Flumazenil Injection - Pharmaceutical Information, Clinical Trials, Detailed Pharmacology, Toxicology
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Flumazenil Injection - Scientific Information

Manufacture: Fresenius Kabi USA, LLC
Country: United States
Condition: Benzodiazepine Overdose, Reversal of Sedation
Class: Antidotes
Form: Liquid solution, Intravenous (IV)
Ingredients: Flumazenil, water, methylparaben, propylparaben, sodium chloride, edetate disodium, acetic acid, hydrochloric acid, sodium hydroxide

Pharmaceutical Information

Drug Substance

Proper Name: flumazenil
Chemical Name: ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4] benzodiazepine-3-carboxylate
Molecular Formula and Molecular Mass: C15H14FN3O3;303.3
Structural Formula:


Description: Flumazenil is a white to off-white crystalline powder with a faint odour. It is slightly soluble in methyl alcohol and practically insoluble in water. Melting range is 198-202ºC. pKa = 1.7

Detailed Pharmacology

Receptor Studies

Using 3H-flumazenil, a benzodiazepine antagonist or 3H-clonazepam, a benzodiazepine agonist as radioligands in in vitro binding studies, a variety of receptor agonists showed very similar potency in inhibiting the binding of either ligand.

Ligand IC50 (nmol/L)
3H-flumazenil binding 3H-clonazepam binding
Agonists
- clonazepam
- flunitrazepam
- diazepam
- zopiclone

1.4
4.8
19.5
50

1.2
5.1
13.5
46
Antagonist
- flumazenil

1.3

2.0

Flumazenil also displaced 3H-flunitrazepam under in vitro conditions. The ED50 was 4.0 mg/kg p.o. when mice were sacrificed 15 minutes after the administration of flumazenil.

Autoradiography studies have revealed that while 3H-flunitrazepam binds to both central and peripheral benzodiazepine receptor sites, 3H-flumazenil binds only to central receptor sites. This suggests that flumazenil will antagonize only those effects of the benzodiazepines that are mediated via the central nervous system.

While flumazenil interacts with the same number of benzodiazepine receptor sites as the benzodiazepines, the mode of interaction of the antagonist and agonists differs. Under conditions which alter receptor affinity for agonists, no change is seen in 3H -flumazenil binding. Specifically, (a) in the presence of GABA, benzodiazepine receptor affinity is enhanced for agonists, but remains unchanged for flumazenil; (b) in the presence of photoaffinity labelling, benzodiazepine receptor affinity is attenuated for agonists, but remains unchanged for flumazenil.

In conclusion, similar to benzodiazepine agonists, flumazenil interacts with central benzodiazepine receptors in nanomolar concentrations, the inhibition of agonist binding by flumazenil being competitive. Unlike the agonists, flumazenil is insensitive to GABA or photoaffinity labeling-induced changes in receptor affinity.

Benzodiazepine Antagonist Activity

Flumazenil potently antagonizes the centrally-mediated pharmacological effects of various benzodiazepines. In the table below, only the minimal effective doses are described, but flumazenil exerts its effects in a dose-dependent fashion.

Table 2 Antagonism of Benzodiazepines in Neurological and Behavioural Studies
Test Performed Species Benzodiazepine Flumazenil
Effect Dose mg/kg                 Effect Minimal Effective Dosemg/kg       
Traction test mice muscle
relaxation
diazepam 3 i.p. reversal 0.2 p.o. (ED50)
Pentylenetrazole-induced convulsions mice antagonism of convulsions diazepam 5 i.p. reversal 2.8 p.o. (ED50) at 15 minutes
7.0 p.o. (ED50) at 45 minutes
Hexobarbital-induced loss of righting reflex mice potentiation meclonazepam 1 i.p. reversal 3 p.o.
Locomotor activity rats hypomotility diazepam 30 p.o. reversal 30 p.o.
Open field behaviour rats reduced
exploration
and rearing
meclonazepam 10 p.o. reversal 1 p.o.
Conflict behaviour rats attenuation of conflict diazepam 5 p.o. reversal 10 p.o.
Behavioural observation dogs ataxia meclonazepam 3 p.o.
meclonazepam 10 p.o.
prevention
reversal
10 p.o.
30 p.o.
Behavioural observation squirrel monkeys sedation, anesthesia flunitrazepam 3 i.v.
midazolam 10 i.v.
reversal
reversal
1 p.o.
0.3 i.v.
Cognition mice induction of anterograde amnesia triazolam 1 p.o. reversal 10 p.o.
Electrophysiological studies
"encéphale isolé”
rats reduced cell firing in several central regions midazolam 0.1-10 μmol/kg i.v.
(0.3-3 mg/kg)
recovery of cell firing 10 μmol/kg i.v.

