Preclinical Safety Assessment of Gadopiclenol: A High-Relaxivity Macrocyclic Gadolinium-Based MRI Contrast Agent

Abstract

Objective

Gadopiclenol is a new high-relaxivity macrocyclic gadolinium-based contrast agent for magnetic resonance imaging of the central nervous system and other body regions. The product has been approved by US Food and Drug Administration and is currently being evaluated by European Medicines Agency. For risk assessment of the single diagnostic use in humans, the safety profile of gadopiclenol was evaluated with a series of preclinical studies.

Materials and Methods

With exception of dose-ranging studies, all safety pharmacology and toxicology studies were performed in compliance with Good Laboratory Practice principles. Safety pharmacology studies were conducted to assess potential effects on cardiovascular (in vitro and in dogs), respiratory (in rats and guinea pigs), neurological (in rats), and renal endpoints (in rats). Toxicology studies were also performed to investigate acute toxicity (in rats and mice), extended single-dose (in rats and dogs) and repeated-dose toxicity (in rats and dogs), reproductive (in rats), developmental (in rats and rabbits) and juvenile toxicity (in rats), as well as genotoxicity (in vitro and in rats), local tolerance (in rabbits), potential immediate hypersensitivity (in guinea pigs), and potential tissue retention of gadolinium (in rats).

Results

Safety pharmacology studies conducted at high intravenous (IV) doses showed a satisfactory tolerance of gadopiclenol in the main body systems. After either single or repeated IV dosing (14 and 28 days) in rats and dogs, gadopiclenol was well tolerated even at high doses. The no-observed-adverse-effect level values (ie, the highest experimental dose without adverse effects) representing between 8 times in rats and 44 times in dogs (based on the exposure), the exposure achieved in humans at the intended diagnostic dose, provide a high safety margin. No or only minor and reversible effects on body weight, food consumption, clinical signs, clinical pathology parameters, or histology were observed at the highest doses. The main histological finding consists in renal tubular vacuolations (exacerbated after repeated exposure), which supports a well-known finding for this class of compounds that has no physiological consequence on kidney function. Reproductive toxicity studies showed no evidence of effects on reproductive performance, fertility, perinatal and postnatal development in rats, or reproductive development in rats or rabbits. The safety profile of gadopiclenol in juvenile rats was satisfactory like in adults. Gadopiclenol was not genotoxic in vitro in the Ames test, a mouse lymphoma assay, and a rat in vivo micronucleus test. There were no signs of local intolerance at the injection site after IV and intra-arterial administration in rabbits. However, because of minor signs of intolerance after perivenous administration, misadministration must be avoided. Gadopiclenol exhibited no signs of potential to induce immediate hypersensitivity in guinea pigs.

Conclusions

High safety margins were observed between the single diagnostic dose of 0.05 mmol/kg in humans and the doses showing effects in animal studies. Gadopiclenol is, therefore, well tolerated in various species (mice, rats, dogs, rabbits, and guinea pigs). All observed preclinical data support the clinical approval.

Magnetic resonance imaging (MRI) is a well-established 3-dimensionnel noninvasive medical imaging technique generating diagnostic anatomical and functional information by using proton nuclear magnetic resonance. The introduction of an exogenous gadolinium-based contrast agent (GBCA) enhances diagnostic sensitivity and specificity, by improving the visibility of the internal body structures.¹ Because of the presence of unpaired electrons, the gadolinium cation (Gd³⁺) drastically shortens the T1 (longitudinal) relaxation time of protons, producing hypersignal intensity on T1-weighted MRI.² Nevertheless, free Gd³⁺ ions are toxic due to interference with calcium ion channels (same ionic radius as Ca²⁺), resulting for example in neurotransmitter inhibition, muscle contraction, and mitochondrial dysfunction.³ It is possible to drastically reduce this toxicity by chelating the Gd³⁺ with a coordination cage (ligand), making the Gd³⁺ ions unavailable to interact with tissues and facilitating elimination and reducing biotransformation and accumulation of the Gd³⁺ in different organs. This chelated agent, which guarantees both safety and efficacy, is known as a GBCA.

The chemical classification of GBCAs depends on the structure of the ligand used to chelate gadolinium. There are 2 types of chelates: macrocyclic and linear ligands. The stability, that is, the strength with which the GBCA chelator holds on to the Gd ion, is a very important property of GBCA and is conditioned by the structure of the GBCA's ligand. Macrocyclic chelators bind most tightly to the Gd³⁺ ion (high dissociation half-life) and therefore have the highest kinetic inertness than linear chelators.⁴ In the 2000s, a causal link was established between the nephrogenic systemic fibrosis (NSF), a very rare but serious disease occurring in patients with severe renal dysfunction, and the previous administration of linear GBCAs, with the highest potential for free Gd³⁺ release by dechelation.^5,6 Consequently, most linear GBCAs were contraindicated for use in specific patient classes considered “at risk” (severe and end-stage renal failure, neonates) in many countries. In 2014, Kanda et al⁷ demonstrated that GBCAs may also cause abnormal long-term brain signal hyperintensities in non–renally impaired patients, raising the questions about the retention of Gd in the body for patients without any reduction of kidney excretion. In the following years, many studies confirmed that linear GBCAs are prone to maximize the dose-dependent long-term Gd body retention versus macrocyclic GBCAs. In the light of all these strong scientific evidences, some health authorities decided to suspend (or restrict) the marketing authorizations of the linear GBCAs due to their higher risk of Gd retention and accumulation in the body.⁸

Gadopiclenol is a new macrocyclic GBCA for contrast-enhanced MRI designed to detect and visualize lesions in the central nervous system (CNS) and in other body regions. Gadopiclenol presents a 2- to 3-fold higher relaxivity in water than the currently approved GBCAs due to its optimized chemical structure (r1 = 12.2 mM⁻¹·s⁻¹ at 1.41 T at 37°C).⁹ Confirmed by the different clinical studies, gadopiclenol can be given at half the conventional dose of Gd compared with other nonspecific gadolinium-containing contrast agents, while providing the same contrast enhancement.^10–13 The recommended dose of gadopiclenol in humans is 0.05 mmol/kg body weight for all indications given in adults and children older than 2 years, by intravenous (IV) bolus injection, that is, single administration.¹⁴ Gadopiclenol has been approved by the US Food and Drug Administration (FDA) in September 2022.¹⁴

The development of gadopiclenol has fulfilled all nonclinical prerequisites in compliance with applicable International Conference on Harmonization (ICH) guidelines and FDA and European requirements to assess the risk to humans of the anticipated single diagnostic dose of 0.05 mmol/kg body weight of gadopiclenol. The nonclinical development program of gadopiclenol was performed in Europe and in the United States, and included pharmacokinetics, safety pharmacology, and toxicity studies (see Fig. 1). The pharmacokinetic profile of gadopiclenol has already been described by Robic et al.⁹ Briefly, the new GBCA exhibited no protein binding (in rats, dogs, and human plasma and red blood cells) and a pharmacokinetic profile similar to that of other extracellular nonspecific GBCAs.

