Cyanokit - Pharmaceutical Information, Clinical Trials, Detailed Pharmacology, Toxicology.
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Cyanokit - Scientific Information

Manufacture: EMD Serono, Inc
Country: Canada
Condition: Cyanide Poisoning, Schilling Test, Transcobalamin II Deficiency, Vitamin B12 Deficiency
Class: Antidotes, Vitamins
Form: Intravenous (IV), Powder
Ingredients: Hydroxocobalamin

Pharmaceutical Information

Drug Substance

Proper name: Hydroxocobalamin
Chemical name: cobinamide, dihydroxide, dihydrogen phosphate (ester), mono
(inner salt), 3’-ester with 5,6-dimethyl-1-α-D-ribofuranosyl-1H-
benzimidazole
Molecular formula and molecular mass: C62H89CoN13O15P
1346.4 atomic mass units
Structural formula:

Physicochemical properties: Hydroxocobalamin is the hydroxylated form of vitamin B12
and is a large molecule in which a cobalt ion is coordinated
in four positions by a tetrapyrol (or corrin) ring. It is a
hygroscopic, odorless, dark red, crystalline powder that is
freely soluble in water and ethanol, and practically
insoluble in acetone and diethyl ether.

When reconstituted in 100 mL (2.5 g vial) or 200 mL
(5 g vial) of diluent, hydroxocobalamin gives a dark red
injectable solution at 25 mg/mL with a pH that ranges from
3.0 to 6.0.

Clinical Trials

Due to ethical considerations, no controlled human efficacy studies have been performed. A controlled animal study demonstrated efficacy in cyanide-poisoned adult dogs [see DETAILED PHARMACOLOGY].

Demographics and Trial Design

Baud Study 1 was a prospective, uncontrolled, open-label study that was carried out in 69 subjects who had been exposed to smoke inhalation from fires. Subjects had to be over 15 years of age, present with soot in the mouth and expectoration (to indicate significant smoke exposure), and have altered neurological status. Cyanide exposure was confirmed a posteriori by assay of the blood sample taken at the fire accident scene. The median hydroxocobalamin dose was 5 g with a range from 4 to 15 g.

Table 1 Summary of Patient Demographics for Baud Study 1 in Cyanide Exposure from Smoke Inhalation
Study # Trial design Dosage, route of
administration and
duration
Number of
study
subjects
Median age
(Range)
Gender
Baud
Study 1
Phase III, prospective,
open label with
subsequent
retrospective
collection of
additional data
5.0 g
hydroxocobalamin
IV infusion (15-30
min)
Repeated dose allowed
up to a max of 15.0 g
N = 69 44 years
(20-94 years)
Female:
n = 36
(52.5%)
Male:
n = 33
(47.8%)

Study Results

Fifty of 69 subjects (73%) survived following treatment with hydroxocobalamin. Fifteen patients treated with hydroxocobalamin were in cardiac arrest initially at the scene; 13 of these subjects died and 2 survived.

Of the 42 subjects whose initial plasma cyanide levels prior to treatment were considered to be potentially toxic, (i.e. ≥39 μmol/L) a total of 28 (67%) survived. Of the 19 subjects whose pretreatment cyanide levels were considered potentially lethal (i.e. ≥100 μmol/L), 11 (58%)survived.

Of the 50 subjects who survived, 9 subjects (18%) had neurological sequelae at hospital discharge. These included dementia, confusion, psychomotor retardation, anterograde amnesia, intellectual deterioration moderate cerebellar syndrome, aphasia, and memory impairment.

In addition to the Baud 1 Study, two retrospective, uncontrolled studies were carried out in subjects who had been exposed to cyanide from fire or smoke inhalation where subjects were treated with up to 15 g of hydroxocobalamin. Survival in these two studies (in patients for whom outcome was known) was 34 of 58 (59%) for one study, and 30 of 72 (42%) for the second. An additional retrospective, uncontrolled study was carried out in 14 subjects who had been exposed to cyanide from sources other than from fire or smoke (i.e., ingestion or inhalation) where subjects were treated with 5 to 20 g of hydroxocobalamin. Ten of 14 subjects (71%) survived following administration of hydroxocobalamin.

Combined Clinical Experience

Across all of the clinical trials, a total of 245 patients with suspected or known cyanidepoisoning were included in the clinical studies of the efficacy of hydroxocobalamin as an antidote. Of the 213 patients in whom the outcome was known the survival was 58%. Of the 89 patients who died, 63 were initially found in cardiac arrest, suggesting that many of these patients had almost certainly suffered irreparable brain injury prior to administration of hydroxocobalamin. Among 144 patients not in initial cardiac arrest whose outcomes were known, 118 (82%) survived. Furthermore, in 34 patients with known cyanide concentrations above the lethal threshold (≥ 100 μmol/l), 21 (62%) survived following treatment with hydroxocobalamin.

