Four weeks i.v. dose range-finding study in rats (CD-98/6288T)

IQB-9302.HCl was intravenously administered to groups of 5 male and 5 female Sprague-Dawley rats during 4 consecutive weeks at doses of 1, 2 and 4 mg/kg given as i.v. bolus. One additional control group was given saline

Results

Mortality
There were no mortalities among the control group animales nor the animals treated with 1 mg/kg/day.
Of the 10 animals administered at the dose of 2 mg/kg, three of them died in the course of treatment (8th, 9th and 25th day of treatment)
Of the 10 animals administered at the dose 4 mg/kg 9 of them died during the treatment period.
In all cases, the deaths occurred in the 6 minute period after administration

Clinical signs
Slightly decreased muscular tone was seen in one control animal and in two animals treated with 1 mg/kg.
All of the animals treated at the 2 mg/kg/day dose showed ataxia, clonic convulsions and dysnea. Most of them also showed salivation, tail rigidity and postration. These symptoms were seen immediately after injection and disappeared in the course of 5 minutes after dosing
All of the animals treated at the 4 mg/kg dose showed clonic convulsions, dyspnoea, pallor, salivation and postration. These clinical signs were observed intermittently during the study, starting immediately after dosing and, in the case of survivors, disappearing in 6 minutes after injection

Body Weight
Body weight increase was similar for all the treatment groups (figure 1 and figure 2). 

Food intake
Food intake was similar in all the treatment groups

Organ weights
No differences were seen between organ weights of control and treated groups

Macroscopic findings
No macroscopic alterations were observed among control animals and animals treated at 1, 2 and 4 mg/kg/day 

Microscopic findings
A slightly increased number of hepatic mitosis (due to a starting hepatic hyperplasia) was observed in two animals treated at 2 and 6 animals treated at 4 mg/kg. No hepatic alterations were observed in samples from the animals treated at the dose of 1 mg/kg/day 
 

4 weeks intravenous subacute toxicity in rats followed by two week recovery

The test substance IQB-9302.HCl was administered intravenously, by bolus, to Crl:CD® (SD) BR Sprague-Dawley rats, for 4 consecutive weeks at the doses of  0.75, 1.25 and 2.25 mg/kg/day.

The rats were distributed in four treatment groups including the Control group.

The Control group and the one treated at 2.25 mg/kg/day consisted of 15 males and 15 males each and the groups treated at 0.75 and 1.25 mg/kg/day consisted of 10 males and 10 females each.

At the end of the four weeks of treatment, the animals were sacrificed, except for five males and five females of the Control and high dose groups, which underwent a two-week observation period.

During this period, whose purpose was to study the evolution of the alterations observed, the animals were not treated. At the end of the period, the animals were sacrificed.

The main results are detailed below:

IQB-9302.HCl, 2.25mg/kg/day

• No mortalities were recorded among the animals treated at this dose. >
• The main clinical sign observed was ataxia. This alteration was accompanied occasionally in most of the animals by clonic convulsions, salivation, prostration, mydriasis, rigidity of the tail and hindquarters, decreased motor activity and pallor. All the clinical signs started immediately after treatment and disappeared two minutes afterwards.
• The bodyweight increase in males and females was similar to that recorded in the Control group.
• The food and water intake in males and females was similar to that recorded in the Control animals.
• No alterations were registered in the ophthalmoscopic examinations carried out.
• No noticeable alterations were recorded in the haematological and biochemical analyses nor in the analyses of urine made at the end of the treatment period. There were no alterations at the end of the recovery period either.
• There were no alterations related to the treatment given in the organ weight at the end of the treatment and recovery period.
• The microscopic examination of the samples taken did not reveal any alteration related to the administration of the test substance

• No mortalities were recorded among the animals treated at this dose.
• The main clinical sign was ataxia. In two animals, this alteration was occasionally accompanied by: decreased muscle tone and pallor (in one male) and prostration, dyspnoea, salivation, mydriasis and rigidity of the tail (in one female). All the clinical signs were observed immediately after administration and had disappeared two minutes after the treatment.
• The bodyweight increase in males and females was similar to that recorded for the Control group.
• The food and water intake in males and females was similar to that recorded in the Control animals.
• No alterations were registered in the ophthalmoscopic examinations carried out.
• No noticeable alterations were recorded in the haematological and biochemical analyses nor in the analysis of urine made at the end of the treatment period. There were no alterations at the end of the recovery period.
• There were no alterations related to the treatment given in the organ weight at the end of the treatment and recovery period.

• No mortality was registered among the animals treated at this dose.
• Only one male sporadically presented ataxia after treatment which disappeared after two minutes post-administration.
• The bodyweight increase in males and females was similar to that registered for the Control animals.
• No noticeable alterations were registered in the food and water intake
• No alterations were observed in the ophthalmoscopic examinations carried out.
• No noticeable alterations were recorded in the haematological and biochemical analyses nor in the analysis of urine made at the end of the treatment period nor at the end of the recovery period.
• There were no alterations related to the treatment given in the organ weight at the end of the treatment and recovery period.