FIGURE 1.

Diagram summarizing the preclinical safety study program for gadopiclenol (studies underlined include toxicokinetic data).

Safety pharmacology studies were conducted in vitro, and in rats and dogs to assess potential effects on cardiovascular, respiratory, neurological, and renal endpoints. Toxicity testing included studies with single and repeated administration (up to 4 weeks) in rodent and nonrodent species, studies into the genotoxic potential in vitro and in vivo, studies into the reproductive and developmental toxicity including juvenile toxicity, and local tolerance. In vivo studies were conducted mainly by IV administration, as this corresponds to the clinical route of administration of gadopiclenol. The reversibility of possible adverse effects was assessed in toxicity studies by recovery groups.

The aim of this article is to summarize the data obtained in preclinical safety program testing of gadopiclenol to provide evidence of the good safety profile of this new MRI contrast agent.

MATERIALS AND METHODS

Contrast Agent

Gadopiclenol (Elucirem; Guerbet, Villepinte, France and Vueway; Bracco Imaging, Milan, Italy) was initially invented by Guerbet with subsequent contribution of Bracco Intellectual Property. It has a molecular weight of 970.11 g/mol, and its molecular formula is C₃₅H₅₄GdN₇O₁₅.⁹

Two different formulations of gadopiclenol were tested: formulation A in early development phase (single-dose toxicity, repeated-dose toxicity 14 days, fertility and early embryonic development, embryofetal development, genotoxicity, local tolerance, and immunotoxicity) and the to-be-marketed formulation B for nonclinical prephase 3 and premarketing studies (repeated-dose toxicity 28 days, prenatal/postnatal development and juvenile toxicity). Both formulations present the same concentration of gadopiclenol, that is, 0.5 mol/L. The initial gadopiclenol formulation A was in water without buffer with 2.5 mmol/L of calcium complex of dodecane tetraacetic acid (DOTA). Formulation B is in trometamol buffer at 10 mmol/L pH 7.5 with DOTA at 1 mmol/L.

Unless otherwise indicated, 0.9% sodium chloride for injection was used as negative control in all in vivo studies.

Animals and Animal Maintenance

The studies were conducted according to international guidelines following standard procedures in mice, rats, dogs, guinea pigs, and rabbits obtained from various licensed breeders. The animals were allocated in conventional cages in air-conditioned rooms with controlled lightening (12 hours day/12 hours night rhythm). Animals were given tap water or demineralized water to drink ad libitum and fed with appropriate standard diets.

Good Laboratory Practice Status

Except for dose-ranging studies, all safety pharmacology and toxicology studies were performed in compliance with Good Laboratory Practice (GLP) principles.

Safety Pharmacology Studies

The safety pharmacology program carried out on gadopiclenol covered the main body systems or functions (CNS, cardiovascular, respiratory, and renal systems), in compliance with the “core battery” defined in ICH S7A and S7B guidelines (see Table 1). All in vivo studies were done by a single IV dose of gadopiclenol.

TABLE 1.

Program of Safety Pharmacology Studies

Study Type and Duration	Route of Administration and Duration of Dosing	Species (Strain)	Tested Concentrations or Dose Levels
Cardiovascular system	In vitro	HEK-293 cells, Purkinje fibers	0, 1.25, 2.5, 5, or 10 mmol/L
	Single IV	Anesthetized and conscious Dog (Beagle)	0, 0.5, 1, or 2 mmol/kg
Respiratory system	Single IV	Rat (SD), Guinea pig (Hartley)	0, 1.25, 2.5, or 5 mmol/kg
Central nervous system	Single IV	Rat (SD and Wistar)	0, 1.25, 2.5, or 5 mmol/kg
Renal system	Single IV	Rat (SD)	0, 1.25, 2.5, or 5 mmol/kg

IV, intravenous.

Cardiovascular System

Preliminary in vitro studies have been conducted in order to assess the electrophysiological effects of gadopiclenol on human ether-à-go-go–related gene (hERG) current, an internationally recognized predictive model of QT prolongation potential.¹⁵ Potential blocking effects of gadopiclenol on hERG tail current were evaluated in vitro on 5 stably transfected HEK-293 cell preparations, using the conventional patch-clamp technique in whole cell configuration, and compared with those generated in the presence of vehicle (extracellular solution with 2% of distilled water). The second in vitro study was performed on 6 Purkinje fibers from female New Zealand white (NZW) rabbits at the same doses as a follow-up of the hERG study.

Effects of gadopiclenol on cardiovascular system have been studied in Beagle dogs. The cardiovascular function (hemodynamic parameters, electrocardiogram [ECG] parameters) was evaluated on 3 male and 3 female anesthetized dogs after IV infusion (slow bolus for 2 minutes) of negative control gadopiclenol. Each animal received each dose level at 30-minute intervals.

The effects on arterial blood pressure, heart rate, ECG, and body temperature were evaluated after IV administration (2 mL/kg per minute) in 3 male and 3 female conscious dogs monitored by telemetry of negative control or gadopiclenol. Each animal served as its own control and received the vehicle and each dose level at 2-day intervals.

Respiratory System

The effects of gadopiclenol on respiratory parameters (respiratory rate, inspiratory, expiratory and relaxation times, tidal and minute volumes, peak inspiratory and expiratory flows, and on the enhanced pause) were studied in the whole-body plethysmography method in Sprague-Dawley (SD) rats (8 males/group) that received gadopiclenol up to 5 mmol/kg or negative control injected intravenously by slow bolus for 2 minutes.

In a second in vivo study, the bronchoconstrictive activity (evaluated by the measure of total pulmonary resistance) was assessed after a single IV infusion of gadopiclenol (in 6 male Hartley guinea pigs), and appropriate negative and positive (histamine) controls were included.

Central Nervous System

The potential neurobehavioral effects of gadopiclenol were evaluated in conscious unfasted SD rats (6 males/group) using the Irwin method¹⁶ (effect on the main central and peripheral nervous system functions) by a single IV administration of negative control (0.9% sodium chloride) or gadopiclenol (at dose levels of 1.25, 2.5, or 5 mmol/kg). For the Irwin test, animals were observed for behavior, at the following time points: 0 (before dosing), 0.5, 1, 2, 5, and 24 hours postdose. Effects on body temperature, body weight, and mortality were also studied.