Administration of hydroxocobalamin was generally associated with a normalisation of blood pressure (systolic blood pressure > 90 mmHg) in 17 of 21 patients (81%) who had low blood pressure (systolic blood pressure > 0 and ≤ 90 mmHg) after exposure to cyanide. Where neurological assessment over time was possible, (96 patients of the 171 patients who presented with neurological symptoms prior to hydroxocobalamin administration), 51 (53%) patients receiving hydroxocobalamin showed improvement or a complete restoration.

Elderly: Approximately 50 known or suspected cyanide victims aged 65 or older received hydroxocobalamin in clinical studies. In general, the effectiveness of hydroxocobalamin in these patients was similar to that of younger patients.

Paediatric Patients

Data are available from a database compiled in France with 56 paediatric patients. The mean age of the paediatric patients was about six years and the mean total dose of hydroxocobalamin was about 120 mg/kg body weight. The survival rate of 41% depended very much on the clinical situation. Out of the 20 paediatric patients without initial cardiac arrest, 18 (90%) survived, of whom four recovered with sequelae. In general, the effectiveness of hydroxocobalamin in paediatric patients was similar to that of adults.

Detailed Pharmacology

Animal Pharmacology

Pharmacodynamics

Efficacy Study in Dogs

Evidence of the effectiveness of hydroxocobalamin for treatment of cyanide poisoning was obtained primarily from studies in animals due to the ethical considerations of performing such controlled studies in humans.

The effectiveness of hydroxocobalamin was examined in a controlled study in cyanide-poisoned adult dogs. Dogs were poisoned by intravenous administration of a lethal dose of potassium cyanide. Dogs then received vehicle (sodium chloride 9 mg/ml), 75 mg/kg or 150 mg/kg hydroxocobalamin, administered intravenously over 7.5 minutes. The 75 mg/kg and 150 mg/kg doses are approximately equivalent to 5 g and 10 g of hydroxocobalamin, respectively, in humans, not only on a body weight basis but also on Cmax basis of total cobalamins-(III).

Survival at hour 4 and at day 15 was significantly greater in the 75 mg/kg and 150 mg/kg hydroxocobalamin dose groups compared with dogs receiving vehicle alone (Table 2):

Table 2 Survival of Cyanide-Poisoned Dogs
Parameter Treatment
Cyanokit
Vehicle
N = 17
75 mg/kg
N = 19
150 mg/kg
N = 18
Survival at Hour 4, n (%) 7 (41) 18 (95)* 18 (100)*
Survival at Day 15, n (%) 3 (18) 15 (79)* 18 (100)*

*p<0.025

Histopathology revealed brain lesions that were consistent with cyanide-induced hypoxia. The incidence of brain lesions was markedly lower in dogs having received 150 mg/kg hydroxocobalamin than in dogs having received 75 mg/kg hydroxocobalamin or vehicle. The rapid and complete recovery of haemodynamics and subsequently of blood gases, pH, and lactate after cyanide poisoning likely contributed to the better outcome of the hydroxocobalamin-treated animals. Hydroxocobalamin reduced whole blood cyanide concentrations from about 120 nmol/mL to 30-40 nmol/mL by the end of the infusion compared with 70 nmol/mL in dogs receiving vehicle alone.

Hemodynamic Effects of Hydroxocobalamin

The possibility of NO-trapping as the cause of the hemodynamic effects of hydroxocobalamin was investigated in anesthetized rabbits. Firstly, rabbits were infused with 75 mg/kg hydroxocobalamin or vehicle in the absence or presence of L-Nω-nitro-L-arginine methyl ester (L-NAME), an inhibitor of endothelial NO-synthase. Then, since NO-synthase inhibition by LNAME itself is known to increase blood pressure, L-NAME was replaced by equi-pressor doses of angiotensin II (ANGII) to elucidate a possible interference of the elevated blood pressure with the hemodynamic effects related to hydroxocobalamin.

Hydroxocobalamin infused at a dose of 75 mg/kg caused moderate hemodynamic effects compared to vehicle alone which had no effect. Mean arterial pressure (MAP) and total peripheral resistance (TPR) increased, whereas heart rate (HR) and cardiac ouptut (CO) decreased. L-NAME caused prominent hemodynamic effects, i.e., an increase in MAP and TPR, and a decrease of CO. HR decreased only slightly. The hemodynamic effects of L-NAME were maintained during the duration of the experiment. Infusion of hydroxocobalamin in the presence of L-NAME caused no further hemodynamic changes.