CONCLUSIONS
No mortalities were registered among the animals treated with the substance IQB-9302.HCl at the doses of 0.75, 1.25 and 2.25 mg/kg/day.

The main clinical signs recorded were ataxia, clonic convulsions, salivation, mydriasis, rigidity of the tail and decreased motor activity. These clinical signs were registered at the doses of 2.25 and 1.25 mg/kg/day. At the dose of 0.75 mg/kg/day only ataxia was observed occasionally in one animal.

No noticeable alterations were recorded in the haematological and biochemical analyses.
There were no alterations related to the treatment given in the organ weight. 

 4 weeks intravenous subacute toxicity in dogs followed by two week recovery.

Groups of 5 male and 5 female dogs were dosed intravenously daily for at least 28 days at levels of 0 (Group BKG1), 1 (Group BKG2), 2 (Group BKG3), and 3 (Group BKG4) mg IQB-9302/kg/day. The test article was dissolved in the vehicle (0.9% Sterile Saline for Injection, USP) at concentrations that allowed the appropriate dose to be delivered in a volume of 1 ml/kg body weight. Initially, the test article dose solutions were infused at a rate of 3 mL/minute but clinical signs necessitated slowing the infusion rate to 1 and/or 2 mL/minute. The control group was administered 0.9% Sterile Saline for Injection, USP at 3 mL/minute. The last 1 dog/sex/group was allowed 14 days for recovery before sacrifice for evaluation. Physical examinations, including ophthalmology, were made during the pretest period and just before scheduled termination of each dog and clinical observations were conducted a minimum of twice daily during the treatment and recovery period. Body weights were taken pretest, weekly during the evaluation, and just prior to sacrifíce. Food consumption was measured weekly. Urine and blood samples were obtained pretest, in Week 4, and from recovery animals in Week 6. Blood samples were obtained on Days 1 and 28 for determination of plasma drug levels. Electrocardiograms and indirect blood pressure measurements were made pretest, in Week 4, and from recovery animals in Week 6. Electrocardiograms were evaluated by a board certified Veterinary Cardiologist. Each dog was euthanized and subjected to a complete gross necropsy in which tissues were collected for histopathologic evaluation by a board certified Veterinary Pathologist and organ weights were taken for statistical evaluation.

Results

There were no test article related effects observed based on physical examinations, ophthalmology, electrocardiograms, indirect blood pressure measurements, clinical pathology, clinical observations (other than dose(postdose), or the macroscopic and microscopic appearance of organs and tissues. Dose/postdose observations revealed clinical signs primarily related to the central nervous system. The effects were generally of short duration and no animals died or were considered moribund during this evaluation. Ataxia was the most frequently noted clinical sign followed by muscle twitching, salivation and seizure activity for both males and females. Muscle twitching and salivation were most cornmonly noted during infusion of the test article while ataxia and seizures were most commonly observed immediately following completion of the infusion. 
Ataxia was observed after dosing at least one or more times in every dog in the mid and high-dose groups and in one female dog in the low-dose group. Muscle twitching was noted on at least one occasion for one low-dose male, three low-dose females, three mid-dose males, four mid-dose females, and all high-dose dogs. Salivation was observed at least once for two mid-dose males, two mid-dose females, three high-dose males and all high-dose females except one. Seizure activity was observed at least once for one male and one female in the mid-dose group. Three high-dose males had at least one seizure episode while one high-dose female had three seizure episodes and another three high-dose females each had at least four seizures. All dosing/post-dosing clinical signs were resolved, and the dogs appeared normal, within 10-15 minutes postdosing. There were no adverse clinical signs noted during the entire 14-day recovery period.

In conclusion, it would appear that daily intravenous administration of up to 3 mg/kg/day of IQB-9302 to beagle dogs had, with the exception of dosing/postdosing clinical signs, no meaningful, distinct or consistent adverse effects relating to drug safety. Clinical signs during dosing and immediately thereafter, primarily related to the central nervous system, were prevalent in mid and high-dose groups but less frequent (primarily one male and three females) in the low-dose group. The initial dose infusion rate of 3 ml/minute was slowed to 1 and/or 2 mL/minute to minimize the severity of the dosing/postdosing clinical signs. With cessation of treatment all remarkable clinical signs ceased and afl other parameters remained normal. Consequently, by strict definition, the no-observed-adverse-effect-level (NOAEL) with regard to clinical signs noted during and immediately after dosing was 0 mg IQB9302/kg body weight/day; however, a dose of 1 mg/kg/day produced virtually no adverse effects in six of ten dogs.
 

Conclusion:
No mortality was registered among the animals treated with the substance IQB-9302.HCl at the doses of 1, 2 and 3  mg/kg/day for 28 consecutive days

The main clinical signs recorded were related to central nervous system. These clinical signs were registered at the doses of 2 and 3 mg/kg/day. At the dose of 1 mg/kg/day only ataxia was observed occasionally in some animals. No ophthalmological or haemodynamic effects were observed after injection

No noticeable alterations were recorded in the haematological and biochemical analyses.
There were no alterations related to the treatment given in the organ weight and no macroscopic or microscopic changes were observed in organs and tissues