The proconvulsant effect was studied after a single IV infusion of negative control, positive control (caffeine at 55 mg/kg), or gadopiclenol (in 6 male and 6 female Wistar rats). Five minutes after the end of administration, each animal was infused with a solution of pentylenetetrazole (PTZ) at 5.5 mg/mL by the IV route at a rate of 0.5 mL/kg per minute to evoke seizures. The time of occurrence of seizures was recorded.

Renal System

The effects of gadopiclenol on renal function (urine output, serum/urine electrolyte balance, serum and urinary biochemistry, and glomerular filtration rate) were evaluated in SD rats (8 males/group) after an oral saline overload given 15 minutes postdosing after a single IV injection (slow bolus for 2 minutes) of negative control, positive control (Furosemide, 10 mg/mL), and gadopiclenol.

Toxicology Studies

The toxicological evaluation of gadopiclenol was carried out according to the program described in Table 2.

TABLE 2.

Program of Toxicology Studies

Study Type and Duration	Route of Administration	Species (Strain)	Tested Concentrations or Dose Levels
Single-dose toxicity	IV	Mouse (CD1)	0, 4, 8, or 12 mmol/kg
		Rat (SD)	0, 2.5, 4, 5, 8, 10, or 12 mmol/kg
		Dog (Beagle)	0, 1, 2 or 4 mmol/kg
Repeated-dose toxicity 14 d	IV	Rat (SD)	0, 2.5, 5, or 10 mmol/kg
		Dog (Beagle)	0, 1, 2, or 4 mmol/kg
Repeated-dose toxicity 28 d	IV	Rat (SD)	0, 2.5, 5, or 10 mmol/kg
		Dog (Beagle)	0, 1, 2, or 4 mmol/kg
Fertility and early embryonic development	IV	Rat (SD)	0, 2.5, 5, or 10 mmol/kg
Embryofetal development	IV	Rat (SD)	0, 2.5, 5, or 10 mmol/kg
		Rabbit	0, 1, 2.5, or 5 mmol/kg
Prenatal/postnatal development	IV	Rat (SD)	0, 2.5, 5, or 10 mmol/kg
Juvenile toxicity	IV	Juvenile rat (SD)	0, 0.6, 1.25, or 2.5 mmol/kg
Genotoxicity in vitro	In vitro	S. typhimurium, L5178Y	Up to 5000 μg/plate
		Mouse lymphoma cells TK+/−	Up to 12,125 μg/plate
Genotoxicity in vivo	IV	Rat (SD)	0, 2.5, 5, or 10 mmol/kg
Local tolerance	Perivascular	Rabbit (NZW)	0.25 mmol/animal
	IV		0.6 mmol/kg
	Intra-arterial		0.6 mmol/kg
Immediate hypersensitivity	SC, IV	Guinea pig (Hartley)	0.5 and 1 mL

IV, intravenous; SC, subcutaneous; SD, Sprague-Dawley; NZW, New Zealand white.

Toxicokinetics Assessment

In toxicology studies (after single and repeated administration), toxicokinetics data were obtained after gadopiclenol concentration measurement in plasma using a validated liquid chromatography–mass spectrometry/mass spectrometry (LC-MS/MS) assay. The toxicokinetic evaluation was performed using Phoenix WinNonlin pharmacokinetic software. A noncompartmental approach consistent with the IV route of administration was used for parameter estimation. Conventional PK parameters were estimated.

Single-Dose Toxicity

A complete and extended single-dose toxicity study program was conducted in rodent and nonrodent species by IV route with gadopiclenol, according to 2 kinds of study plans:

• acute toxicity studies in rats and mice, and

• pivotal “expanded” single-dose toxicity studies (toxicokinetics, hematology, clinical chemistry, necropsy, and histopathology data) in rats and dogs.

The design of these studies is described in Table 3. Animals in the control groups were treated with the respective volume of physiological saline under the same experimental conditions. A full postmortem examination was performed on all surviving animals at the end of the observation period (14 days), and on animals that died prematurely.

TABLE 3.

Design of Single-Dose Toxicity Studies With Gadopiclenol

Species (Strain)	No. M and F Per Group	Administration Route (Vehicle/Formulation)	Dose Levels, mmol/kg	In-Life Study Parameters	Pathology	Laboratory Investigations
Mice (CD1)	n = 5 M/5 F	Intravenous (0.9% NaCl and solution of excipients)	0, 4, 8, or 12*	Clinical signs, body weight, food consumption	Necropsy	None
Rats (SD)	n = 5 M/5 F	Intravenous (0.9% NaCl and solution of excipients)	0, 4, 8, or 12*	Clinical signs, body weight, food consumption	Necropsy	None
Rats (SD)	n = 15 M/15 F†	Intravenous (0.9% NaCl and solution of excipients)	0, 2.5, 5, or 10	Clinical signs, body weight, food consumption, ophthalmoscopy, ECG	Macroscopic examination of organs, injection site, organ weights, histopathology	Toxicokinetic, hematology, serum chemistry, urinalysis
Dogs (Beagle)	n = 5 M/5 F‡	Intravenous (0.9% NaCl and solution of excipients)	0, 1, 2, or 4	Clinical signs, body weight, food consumption, ophthalmoscopy, ECG	Macroscopic examination of organs, injection site, organ weights, histopathology	Toxicokinetic, hematology, serum chemistry, urinalysis

*Maximum acceptable injectable dose volume (25 mL/kg) in mice and rats.¹⁷

†Ten animals/sex/group were killed on day 2 postdosing and 5 animals/sex/group on day 15 postdosing. Additional 6 males and 6 females/group (except for the sponsor-supplied vehicle group) were used for toxicokinetic evaluation and were sampled at predose and 0.08, 0.25, 0.5, 1, 3, and 6 hours postdosing on day 1.

‡Three animals/sex/group were killed on day 2 postdosing and 2 animals/sex/group on day 15 postdosing. For toxicokinetic evaluation, all animals were sampled at predose and 0.5, 1, 3, and 6 hours postdosing on day 1.

SD, Sprague-Dawley; M, male; F, female; ECG, electrocardiogram.