The infusion of ANGII increased MAP and TPR, while CO decreased. HR did not change. The hemodynamic effects of ANGII were maintained during the duration of the experiment. The increases in MAP caused by L-NAME and ANGII were comparable. However, in contrast to LNAME, ANGII did not prevent the hemodynamic effects of hydroxocobalamin, i.e., MAP and TPR increased again, whereas CO decreased. HR decreased only slightly.

In summary, in anesthetized rabbits, in the presence of NO-synthase inhibition by L-NAME, the moderate hemodynamic effects of hydroxocobalamin, in particular the increase in MAP and TPR, were prevented. The hemodynamic changes produced by L-NAME itself were not of importance for the missing effect of hydroxocobalamin, because an equi-pressor dose of ANGII applied instead of L-NAME did not prevent the hemodynamic changes related to the hydroxocobalamin. Thus, it can be concluded, that in anesthetized rabbits the moderate hemodynamic effects of hydroxocobalamin are the consequence of the NO-scavenging property of the compound, which may lead to vasoconstriction of conductance and resistance vessels.

Pharmacokinetics

A dose proportional increase in AUC was demonstrated for both free cobalamins-(III) and total cobalamins-(III) in dogs and humans following IV administration. The mean predominant halflives were 6 and 8 hours in the dogs, and 28 and 31 hours in humans for free and total cobalamins-(III), respectively. Total body clearance of free cobalamins-(III) in dogs was 0.38 to 0.50 L/h/kg and was approximately 6 to 7-fold higher compared to total cobalamins-(III).

As hydroxocobalamin is administered directly into the compartment where it primarily exerts its detoxifying action, i.e., into the blood, distribution studies were focused on plasma protein binding and organ distribution of hydroxocobalamin was not investigated.

Distribution of hydroxocobalamin is largely influenced by the coordinative binding to proteins. During the plasma protein binding, hydroxocobalamin reacts by replacing the coordinatively bound hydroxo ligand by accessible histidine- and thiol-groups of the proteins to form various cobalamin-(III) complexes. Equilibrium concentrations were reached only within 1 to 2 hours after infusion.

The protein binding of hydroxocobalamin was investigated ex vivo during toxicokinetic studies in rats and dogs and during a clinical Phase I study by quantification of free cobalamins-(III). The protein binding at equilibrium exhibited a pronounced species-dependent difference, as evaluated from the average free fraction of cobalamins-(III), with humans showing the lowest free fraction of about 5%, rats showing the highest free fraction in the range of 24% and dogs in the range of 16%. This species difference may be related to the reactivity of accessible histidine and cysteine residues that is dependent on the tertiary structure of the respective plasma proteins.

Metabolism of hydroxocobalamin is mainly characterized by exchange reactions of the hydroxoligand with other physiological ligands that exhibit high affinity to the cobalamin-(III). Thus, the binding of hydroxocobalamin to plasma proteins may be regarded as reversible metabolism. In addition, hydroxocobalamin forms low molecular derivatives with coordinating physiological compounds such as thiols, histidine, thiocyanate, and others. Several unidentified high pressure liquid chromatography (HPLC) peaks have been detected in in vitro incubates of plasma with hydroxocobalamin.

Hydroxocobalamin is known to react with cyanide forming cyanocobalamin in vivo, even at the very low physiological concentrations of both reactants. Due to the extremely high complex stability (Kdiss ≈ 10-12 M-1), cyanocobalamin is regarded as a physiological end product of hydroxocobalamin especially during cyanide intoxication. This reaction is known to proceed very rapidly with reaction rates of 660 M-1s-1 and 320 M-1s-1 for CN- and HCN, respectively.

Excretion of hydroxocobalamin occurs mainly via the renal route. In the literature, it is reported that hydroxocobalamin is excreted in the urine in dogs. In human volunteers, the mean renal clearance of hydroxocobalamin (2.5 g to 10.0 g) amounted to 58% to 74% of total clearance.

