin / Na,K-ATPase: 1 h

Blood anti-toxin concentration should exceed bound toxins during a predicted period of time to allow toxin efflux

Anti-toxin with slow clearance (IgG, Fab ’2 < Fab) should be preferred to allow

redistribution over a prolonged period

Involve the removal of toxin from the toxin receptor compartment to the antibody distribution space

Toxin eliminationToxin elimination

Extra-renal clearance: = 60-70%Reticulo-endothelial tissues gut, liver, spleen, lymph nodes

Renal clearance:• Fab are filtrated through glomeruli, rapidly and extensively reabsorbed and catabolized by the proximal tubule cells.• Fab increases the renal clearance of toxins: + 20-30%However: Possible tubular re-absorption

Re-circulation of free toxin molecules Reduction in glomerular filtration 

Low MW-toxin bound to anti-toxin adopt anti-toxin elimination properties

Pharmacokinetic aspects of basic mechanisms

Free toxin level

Total toxin level

Fab level

Phase 1 Phase 2 Phase 3

Developmental requisites for Developmental requisites for immunotoxicotherapyimmunotoxicotherapy

1- Severe poisonings with a high risk of death and short-term effects

2- Toxicity in the milligram range, allowing stoechiometric neutralization

3- Efficient production of antibody in animals after conjugation to a protein

4- High affinity antibody (2. 1010 M-1)

5- Distribution volume much greater than its corresponding Fab with the possibility of a rapid redistribution from tissue to blood

Colchicine is a good candidate for the development of a successful immunotherapy, as it perfectly answers to the toxin-dependent requisites.

Limitations in colchicine poisoning (1)Limitations in colchicine poisoning (1)

Whereas cardiac glycosides

are membrane-associated

toxins, efficiently reversed

with an equimolar dose of

specific Fab colchicine is low-

MW acting intracellular

poison, needing to consider

an expanding

immunotherapy model to

intracellular toxins.

Colchicine characteristics may theoretically limit the potential benefits

Limitations in colchicine poisoning (2)Limitations in colchicine poisoning (2)

By opposite to digitalis poisonings where only one organ, the heart, is at vital risk, severe colchicine poisonings induce multi-organ failure with not always reversible structural injuries.

• There is no evidence that antibodies can reverse organ injuries.

• In order to consider any therapeutic interest of antibodies, it is necessary to hypothesize that neutralization of residual unbound colchicine may convert a lethal intoxication into a disease compatible with survival, even if a greater part of the toxin has already damaged the organism.

Assessment of the efficacy of toxin detoxification Assessment of the efficacy of toxin detoxification using immunotoxicotherapyusing immunotoxicotherapy

Scherrmann JM. Clin Toxicol 1989

To test the reversal of toxicity

To test the sequestration effect

To test the best fragments and administration conditions

In vitroIn vitro experimental studies (1) experimental studies (1)

Wolf AD. J Biol Chem 1980

Partial reversion of colchicine-induced inhibition of tubulin polymersiation using colchicine-sepcific antibodies

In vitroIn vitro experimental studies (2) experimental studies (2)

• A tightly bound intracellular toxin was extracted with high-affinity antibodies

at a rate depending on its dissociation rate from its receptors. Kinetics was of

first-order decline with t1/2 ranging from 15.5 to 16.4 h

Colchicine neutralization with antibodies has proven to be effective. Although intracellular binding of colchicine to microtubules was expected to limit immunotoxicotherapy efficacy, a reversible effect on microtubules was found.

• Colchicine-induced polyploidy and chromosomal aberrations in a model of

Chinese hamster ovary cell were reversible with a specific high-affinity

monoclonal antibody, even when administered up to 6 hours after colchicine

exposure Rouan SE. Am J Pathol 1990 

Chappey ON. J Pharmacol Exp Ther 1995

In vivoIn vivo experimental studies (1) experimental studies (1)

Anti-colchicine active

immunization of rabbits:

- Protective effects against 3-

mg/kg colchicine (> LD50), with an

antibody titer-dependent

response.

- Effective trapping of colchicine

Scherrmann JM. Clin Toxicology 1989

Biexponential - t1/2: 12h

Unbound colchicine < 0.5%

In mice, colchicine-specific goat IgG (1/2-1/8 molar dose) favorably

improved outcome when previously (90 min) receiving IP 3.8-mg/kg

colchicine lethal dose.

In vivoIn vivo experimental studies (2) experimental studies (2)

In mice, colchicine-specific IgG administration, even after colchicine

distribution phase, significantly decreased mortality rate.

Terrien N. Toxicol Appl Pharmacol 1990

Sabouraud AE. Toxicology 1991

Colchicne Fab fragments-related alteration in kineticsColchicne Fab fragments-related alteration in kinetics

In rabbits: Fab infusion 1.5 h after 0.1-mg/kg colchicine, over 0.25 h, 1/2-molar dose

Colchicine concentration x10-16 within 15 min Total plasma AUC x20

Undetectable free plasma fraction over a period of 2 h

• Decrease of the VD /24

• Decrease of total clearance: /17• Reduction of biliary excretion: - 80% • Reduction of the dose fraction excreted in urine: 9% versus 38%

In mice: Fab induced

• Decrease in colchicine VD

• Colchicine sequestration in intravascular spaces • Decrease of colchicine concentrations in most tissues

Tissue extraction & elimination:

Sabouraud AE. J Pharm Pharmacol 1992

Sabouraud AE. J Pharmacol Exp Ther 1992

Case report of a severe human colchicine Case report of a severe human colchicine overdose overdose

treated with colchicine-specific Fab fragments (1)treated with colchicine-specific Fab fragments (1)