Repeated-Dose Toxicity

Systemic tolerance studies with repeated IV administrations were conducted in rats and dogs with a maximum dosing period of 14 days and 28 days. The studies evaluated (1) the toxicity, (2) the toxicokinetics of gadopiclenol after repeated daily IV injections, and (3) the reversibility of any toxic effect (recovery period of 14 days or 28 days with additional animals to investigate the reversibility of effects). To determine the dose levels used in these studies, preliminary non-GLP studies were performed in each species. Details of the GLP studies are given in Table 4. In addition to the usual endpoints, the 28-day toxicity study in rats also investigated behavioral alterations (functional observation battery, motor activity, locomotor activity) that could potentially result from repeated exposures to a GBCA: related to concerns on Gd tissue deposition (especially the brain) after multiple administrations of GBCAs. Immunochemistry of transforming growth factor β (TGF-β) was also investigated in the skin of animals, additionally to the conventional histopathology, to assess early markers of skin fibrosis (relating to the potential risk of NSF after GBCA exposure).

TABLE 4.

Design of Repeated-Dose Toxicity Studies With Gadopiclenol

Species	Rats (SD)	Rats (SD)	Dogs (Beagle)	Dogs (Beagle)
Duration of treatment	14 d	28 d	14 d	28 d
Dose levels (mmol/kg per day)	0, 2.5, 5, 10	0, 2.5, 5, 10	0, 1, 2, 4	0, 1, 2, 4
Negative control (control group)	Sterile physiological saline (0.9% NaCl): 20.4 mL/kg	Sterile physiological saline (0.9% NaCl): 20.4 mL/kg	Sterile physiological saline (0.9% NaCl): 20.4 mL/kg	Sterile physiological saline (0.9% NaCl): 20.4 mL/kg
Administration route	Intravenous	Intravenous	Intravenous	Intravenous
No. animals	n = 10/sex/group	n = 10/sex/group	n = 3/sex/group	n = 3/sex/group
Duration of recovery	14 d	28 d	14 d	28 d
No. animals in recovery groups	n = 5/sex in control and high-dose groups	n = 5/sex in control and high-dose groups	n = 2/sex in the control and high-dose groups	n = 2/sex in the control and high-dose groups
In-life study parameters	Clinical signs, body weight, food consumption, ophthalmology	Clinical signs, body weight, food consumption, ophthalmology and behavioral alterations (functional observation battery, motor activity, locomotor activity)	Clinical signs, body weight, food consumption, ophthalmoscopic and electrocardiographic examinations	Clinical signs, body weight, food consumption, ophthalmoscopic and electrocardiographic examinations
Pathology	Macroscopic examination, organ weight, and histopathology	Macroscopic examination, organ weight, and histopathology	Macroscopic examination, organ weight, and histopathology	Macroscopic examination, organ weight, and histopathology
Laboratory investigations	Hematology, serum chemistry, and urinalysis	Hematology, serum chemistry, and urinalysis	Hematology, serum chemistry, and urinalysis	Hematology, serum chemistry, and urinalysis
No. animals in satellite TK	n = 7/sex/group	n = 6/sex/group [3 for control]	NA	NA
TK evaluation	Sampling at 0.08, 0.25, 0.5, 1, 3, and 6 hours postdose on days 1 and 14	Sampling at 0.167, 0.33, 0.5, 1, 3, and 6 hours postdose on days 1 and 28	Sampling at 0, 0.5, 1, 3, 6, and 8 hours postdose on days 1 and 28	Sampling at 0, 0.5, 1, 3, 6, and 8 hours postdose on days 1 and 28

SD, Sprague-Dawley; NA, not applicable.

Reproductive and Developmental Toxicity

A fertility study was conducted in male and female rats, embryofetal development studies were performed in pregnant rats and rabbits, and a prenatal/postnatal development toxicity study was conducted in rats. Furthermore, a juvenile toxicity study in rats and a study comparing Gd tissue retention in juvenile and adult rats were carried out. For each reproductive and developmental toxicity study, dose levels of gadopiclenol were chosen based on a previous dose-range finding study. The study designs are summarized in Table 5.

TABLE 5.

Design of Reproductive Toxicity Studies of Gadopiclenol

Study Type	Fertility Including Early Embryonic Development	Embryotoxicity	Embryotoxicity	Prenatal and Postnatal Development	Juvenile Toxicity	Gd Retention Juvenile vs Adults
Species	Rats (SD)	Rats (SD)	Rabbits (NZW)	Rats (SD)	Juvenile rats (SD)	Juvenile or adult rats (SD)
Treatment period	M: 28 d before pairing and continued through euthanasia F: 14 d before pairing and continued through GD 7	GD 6 to 17	GD 6 to 19	GD6 to LD20	Single: PND10 Repeated: every 4 d from PND10 to PND30	Single dose or repeated dose: every 4 d during 8 wk
Dose levels (mmol/kg per day)	0, 2.5, 5, 10	0, 2.5, 5, 10	0, 1, 2.5, 5	0, 2.5, 5, 10	0, 0.6, 1.25, 2.5	0.6
Negative control (control group)	Sterile physiological saline (0.9% NaCl)	Sterile physiological saline (0.9% NaCl)	Sterile physiological saline (0.9% NaCl)	Sterile physiological saline (0.9% NaCl)	Sterile physiological saline (0.9% NaCl)	Reference item for adults (Omniscan, gadodiamide)
Administration route	Intravenous	Intravenous	Intravenous	Intravenous	Intravenous	Intravenous
No. animals	n = 22/sex/group	n = 25 F/group	n = 23 F/group	F0: n = 22 F/group F1: n = 20/sex/group	n = 15/sex/group	n = 6/sex/group
Sacrifice or cesarean (day)	M: day 44 F: GD13	GD20	GD29	F0: after weaning F1 male: after necropsy of the majority of the F1 females F1 female: GD13	2 d or 9 wk after dosing (ie, PND11, PND31, PND71, or PND 91)	1 day or 8 wk after dosing
In-life study parameters	Clinical signs, body weights, food consumption	Clinical signs, body weights, food consumption	Clinical signs, body weights, food consumption	F0/F1: clinical signs, body weights, food consumption F1: postweaning development and behavioral tests	Clinical signs, body weights, food consumption, ophthalmology, tibia length, reflex and physical development, neurobehavioral tests	Clinical signs, body weights, food consumption
Reproductive parameter	M/F: mating, fertility, and fecundity indices F: estrous cyclicity, uterine and ovarian examinations M: sperm analyses	Uterine and ovarian examinations, fetal examinations (external, visceral, and skeletal observations)	Uterine and ovarian examinations, fetal examinations (external, visceral, and skeletal observations)	F0: number of implantations F1: uterine and ovarian examinations, litter parameters (including number of pups born, pup survival, and pup weights)	M/F: sexual maturation F: estrous cyclicity M: sperm analysis	NA
Pathology	Macroscopic examination, reproductive organ weight	Macroscopic examination	Macroscopic examination	Macroscopic examination, organ weight	Macroscopic examination, organ weight, and histopathology	Macroscopic examination, organ weight, and histopathology in selected tissues
Laboratory investigations	NA	NA	NA	Hematology, serum chemistry, and urinalysis (1 F1 pup/sex/litter on PND21 and all surviving adult F1 animals before necropsy), Gd determination in tissues, and in plasma (6 F0 F/group and 1 F1 pup/sex/litter on PND21)	Hematology, serum chemistry and urinalysis, Gd determination in tissues on PND11, PND31, PND71, or PND 91 (6 animals/sex/group)	Gd determination in tissues and in plasma on PND11/D1, PND67/D57, or PND123/D113
No. animals in TK	n = 6/sex/group	n = 9 F/treated group n = 3 F in negative control group	n = 4 F/group	F0: n = 3 F/group F1: n = 1 pup/sex/litter	n = 18/sex/treated group n = 9/sex in negative control group	NA
TK evaluation	Sampling at 0.083, 0.25, 0.5, 1, 3, and 6 hours post the start of injection on day 1, GD 7 (females), and days 44 or 45 (males)	Sampling at 0.167, 0.333, 0.583, 1.083, 3.083, and 6.083 hours after the start of injection on GD 6 and 17	Sampling at 0.167, 0.333, 0.583, 1.083, 3.083, and 6.083 hours after the start of injection on GD 6 and 19	F0: sampling at 0, 17, 0.5, 2, 6, and 24 hours postdose on GD6 and LD20 F1: PND 20	Sampling at 0, 17, 0.5, 2, 6, and 24 hours postdose PND10 or PND30	NA