(3 mg/kg)
Acute spinal cats enhancement & prolongation of dorsal root potentials meclonazepam 0.1 i.v. prevention & reversal 1 i.v.
Respiratory study rabbits reduced respiratory min. volume & rate of respiration diazepam 0.1 i.v.
flunitrazepam 0.03 i.v.
reversal
reversal
0.1 i.v.
0.1 i.v.

The ability of flumazenil to antagonize benzodiazepine agonists is specific for this class of drugs. The muscle relaxant, anticonvulsant, and anticonflict effects of phenobarbital, meprobamate, and ethanol were not antagonized by flumazenil. Flumazenil was also inactive against scopolamine or hypercapnia-induced anterograde amnesia and morphine-induced respiratory depression.

Flumazenil-induced Withdrawal

Flumazenil, administered i.v., i.m., or p.o., elicited typical benzodiazepine withdrawal signs in mice, rats, cats, and squirrel monkeys, following chronic administration of benzodiazepines (12 to 35 days). The symptoms included emesis, vocalization, tremors, rigidity, and convulsions.

The type of withdrawal symptoms and their intensity depended upon the dose and duration of benzodiazepine treatment, as well as the time of flumazenil administration vis à vis the last dose of the benzodiazepine.

Intrinsic Activity

Flumazenil did not affect normal behaviour in rats, dogs, or squirrel monkeys in doses of up to 100 mg/kg. Similarly, motor activity, conditioned avoidance behaviour, conflict behaviour in rats and continuous avoidance behaviour in squirrel monkeys remained unchanged when flumazenil was given in doses of up to 100 mg/kg p.o. These findings indicate that flumazenil is devoid of benzodiazepine agonist activity at doses that are substantially higher than those which exert antagonist activity.

Inverse Agonist Activity

A group of benzodiazepine receptor ligands, classified as "inverse agonists" cause opposite effects to those of the benzodiazepine receptor agonists, namely they produce convulsions and anxiety in appropriate animal models.

Flumazenil did not induce convulsions, except at sublethal doses. However, it did exert weak anxiogenic activity in several behavioural animal models, namely in the “social interaction” and “conditioned spatial aversion” tests, as well as in various conflict situations. Active doses ranged from 4 to 30 mg/kg i.p. or p.o.

Cardiovascular Effects

Flumazenil did not affect blood pressure or heart rate either in spontaneously hypertensive rats (maximum dose 100 mg/kg p.o.) or in renal hypertensive dogs (maximum dose 30 mg/kg p.o.).

Toxicology

Acute Toxicity
Route of Administration Species Sex           LD50 (mg/kg) Symptoms
i.v. mice

rats
male
female
male
female
159-168
132-159
119-134
161-182
Deaths, preceded by tonic-clonic convulsions, occurred within 30 minutes of dosing. Surviving animals were hypoactive, and manifested respiratory depression and increased muscle tone.
i.p. mice

rats
male
female
male
female
> 2,000
1,500
2,200
Deaths occurred within three days of dosing. Most animals were hypoactive, and manifested catatonia, tremors, salivation, lacrimation and respiratory depression.
s.c. mice male & female > 1,000
p.o. mice

rats
rabbits
male
female
male & female
male & female
2,500
1,300
4,200
2,000

Intravenous Pyramiding Dose Toxicity in Dogs

Three groups of four dogs each (2/sex) received (a) flumazenil at doses of 0.01, 0.03, 0.1, and 0.3 mg/kg; (b) pyramiding doses of the vehicle (0.1, 0.3, 1.0, and 3.0 mL/kg) and; (c) equivalent volumes of physiological saline. The dogs were dosed twice a week over a two-week period. All dogs survived the pyramiding doses of flumazenil and were essentially asymptomatic throughout the study. Flumazenil did not affect body weight, food intake, hematological or clinical chemical parameters.

Long-Term Toxicity Studies

Two-Week Intravenous – Rats

Flumazenil was injected i.v. into the tail vein of rats (8/sex/group) at doses of 0, 1, 3, and 10 mg/kg/day.

Tissue irritation at the injection site was pronounced and dose-related. At the low dose, local tolerance was acceptable. At the mid-dose, all rats had swollen and reddened tails beginning with the third dose and continuing to the end of the study. At the high dose, intolerance at the injection site was severe. Hematomas occurred in five of eight males and one female rat. Two of eight male rats in the high-dose group also showed ulceration of the tail, and in three male rats the administration of the drug had to be performed intraperitoneally on the fifth day. In general, female rats tolerated the treatment better than male animals. Pronounced bleeding at the injection site was also observed in most of the mid- and high-dose animals beginning about the fifth day of the study.

At the 10 mg/kg dose, male rats gained weight at a slower rate than male controls; this effect was considered to be treatment-related. A similar effect in female rats was equivocal.