Toxicology

The toxicological profile of hydroxocobalamin has been investigated in rats and dogs. The studies included single dose studies in rats and dogs, repeat dose toxicity studies in dogs comprised a 3-day study and a 4-week study. Additionally, cyanocobalamin, the detoxification product of cyanide upon reaction with hydroxocobalamin, was tested in a 14-day repeat dose study in dogs. The significant toxicity studies are presented in Table-3

Table 3 Summary of Toxicology Studies
Species/
Strain
Sex
No. per
group
Route/
Duration
Doses
(mg/kg)
Key Results
Single-Dose Toxicity in Dogs
Dog /
Marshall
beagle
4M /4F
IV/
single
dose
150
300
1200
NOAEL = 300 mg/kg
Clinical symptoms: reddish urine, skin and mucous membranes. Wrinkles and/or wheals in head region and swollen ears seen transiently in 1 low dose M and most dogs in 300 or 1200 mg/kg groups. Sharp drop in platelets in some of 1200 mg/kg group. Transient increase in RBC, hematocrit and hemoglobin all groups for up to 1 h after end of dosing. Minor increase in ALT and AST in 2 low dose males and 1 mid dose M. Slight to pronounced increases in ALT and AST in individual dogs in 1200 mg/kg group. Minor increase in ALP at 300 mg/kg and higher. All hematological and clinical chemistry effects were reversible at the end of 14 day recovery period.
Histopathology: liver: sinus edema, activated Kupffer cells, acute small necroses, and a slightly increased incidence of microgranulomas seen 24 h after dosing at 300 or 1200 mg/kg. Kidneys: tubular dilation, tubular cast formation, and focal interstitial and medullary hemorrhages seen in the kidneys in high dose dogs 24 hours after dosing, crystalline intracytoplasmic depositions seen in high dose M and 1 low dose F. Other organs: vasculitis in the fat tissue around the oviduct in one high dose F
At the end of two week treatment free period following changes were observed: liver: deposition of an eosinophilic material in Kupffer cells in all treated groups, kidneys: tubular dilation, tubular casts in high dose dogs, bone marrow: single cell necrosis in most likely macrophages in all treated groups., liver and bone marrow changes were evaluated to be due to excessive storage
Repeat-Dose Toxicity in Dogs
Dog /
Marshall
beagle
2M/2F
IV/ 3
days
300
600
1200
Clinical symptoms: reddish urine, skin and mucous membranes. Wrinkles and/or wheals in head region and swollen ears seen in all treated dogs. Urticaria like signs observed for up to 6 h in high dose dogs. Slight decrease in lymphocytes in 1 low dose F, 1 mid dose F and all high dose dogs. Decreased platelet count in 3 high dose dogs, marked increased in erythrocyte sedimentation rate after 1 and 2 h in 2 high dose F. Moderate increase in ALT and AST in all groups overall but pronounced in individual dogs. Marked increase in ALP in 1 low dose and 2 high dose F. Potassium levels slightly decreased in 1 low dose F and all mid and high dose dogs.
Necropsy: discoloration of tissues rich in elastic and collagen fibers. Histopathology: Hepatocellular necroses seen in all dose groups. Dose dependent deposition of eosinophilic material in Kupffer cells in all dose groups. Deposition of a brown pigment in 3/4 dogs in each dose group. Dilation of kidney tubules with proteinaceous cast formation and necrosis of single tubular cells in 600 and 1200 mg/kg groups. Focal, segmental formation of thrombi in the glomeruli, focal plasma leakage of arteries in kidney cortex, edema of tubular cells and focal hemorrhages in the 1200 mg/kg group. A few single cell necroses seen in bone marrow in 1/4 , 3/4 and 4/4 dogs in low, mid and high dose groups respectively. Minimal to mild focal degeneration of myofibers diagnosed in the left heart ventricle in 2 high dose dogs. Early formation of thrombi in spleen in 3 high dose dogs and perivascular hemorrhages affecting vessels in the hilus region in 2 dogs in the 1200 mg/kg group. Early formation of thrombi in 1 mid dose dog unilaterally in the ciliary body of the eye and 2 high dose dogs in the gall bladder and the fat tissue around the oviduct. Minimal deposition of an eosinophilic material in sinus macrophages of lymph nodes in some low, mid, and high dose animals.
Dog/
Marshall
beagle
3M/3F
(75, 150)
5M/5F
(300)
IV/ 4
weeks
75
150
300
Clinical symptoms: reddish urine, skin and mucous membranes in all dose groups throughout the treatment period but reversible by end of 8 week recovery period. Swollen ears, wrinkles and/or wheals in the head region in the mid and high dose groups. Isolated cases of vomiting and/or salivation seen in some high dose dogs. Reduced platelet count in 1 low dose F and 1 high dose M in week 4.
Dose-dependent increase in ALT in all dose groups. Increased AST in all treated M and 1 high dose F. Increased ALP in 2 high dose M. A higher incidence of oxalate crystals observed in the urine of all treated compared to control dogs in week 4. All of these changes were reversible by end of 8 week recovery period.