GD, gestation day; LD, lactation day; PND, postnatal day.

Genotoxicity

The genotoxic potential of gadopiclenol was studied both in vitro and in vivo as required by ICH guidelines.^18,19 Appropriate negative and positive controls were included.²⁰

A bacterial reverse mutation assay (Ames test) was carried out using both plate incorporation and preincubation methods (37°C ± 2°C for approximately 25 minutes under stirring) on 5 strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535, and TA 1537). Each strain was exposed to 5 concentrations of gadopiclenol up to the maximum recommended dose level of 5000 μg/plate, with and without metabolic activation (S9-mix). Each experimental point was tested in triplicate.

A second in vitro test consisting in cytogenetic evaluation of chromosomal damages in a mammalian cell gene mutation test (L5178Y mouse lymphoma cells TK^+/−) was carried out. Short treatments (4 hours) were performed both with (the dose levels ranging from 155 to 5000 μg/mL) and without (the dose levels ranging from 155 to 12,125 μg/mL) metabolic activation, whereas long treatments (24 hours) were performed only without metabolic activation (the dose levels ranging from 1.7 to 5000 μg/mL). Duplicate cultures were performed for each experimental point.

An in vivo mammalian erythrocyte micronucleus assay was performed. Five male and 5 female SD rats per group received intravenously gadopiclenol at 2.5, 5, and 10 mmol/kg per day during 2 consecutive days. The animals were killed between 18 and 24 hours after the second injection for bone marrow harvesting. Saline (0.9% NaCl) was used as the negative control item and administered at a dose volume of 20.4 mL/kg per day for 2 days. Cyclophosphamide was used as a positive control by single intraperitoneal injection of 15 mg/kg.

Local Tolerance

In addition to the satisfying observations in single and repeated-dose toxicity studies, a local tolerance study was carried out in NZW rabbits receiving gadopiclenol by IV, perivenous (PV), or intra-arterial (IA) administration at dose levels of 0.6 mmol/kg (IV and IA) and 0.25 mmol/animal (PV). One group of 10 animals received gadopiclenol, whereas another group of 10 animals received 0.9% saline as a negative control. Five animals in each group were killed 24 hours postdosing, and the remaining animals 96 hours postdosing. All injection sites were examined histopathologically.

Immunotoxicity

The potential of gadopiclenol to induce immediate hypersensitivity reactions was evaluated in 10 male Hartley guinea pigs, which received 2 subcutaneous injections of 0.5 mL of gadopiclenol on days 0 and 7 (induction phase) and then an IV injection of 1 mL of gadopiclenol on day 21 (challenge phase). In 5 animals/group, negative (0.9% NaCl) and positive (ovalbumin) controls were also given in the same conditions. Aluminum hydroxide was given on day 0 to stimulate the immune response.

Gd Tissue Retention Studies

Gd presence measured in selected tissues (brain/cerebellum, skin, kidney, femur, liver) by a validated method using inductively coupled plasma–mass spectrometry was investigated in 3 studies from the preclinical program described previously: (a) juvenile toxicity study in rats, (b) Gd tissue retention in juvenile and adult rats, and (c) prenatal/postnatal development study in rats.

RESULTS

Safety Pharmacology

No toxicokinetics assessment was done in the safety pharmacology studies, because this information is available in rats and dogs from toxicology studies at the same dose levels. When dose levels used in animal studies are compared with the human dose, it is based on body surface area conversion factors.²¹

Cardiovascular System

In vitro, no statistically significant effect on the hERG tail current was observed up to the concentration of 2.5 mmol/L. At higher concentrations, gadopiclenol induced a concentration-dependent inhibition of hERG tail current amplitude at all tested doses, statistically significant at 5 and 10 mmol/L (18.9% and 31.9%, respectively, with P < 0.01 vs vehicle). These effects did not reverse after the washout period with the vehicle.

In the second in vitro study on rabbit Purkinje fibers, gadopiclenol did not show significant effects on action potentials when compared with the negative control group.

In anesthetized dogs, slight and transient variations of hemodynamic parameters were observed at 1 and 2 mmol/kg. QT and QTc interval durations were increased at 2 mmol/kg, over the first 10 minutes postdosing, reaching a peak value at 3 minutes postdosing (approximately +10% mean variations from predose, at 3 minutes postdosing, for QT and QTc values). This effect was mainly marked in only 1 of the 6 animals and was not confirmed in the study in conscious dogs described hereafter, which is considered as a better model to predict QT prolongation potential. The IV no-observed-effect level (NOEL) in anesthetized dogs was established at 0.5 mmol/kg (ie, approximately 5 times the human equivalent dose [HED] after adjustment for body surface area).