Two-Week Oral – Rats

Flumazenil was administered by gavage to rats (8/sex/group) at doses of 0, 5, 25, and 150 mg/kg/day. The drug was given for 15 or 16 consecutive days to female and male rats, respectively.

Flumazenil was devoid of toxic effects at the doses studied.

Two-Week Intravenous – Dogs

Flumazenil was injected i.v. to beagle dogs (2/sex/group) at doses of 0, 1, 3, and 10 mg/kg/day.

The mid-dose produced drowsiness and the high dose produced drowsiness and ataxia; both effects were observed following dosing. In the course of the study, a slight tolerance did develop to these effects.

Injections were poorly tolerated in the dogs receiving 10 mg/kg of flumazenil, due to hardened and thrombosed veins and a strong defence reaction from the animals.

In the high-dose group, reticulocytes were significantly increased in the second week of the study in comparison to the control group. Platelets were decreased in all treated groups vis à vis baseline values, but the changes were not dose-related. A statistically significant increase in the relative liver weights was noted in the high-dose group, this effect was considered to be treatment-related.

Two-Week Oral – Dogs

Flumazenil was administered in capsules to beagle dogs (2/sex/group) at doses of 0, 5, 20, and 80 mg/kg/day for 15 consecutive days.

Slight diarrhea was noted at the 20 mg/kg dose and marked diarrhea (sometimes bloody) at the 80 mg/kg dose. Mean spleen weights were decreased and mean liver weights increased in all flumazenil-treated dogs. At necropsy, the tunica mucosa of the colon was more convoluted in high-dose animals than in controls.

Four-Week Intravenous – Rats

Flumazenil was injected i.v. into the tail vein of rats (12/sex/group) at doses of 0, 1, 3, and 10 mg/kg/day.

Local tolerance to the i.v. injections was poor and the degree of swelling and pain was dose-related. At the high-dose, the route of administration had to be switched from intravenous to intraperitoneal about 15 days after the beginning of the study.

In male rats, there was a dose-related weight gain deficit. Although the mean values remained within the normal range, WBC counts decreased in male rats in a dose-related fashion at week 4. The decrease seen in the high-dose group was statistically significant. Both absolute and relative liver weights were increased in high-dose female rats. Perilymphadenitis was seen in high-dose males and females, this might have been due to the intraperitoneal injections. One high-dose female rat had a moderate degenerative change in the retina.

Four-Week Intravenous – Dogs

Flumazenil was injected i.v. to beagle dogs (2/sex/group) at doses of 0, 1, 3, and 10 mg/kg/day.

Mid-dose dogs were sedated and high-dose dogs showed both sedation and ataxia. Tolerance did not develop to these effects. WBC counts decreased slightly. While the mean values were within normal range, in a few dogs they fell below normal. Local tolerance (injection site) was poor in the high-dose groups as also shown by high inflammation scores.

Thirteen-Week Oral – Rats

Flumazenil was administered in the diet to rats (18/sex/group) at doses of 0, 5, 25, and 125 mg/kg/day.

In female rats, liver weights were somewhat elevated in the high-dose group, and thyroid weights slightly decreased in a dose-related fashion.

Thirteen-Week Oral – Dogs

Flumazenil was administered in capsules to beagle dogs, (3/sex/group), seven days/week, at doses of 0, 5, 20, and 80 mg/kg/day.

Slight sedation, lasting 1-3 hours following dosing was noted in the high-dose group; tolerance did not develop to this effect. Weight gain in high-dose animals was somewhat attenuated when compared to controls. At week 12, heart rate was increased in high-dose dogs, vis à vis both baseline and control dogs. There was a dose-related decrease in both absolute and relative spleen weights.

Twelve-Month Oral – Rats

Flumazenil was administered in the diet to rats (20-30/sex/group) at doses of 0, 6, 20, and 125 mg/kg/day. An interim sacrifice of ten control rats (5/sex) and ten rats from the high-dose group (5/sex) was carried out at six months.

Hemoglobin, erythrocyte and hematocrit values were slightly lower in treated male animals than in controls, throughout the study. In females, these parameters were reduced only at six months.

At six months, both absolute and relative thyroid weights in high-dose males, and liver weights in high-dose females were significantly increased. Histopathological evaluation revealed a slight to moderate congestion in the liver of all females treated with the high dose.

At twelve months, absolute and relative thyroid weights were slightly decreased in low- and mid-dose males, but increased in high-dose animals. Liver weights and histopathological findings were similar in treated and control rats.

Mutagenicity

Flumazenil had no mutagenic activity in six out of seven mutagenicity assays (Ames test, Treat and Plate test, gene mutation, in vivo and in vitro clastogenicity and in vivo DNA repair). In a UDS assay, there was a dose-dependent unscheduled incorporation of 3H- thymidine in nuclear DNA of rat hepatocytes after treatment with flumazenil concentrations of 252, 504, and 1,010 mcg/mL for eighteen hours. However, the increase could only be shown at substance concentrations that were also cytotoxic. Since there were no effects in the absence of cytotoxicity, interactions between cytotoxic, and DNA-damaging effects, resulting in repair processes, cannot be excluded.