Necropsy: discoloration of tissues rich in elastic and collagen fibers in 3/6 low dose dogs and in all mid and high dose dogs, reversible by the end of the 8 week recovery period. Liver, spleen, and kidney weights increased in all groups at the end of the treatment period. Kidney and spleen weight changes comparable to the control group by the end of the recovery period but liver weights in high dose F remained high.
Histopathology: deposition of eosinophilic material in sinusoidal Kupffer cells and in hepatocytes in all dose groups accompanied by focal perivascular inflammatory cell infiltration. Minimal to moderate degeneration of hepatocytes, mild single cell necrosis, bile duct proliferation, and fibrosis seen in high dose groups. After 8 week recovery liver changes were not completely resolved but incidence and severity were lower. In the kidneys intracytoplasmic deposition of an eosinophilic material seen in the proximal tubules in most treated dogs. Focal basophilia of cortical tubules associated with single cell necrosis in high dose groups. Kidney findings were completely resolved by the end of the recovery period. Dose-dependent single cell necrosis in the bone marrow seen in all dose groups at the end of the treatment period and at a lower incidence and severity at the end of the 8 week recovery period. Activation of lymph follicles seen in the spleen in some high dose dogs, and deposition of an eosinophilic material seen in sinus macrophages in lymph nodes. This was not seen in the recovery group. Focal mononuclear cell infiltrates in the heart seen in 1/6, 2/6, and 4/6 dogs of the low, mid, and high dose groups, respectively and in 2/4 recovery animals. There was no difference in the severity of the alteration. Toxicological significance of the finding is not known..
Reproductive and Developmental Toxicity
Rat /
Wistar
6 F
IP / 12
days
(G6-
G17)
75, 150,
300
Maternal NOAEL < 75 mg/kg, Developmental NOEL = 75 mg/kg.
Dose dependent minimal to mild maternal toxicity in all treated rats. Mortality in some mid dose animals but all high dose animals survived. Reddish urine in all groups and reddening of the skin in two high dose rats. Swelling of the skin in one mid dose rat and five high dose rats. Dose dependent decrease in body weight gain and food consumption in mid and high dose rats.
No apparent treatment-related effect on number of dams pregnant on day 20 of gestation. The mean number of corpora lutea and implantations in all groups were comparable to control. The sex distribution of fetuses was not impaired. Live litter size was slightly reduced in the high dose group. There was a significant increase in the incidence of post-implantation loss. Slightly retarded fetal skeletal ossification and reduced fetal weight in the high dose group. Two high dose and two intermediate dose fetuses, each from a different dam, with treatment related shortened extremities. Both of the intermediate dose fetuses had microcephaly and one also had microphthalmia and micrognathia.
Rabbit /
New
Zealand
white
20F
IV/ 14
days
(G6-
G19)
75, 150,
300
Maternal NOAEL < 75 mg/kg, Developmental NOEL = 75 mg/kg.
Red/purple discoloration of the urine, periorbital membrane and/or injection site and reductions in feed consumption during the postdose period with all dose groups. Body weight gains were reduced during the postdose period at 150 and 300 mg/kg. No treatment related differences in the mean numbers of implantations and live litters size, nor in pre- or post-implantation loss or sex ratio of fetuses. Two dead fetuses in 300 mg/kg group. Increases in fetal gross external, visceral and soft tissue malformations and variations at 150 and 300 mg/kg. Multiple skeletal alterations occurred in increased number of fetuses at 150 and 300 mg/kg group.

Abbreviations: M= male; F = female; IP = intraperitoneal; ALAT = alanine aminotransferase; ASAT = aspartate aminotransferase; IV = intravenous; RBC = red blood cell; ALP = alkaline phosphatase;NOAEL = no observable adverse effect level; NOEL= no observable effect level

Carcinogenesis

Hydroxocobalamin has not been evaluated for carcinogenic potential.

Mutagenesis

Hydroxocobalamin was negative in the following mutagenicity assays: in vitro bacterial reverse mutation assay using Salmonella typhimurium and Escherichia coli strains, an in vitro assay of the tk locus in mouse lymphoma cells, and an in vivo rat micronucleus assay.

Phototoxicity

A single intraperitoneal administration of 75, 150 or 300 mg/kg of hydroxocobalamin to pigmented Long-Evans rats followed one hour later by a single exposure to solar-simulated ultraviolet radiation, did not elicit skin responses or ocular responses indicative of cutaneous or ocular phototoxicity