In conscious dogs, gadopiclenol at all tested dose levels did not induce any modification of arterial blood pressure and heart rate and did not significantly modify the duration of RR and PR intervals, of the QRS complex, or of the QT and QTc intervals, irrespective of the formula used for heart rate correction. No treatment-related ECG abnormalities were observed. The IV NOEL in conscious dogs was established at 2 mmol/kg (ie, 22 times the HED after adjustment for body surface area).

Respiratory System

In rats, compared with the negative control, gadopiclenol did not exert any relevant effect on respiratory parameters. A NOEL was thus established at 5 mmol/kg (ie, 16 times the HED after adjustment for body surface area).

In anesthetized guinea pigs, there was no sign of bronchospasm up to 2.5 mmol/kg, except in 1 animal showing a transient (approximately 30 seconds) with a decrease of −14% in tidal volume. At the dose level of 5 mmol/kg, gadopiclenol induced the following changes: a decrease from −14% to −19% in tidal volume, lasting at least 3 minutes in 2 animals out of 6; and a decrease of −88% in tidal volume associated with transitory episodes of muscular spasms in 1 animal out of 6. Under these experimental conditions, a NOEL was thus established at 1.25 mmol/kg and a no-observed-adverse-effect level (NOAEL) at 2.5 mmol/kg (ie, approximately 10 times the HED after adjustment for body surface area).

Central Nervous System

In conscious unfasted rats, no effect on the main central and peripheral nervous system functions was attributable to gadopiclenol. Only a decrease of −0.7% (on average vs pretest) in rectal temperature was noted 0.5-hour postdosing at 5 mmol/kg. This change was considered biologically relevant but not adverse. Therefore, a NOEL was established at 2.5 mmol/kg (ie, 8 times the HED after adjustment for body surface area) and a NOAEL at 5 mmol/kg (ie, 16 times the HED after adjustment for body surface area).

A decrease in the time of onset of PTZ-induced seizures of −23% (vs control) was observed at the highest tested dose of 5 mmol/kg in PTZ-treated rats. Therefore, the NOEL/NOAEL was 2.5 mmol/kg (ie, 8 times the HED after adjustment for body surface area).

Renal System

In healthy rats, gadopiclenol had no effect at 1.25 mmol/kg on renal system. A dose-related antidiuretic effect at doses of 2.5 and 5 mmol/kg (a decrease in free water clearance of −26% to −73%, compared with controls, respectively) and an antinatriuretic effect (at 5 mmol/kg, a decrease in sodium [−19%] and chloride [−18%] urinary concentrations) were observed up to 6 hours after the 2-minute infusion of gadopiclenol. Under these experimental conditions and based on the effects on urine osmolality and the free water clearance, a NOEL/NOAEL can be established at 1.25 mmol/kg (ie, 4 times the HED after adjustment for body surface area).

Toxicology

A summary of NOAELs determined in general and reproduction toxicity studies and the safety margin are shown in Table 6.

TABLE 6.

NOAELs Compared With the Expected Therapeutic Dose in Humans

Study	NOAEL, mmol/kg per d	Ratio Between NOAEL in Animal and Intended Human Dose of 0.05 mmol/kg
		Based on AUC* Ratio	Based on Body Surface Area†
Safety pharmacology
Hemodynamics in anesthetized dogs	0.5	—	5
Cardiovascular in conscious dogs	2	—	22
Respiration (plethysmography) in rats	5	—	16
Bronchospasm in guinea pigs	2.5	—	10
CNS (Irwin) in rats	5	—	16
Proconvulsant effect in rats	2.5	—	8
Renal function in rats	1.25	—	4
Toxicology
Acute toxicity in mice	12	—	20
Acute toxicity in rats	12	—	39
Single-dose toxicity in rats	10	59	32
Single-dose toxicity in dogs	4	25	44
14-d toxicity in rats‡	5	16	16
14-d toxicity in dogs‡	4	26	44
28-d toxicity in rats‡	2.5	9	8
28-d toxicity in dogs‡	2	15	22
Fertility in rats‡	5 (parenteral toxicity)	M: 63	32
	10 (reproductive performance and fertility)	F: 62
Embryofetal development in rats‡	5 (maternal toxicity)	26 (maternal toxicity)	16 (maternal toxicity)
	10 (developmental toxicity)	52 (developmental toxicity)	32 (developmental toxicity)
Embryofetal development in rabbits‡	2.5 (maternal toxicity)	24 (maternal toxicity)	16 (maternal toxicity)
	2.5 (developmental toxicity)	24 (developmental toxicity)	16 (developmental toxicity)
Prenatal and postnatal development in rats‡	5 (maternal toxicity)	19 (maternal toxicity)	16 (maternal toxicity)
	10 (postnatal developmental toxicity)	55 (postnatal developmental toxicity)	32 (postnatal developmental toxicity)
Juvenile toxicity in rats‡	2.5	8	8

*From toxicokinetics, gender averaged means of AUC (except for fertility study). Calculated from AUC in (adults) healthy volunteers at 0.05 mmol/kg.¹²

†Using the conversion factor according to FDA guidance.²¹

‡For repeated administrations, AUC based on end of treatment period values.

NOAEL, no-observed-adverse-effect level; CNS, central nervous system; AUC, area under the curve; FDA, US Food and Drug Administration.

Toxicokinetics in Toxicity Studies

Systemic exposure (mean area under the curve [AUC], shown in Fig. 2) increased in a generally proportional manner across the tested dose range in toxicity studies, indicative of linear kinetics with increasing doses. In rats and rabbits, the increase was often slightly greater than proportional between the intermediate and the high dose. No gender difference was evidenced on the pharmacokinetic profile of gadopiclenol.

FIGURE 2.

Systemic exposure (area under the curve) versus the increasing doses. AUC _0-inf, area under the plasma concentration versus time curve from time 0 to infinity.

After repeated dose administration, no difference was seen between sexes except in the juvenile rats, for which the exposure was higher for males compared with females (n = 3/sex) only at the lowest dose level for both dose occasions (postnatal day [PND] 10 and PND 30).

After repeat-dose administration, plasma exposures to gadopiclenol were similar on day 1 and at the end of the treatment period, indicating no accumulation of the product.

Single-Dose Toxicity

In acute toxicity studies in rats and mice, no mortality was observed up to the highest dose level of 12 mmol/kg. In both species, gadopiclenol induced some swelling of the face or limbs on day 1, lasting 3 to 4 hours postdosing but totally resolving thereafter. The highest dose tested (12 mmol/kg) was considered as a NOAEL in rats and mice, when given as a single IV slow bolus injection and 8 mmol/kg as NOEL in rats and mice.