Reproduction and Teratology

Fertility and General Reproductive Performance

In a Segment I study, flumazenil was administered by gavage to rats in doses of 0, 15, 45, and 125 mg/kg/day. Thirty-two males per group were treated for 10 weeks prior to mating and during the mating period. The treatment of 32 females per group started two weeks prior to mating and continued through the gestation and lactation periods. No mortality or adverse effects were observed on parental animals.

Mating success, gestation length, and outcome of pregnancy was not influenced by treatment either in the parental or in the Fl-generation.

Gestational parameters such as the number of corpora lutea, implantations, resorptions, and number of pups born alive were comparable to concurrent and historical control data in the parental as well as in the Fl-generation.

Weight gain of Fl-pups was normal in the low- and mid-dose groups but slightly decreased in the high-dose group. This decrease became statistically significant at weaning (lactation day 23). The viability of pups from the F1- and F2-generation was not affected by treatment.

Teratology – Rats

In a Segment II study, flumazenil was administered by gavage to rats at doses of 0, 15, 50, and 150 mg/kg/day. The test drug was administered to 40 mated female rats/dose from day 7 to day 16 of gestation inclusively; control rats received a similar volume of the vehicle. The study included rearing of the offspring until weaning in order to determine the postnatal effects of prenatally administered flumazenil.

Weight gain by the dams was not impaired and there was no evidence for an adverse effect on the various reproductive parameters (i.e., resorption rate, number of dead fetuses, mean body weight of fetuses, mean crown/rump length and duration of the gestation period). During the postnatal period, the body weight of pups increased uniformly in all dose groups and the incidence of pup mortality was not increased in any of the treatment groups.

External, skeletal, and soft tissue examinations of the fetuses gave no indication of treatment-related teratogenicity. Five fetuses from a single litter in the high-dose group showed multiple skeletal abnormalities (i.e., shortened, poorly ossified, and deformed long bones in fore and hind limbs, missing toes, and enlarged heads).

Teratology – Rabbits

In a Segment II study, flumazenil was administered by gavage to rabbits at doses of 0, 15, 50, and 150 mg/kg/day. The test drug was administered to 20 mated female rabbits/dose from day 7 to day 19 of gestation, inclusively; control rabbits received a similar volume of the vehicle. Weight gain of the does during the gestation period, mating success, mean number of corpora lutea, and mean number of implantations were not impaired in any of the groups. The resorption rate noted in the high-dose group (1.6 per pregnant female), was significantly greater than that for the concurrent control (0.7 per pregnant female), but was within the range for historical controls. Examinations of fetuses for malformations revealed no evidence for a teratogenic effect of flumazenil up to a dose of 150 mg/kg/day.

Perinatal and Postnatal – Rats

In a Segment III study, flumazenil was administered by gavage to rats at doses of 0, 5, 25, and 125 mg/kg. The test drug was administered to 24 mated females/group from day 16 of gestation until weaning, on day 22 of lactation. A control group received the vehicle.

There were no significant dose- or drug-related differences between the groups in the number of intrauterine and perinatal deaths. Mortality during lactation was increased in the high-dose group (14% versus 7.8% in the control group). From the weanlings in which the organs were weighed, a slight but dose-related increase of liver weights was noted in the mid- and high-dose groups. The physical and functional development of neonates was normal, although there was a slight but statistically significant delay of incisor eruption, ear opening, and auditory startle response in the offsprings of high-dose-treated dams.

Irritation Studies

Venous Irritation – Rabbits

A single injection of 1.0 mL of flumazenil (1 mg/mL, mixed micelles formulation) into the marginal ear vein of 6 New Zealand rabbits did not cause significant irritation of the veins.

Local Tolerance – Rabbits

Five rabbits were given an i.v. injection of 0.5 mL of flumazenil (0.5 mg/5 mL, aqueous formulation) in the direction of the venous flow for the marginal ear veins. Intravenous tolerance was rated as good; only one animal had some reddening in the vicinity of the injection site (without any effect on the vein) on days 1 and 2.

Local Tolerance - Rat Hindquarter Muscle

Intramuscular tolerance was rated as good in 10 rats receiving 0.1 mL of flumazenil

(0.5 mg/5mL, aqueous formulation) into the gastrocnemius muscle of each hind limb. Creatinine phosphokinase was elevated relative to baseline at 24 hours after injection in both treated and control animals. The elevations seen in treated rats were somewhat larger than those observed in the control group.

Hemolysis Testing – Dogs

Intravenous administration of 1.0 mL of flumazenil (1 mg/mL, mixed micelles formulation) to 12 dogs did not produce any significant hemolysis.