25-year-old woman, suicidal attempt, 0.96 mg/kg (60 mg) colchicine. - Call: 24 h after ingestion due to severe GI pain- On the scene: HR 110 /min, unrecordable BP

500 ml colloid and 10 mg/kg/min dobutamine

- ICU: 38.8 °C, BP 110/80 mmHg, HR 110 /min, RR 60 /min, Creatinine 140 µmol/l, WBC 69,300 /mm3, platelet 268,000 /mm3, PT 14%, V <5%

pH 7.38, PaCO2 28 mmHg, PaO2 86 mmHg (O2 3 l/min)

Chest X-Rays: pulmonary edemaPlasma colchicine: 24 ng/ml (RIA)

Gastric lavage + activated charcoalCrystalloid (4400 ml) and colloid (1100 ml) Deterioration of hemodynamic status despite dobutamine

Baud FJ. N Engl J Med 1995

Case report (2)Case report (2)

Baud FJ. N Engl J Med 1995

-45 min -15 min +1H +12H

PAP (mmHg) 23 18 13 11PCWP (mmHg) 11 11 1 3SBP (mmHg) 76 78 88 90HR (/min)Cardiac Index (l/min/m2) 2.01 2.21 5.70 4.52Resistance (dyn.sec.cm-5/m2) 2070 2066 954 1227

AV difference in O2 11.3 10.1 4.2 4.7

Plasma lactate (mmol/l) 5.4 ND 4.3 2.7

Dobutamine (µg/kg/min) 8 16 24 8Dopamine (µg/kg/min) - - - 3Norepinephrine (mg/h) - - - 0.5

Case report (3)Case report (3)

Infusion of goat colchicine-specific Fab fragments H36 after ingestion

• Antiserum derived from goats immunized with a conjugate of colchicine + serumalbumine

• Affinity: 2 x 1010 M-1

• No separation of colchicine-specific Fab fragments (7.5%) from others

• Dosage: 480 mg (6.4 g Fab fragments) in 160 ml240 mg over 1-hour period240 mg over 6 hours

Baud FJ. N Engl J Med 1995

Case report (4)Case report (4)

Baud FJ. N Engl J Med 1995

-45 min -15 min +1H +12H

PAP (mmHg) 23 18 13 11PCWP (mmHg) 11 11 1 3SBP (mmHg) 76 78 88 90HR (/min)Cardiac Index (l/min/m2) 2.01 2.21 5.70 4.52Resistance (dyn.sec.cm-5/m2) 2070 2066 954 1227

AV difference in O2 11.3 10.1 4.2 4.7

Plasma lactate (mmol/l) 5.4 ND 4.3 2.7

Dobutamine (µg/kg/min) 8 16 24 8Dopamine (µg/kg/min) - - - 3Norepinephrine (mg/h) - - - 0.5

Case report (5)Case report (5)Toxicodynamics:

• Significant arterial vasodilatation within 30 min.• Marked improvement in all indexes of tissue perfusion.• Withdrawal of inotropic drugs within 36 h.

• ARDS took longer to subside.•  No prevention of the delayed occurrence of bone marrow aplasia, complete hair loss, and transient peripheral neuropathy.

• No adverse effect (hypersensitivity, serum sickness) or recurrence of toxic signs

Survival with the well-known sequential phases however with a shortened duration

Baud FJ. N Engl J Med 1995

Case report (6)Case report (6)

Baud FJ. N Engl J Med 1995

Toxicokinetics: •  total plasma concentration (x6: 12 to 122 ng/ml), as soon as 10 min after infusion, whereas free concentration became undetectable

• urinary excretion (x6), initially bound to Fab fragments Renally-excreted colchicine: 5.2 mg

• Neutralized colchicine = 3.7 mg / 9 mg of present colchicine

•  Fab/total colchicine ratio: 4 (0-1 h), 2 (1-7h) and <1 (>7h)

T1/2: 25 h

Case report (7)Case report (7)

The patient’s clinical improvement was explained by the direct effects of colchicine-specific Fab fragments:

• Redistribution from tissues into plasmaIncrease in plasma concentration

• Sequestration into plasma compartmentHigh affinity of Fab to colchicine

• Immunoneutralization of the effectsDecrease of protein-unbound colchicine (during 7h-infusion) and subsequent partial rebound (H12)

Baud FJ. N Engl J Med 1995

The situation today?The situation today?

Solved problems:• Specific antibody development is difficult• Fragmentation technique is a complex procedure• Large amount of fragments is required for a stoechiometrical neutralization

To date, no commercial preparation of colchicine-specific Fab fragments is available.

Why ?

Unsolved problems:- Inadequation between development costs and potential prescriptions (orphan)- Difficulties (legislation and bureaucracy) to organize an European trial

Further considerations for colchicine Further considerations for colchicine immunotoxicotherapyimmunotoxicotherapy

Regarding the indications and time to administer:• Immediate or potentially life-threatening intoxication• Concept of critical dose (when a risk of mortality occurs)• Challenge the classical thinking of the necessity of rapid administration of the total Fab dose to improve poisoning.

Regarding the minimal efficient dose to administer:• Optimal amount to neutralize = total ingested dose - critical dose• Inframolar neutralization may be efficient as in animal reports. • Removal of a modest portion of colchicine may dramatically improve outcome.

Regarding the therapeutic action:Further studies are needed to determine the Fab effect on bone marrow aplasia.

Baud FJ. Arch Toxicol Suppl 1997

Conclusions:Conclusions:

• Coclchicine-specific Fab fragments may be useful to complete the supportive care in the most severe colchicine poisonings.

• However, to date, these fragments are still not commercially available.

• Until issues of cost and supply are worked out, coclchicine-specific Fab fragments remain a dream in the areas where poisonings are frequent and specific therapy urgently required.