In “expanded” single-dose toxicity studies, the treatment with gadopiclenol was well tolerated in rats and dogs. Transient swelling of the face and/or limbs (reversible in a few hours) in rats was only observed at the highest injected doses of gadopiclenol, and a swelling of the optic nerve was observed in 2 male dogs of the high-dose group, but the relationship to the treatment was doubtful.

On day 2, minimal to mild tubular cell vacuolations were observed in the kidneys, with a dose-related incidence and severity but with a partial recovery (rats) or total recovery (dogs) over 14 days. Based on these results, the NOAEL was established at the highest dose tested, 10 mmol/kg in rats and 4 mmol/kg in dogs given as a single IV slow bolus injection.

Repeated-Dose Toxicity

Regarding repeat-dose toxicity, gadopiclenol was well tolerated in both species, rats and dogs, even at very high repeated dose levels and no treatment-related death occurred. Similar renal histopathological changes than that observed after single administration were seen in both species, without any associated clinical pathology changes. Renal findings to gadopiclenol were mainly characterized by an increase in weight and the development of cytoplasmic vacuolations in the tubules (mainly in rats) and urinary bladder but without relevant and persistent functional consequences. Compared with single-dose toxicity, some additional treatment-related adverse effects were seen in rats only, on body weight, food consumption, clinical signs, clinical pathology parameters, and histology (vacuolated/granular macrophages), but these changes were always partially or totally reversible (see Table 7). The microscopic findings were, as expected, somewhat more exacerbated in incidence and severity after 28 repeated administrations compared with 14 repeated injections due to the prolonged dosing period. No additional changes due to repeat dosing were seen in dogs.

TABLE 7.

Toxicological Findings in Rats and Dogs After Single (“Expanded”) and Repeated IV Administrations of Gadopiclenol

Species	Duration of Treatment	Dose Level, mmol/kg	Findings	Reversibility
Rat	1 d	0, 2.5, 5, 10	• At 10 mmol/kg (1 M), swelling of the limbs	Reversible
			• At 10 mmol/kg (M), increase in kidney weights and epithelial tubular cells vacuolation	Partially reversible
Rat	14 d	0, 2.5, 5, 10	• Dose-related decrease in body weight gain and food consumption	Partially reversible
			• Dose-related decreases in RBC, hematocrit, hemoglobin, and triglyceride levels	Reversible
			• Mild to severe tubular cell vacuolations in the renal cortex and urinary bladder	Partially reversible
			• At 5 and/or 10 mmol/kg per d, cell vacuolation findings present in liver, lungs, lymph nodes, and in tissues throughout the body, mainly as vacuolated/granular macrophages	Reversible
Rat	28 d	0, 2.5, 5, 10	• At highest dose level, swelling of the limbs and/or nose/muzzle, decreased activity, salivation, urination, and purple discoloration of the tail	Reversible
			• Dose-related decreases in RBC, hematocrit, hemoglobin, and triglyceride levels	Reversible
			• Minimal decreases in ALT in both sexes at high-dose level	Nonreversible
			• Mild to severe tubular cell vacuolations in the renal cortex and urinary bladder	Partially reversible
			• At 5 and/or 10 mmol/kg per d, cell vacuolation findings present in liver, lungs, lymph nodes, and in tissues throughout the body, mainly as vacuolated/granular macrophages	Reversible
Dog	1 d	0, 1, 2, 4	• At 4 mmol/kg (2 M), with peripapillary conus or swelling of the optic nerve	Reversible
			• Dose-dependent vacuolation of epithelial tubular cells	Reversible
Dog	14 d	0, 1, 2, 4	• At all tested doses, increase in kidney weight	Reversible
			• At all dose levels, mild to marked epithelial cell vacuolation of the cortical and medullary tubules	Reversible
Dog	28 d	0, 1, 2, 4	• At the highest dose, swelling of the face and ears during week 2 through 4 (1 M)	Reversible
			• Tan discoloration of kidneys at the high-dose (2 M and 1 F)	Reversible
			• At all tested doses, kidney weight increase	Reversible
			• At all dose levels, mild to marked epithelial cell vacuolation of the cortical and medullary tubules	Reversible
			• Minimal vacuolation in urinary bladder	Reversible

Note: The corresponding NOEL among the tested dose levels is highlighted. The corresponding NOAEL among the tested dose levels is indicated in bold and underlined.

IV, intravenous; ALT, alanine aminotransferase; F, female; M, male; RBC, red blood cell; NOEL, no-observed-effect level; NOAEL, no-observed-adverse-effect level.

In the rat 28-day toxicity study, no changes were noted in the functional observational battery evaluations, locomotor activity, or during the rotating rod system assessment. Histologically, neither fibrosis nor an increase in TGF-β immunostaining in the skin was evidenced after repeated administrations of gadopiclenol.

Based on these results, the NOAEL was considered to be 5 mmol/kg per day when given as daily IV injection for 14 days in rats, to be 2.5 mmol/kg per day when given as daily IV injection for 28 days in rats, to be 4 mmol/kg per day when given as daily IV injection for 14 days in dogs, and to be 2 mmol/kg per day when given as daily IV injection for 28 days in dogs.

Reproductive and Developmental Toxicity

Rat, Fertility Including Early Embryonic Development

In the fertility study, no toxicity was observed on body weight, sperm analysis, reproductive performance indices, gestation day 13 uterine implantation data, or organ weights at any of the dose levels evaluated.

Transient and nonadverse clinical signs (decreased activity and, in males, swollen forehand hind-limbs and swollen nose/muzzle) were observed during the study, as observed during the repeat-dose toxicity studies in rats. In males and females at 10 mmol/kg per day, a lower body weight gain was observed for the majority of the treatment period (up to −49% lower) and correlated with a statistically lower food consumption (P < 0.01). At 10 mmol/kg per day, the mean cycle length was significantly increased (+52.3%, P < 0.05), and the number of cycles was significantly reduced compared with control females (−22.2%, P < 0.05). However, these effects did not correlate with the effects on reproductive and fertility indices and were not considered adverse. At necropsy, a dose-dependent increase in tan discolored kidneys and enlarged kidneys was observed in gadopiclenol-treated males, not seen in females. These kidney findings correlate with the microscopic findings noted in the previous toxicology studies.

Based on these results, the NOAEL for parenteral toxicity in males and females was 5 mmol/kg per day based on lower body weight change, lower food consumption, and macroscopic kidney findings (males only).

The NOAEL for reproductive performance and fertility in males and females was 10 mmol/kg per day.

Rat and Rabbit, Embryotoxicity

In the embryofetal development studies in rats and in rabbits, some signs of maternal toxicity were shown at highest dose level during the organogenesis period. This was characterized by swelling, decreased activity, lower gestation weight gain, and food consumption. In both species, no treatment-related effects were found on uterine implantation data, fetal sex ratios, and fetal external, visceral, and skeletal examinations. In rabbits only, a lower mean fetal body weight (10%–12% lower than controls) was observed at the high dose, and this was attributed to the maternotoxicity. An increased incidence of tan discolored kidneys at necropsy was also observed, which is consistent with the findings from the general toxicity studies after repeat dosing.

Based on these results, NOAELs for maternotoxicity were established at 5 and 2.5 mmol/kg per day in rats and rabbits, respectively.

The NOAELs for developmental toxicity were established at 10 and 2.5 mmol/kg per day in rats and rabbits, respectively.

Rat, Prenatal and Postnatal Development

Gadopiclenol administrations to pregnant/lactating rats were associated with transient clinical observations in all groups receiving gadopiclenol (considered nonadverse at 2.5 and 5 mmol/kg per day), reduced food consumption during the whole gestation period at 5 and 10 mmol/kg per day, and transient reduced body weight gain at 10 mmol/kg per day (with only a transient and minor impact on absolute body weight). In addition, there was a higher incidence of moribund females and with injection site observations at 10 mmol/kg per day related to the administration procedure but exacerbated by gadopiclenol. The NOAEL for maternal toxicity was therefore considered to be 5 mmol/kg per day.

Effects on subsequent postnatal development of the F1 (first filial) offspring included nonadverse reduced pup body weight gain from birth, leading to reduced body weight up to 4 weeks of age for females and termination (15 weeks of age) for males in the intermediate- and high-dose groups. Despite the effect on growth, there was no influence of maternal treatment on preweaning functional development, postweaning behavior, or reproductive performance.

The NOAEL for postnatal development of the offspring, including reproductive performance, was, therefore, considered to be 10 mmol/kg per day.

Rat, Juvenile Toxicity

Single or repeat IV administration of gadopiclenol to juvenile rats at all tested doses was well tolerated and did not induce any adverse effects (including behavioral testing).

Non–dose-related, decrease in ferritin in males in all treated groups and in females treated at 1.25 and 2.5 mmol/kg per occasion was observed after repeated administrations and was completely reversible after the treatment-free period. In addition, dose-related decrease of serum iron in females, associated at the high dose with increased iron urinary excretion, was observed at all doses after repeated administrations and was completely reversible after the treatment-free period. These effects on iron balance were not observed in repeat-dose toxicity studies performed in adult animals. However, these effects were of low magnitude, not always related to the dose, observed in an immature renal system, and fully reversible. They were not considered as adverse, although relationship to gadopiclenol treatment cannot be ruled out.

Histologically, only nonadverse cortical tubular vacuolation in the kidneys was noted after repeated administrations at all dose levels and was completely reversible after the treatment-free period. These microscopic findings in kidneys were also observed in adult rats after single or repeated administrations.

Based on these findings, the NOAEL was 2.5 mmol/kg per occasion for male and female juvenile rats.

Juvenile Versus Adult Toxicity in Rats

In the study comparing juvenile and adult rats, there was no unscheduled death or treatment-related clinical sign or effect on body weight gain in any group and subgroup. With single or repeated administration of either gadopiclenol or gadodiamide, the linear GBCA was used as comparator, induced at terminal sacrifice, minimal cytoplasmic vacuolation in the proximal convoluted renal tubule epithelium both in adults and juvenile animals. In the absence of degenerative or necrotic changes, it was considered nonadverse. After 8 weeks of recovery period, the reversibility of this finding was complete in all adult and juvenile groups.

Genotoxicity

Gadopiclenol was not genotoxic in vitro in the Ames test and in a mouse lymphoma assay, with or without metabolic activation. A micronucleus test performed in rats (IV route) was negative.

Local Tolerance

Local tolerance at the injection site was satisfactory in the single- and repeat-dose toxicity studies in rats and dogs described previously. When observations were made, they concerned both control and treated groups and therefore were related to the administration procedure and were not indicative of a local toxicity of gadopiclenol. There was no difference between the various formulations used.

In the dedicated study performed in rabbits, there were no deaths or systemic treatment-related clinical signs throughout the study, and there was no effect of treatment on body weight in the treated group.

After IV or IA administration, a transient very slight to moderate erythema was observed (mean irritation index up to 0.7 and 1.8, respectively, on a scale of 0–8), which totally reversed on day 2. This was associated with histopathological findings (moderate vascular intimal necrosis of the arterial wall or minimal erosion of the vascular intima) on day 2 after IA administration, no longer seen on day 4.

After PV administration, very slight to moderate erythema with very slight to moderate edema was noted (mean irritation index up to 2.3), associated with histological changes in dermis and epidermis (dermal hemorrhage, dermal edema, inflammation, dermal and epidermal necrosis, acanthosis), which were still observed on day 4.

Immunotoxicity

In guinea pigs, an IV injection of gadopiclenol did not induce any signs of immediate hypersensitivity in the animals previously exposed to the test item.

Gd Tissue Retention Studies

Rat, Prenatal and Postnatal Development

The results of gadolinium concentration for this study appear in the Supplementary Data, http://links.lww.com/RLI/A876.

Rat, Juvenile Toxicity

Gd concentrations in evaluated tissues (kidneys, liver, bone, skin, cerebellum and brain) increased with increasing doses in all tissues after single and repeated doses, at the end of the treatment period or after a 9-week recovery period. At the end of the 9-week recovery period, the tissues with highest Gd concentrations were the kidneys, followed by the femur, brain, cerebellum, liver, and finally the skin. Overall, higher Gd concentrations were observed after repeat versus single dosing in all tissues from both sexes. No sex-related difference was observed (Figs. 3, 4).

FIGURE 3.

Washout of Gd in different tissues (brain, cerebellum, femur, kidneys, liver, skin) of juvenile rats after single or repeated administrations of gadopiclenol at different dosages and after a recovery period (9-week treatment-free period).

FIGURE 4.

Mean Gd concentration in different tissues in juvenile rats after single or repeated administrations (total of 6 administrations) of gadopiclenol at different dose levels with a recovery period (9 weeks) or without (up to 2 days after the last day dosing).

After single dose and 9-week recovery period, the Gd concentrations were very low and often below or close to the lower limit of quantification (LLOQ) in all tissues, except in the kidneys of all groups in both sexes. The clearance of Gd was nearly complete (above 96% and up to 100%), compared with the end of the dosing period (Figs. 3, 4).

After repeated dose and 9-week recovery period, Gd concentrations were low and often below or close to the LLOQ in all tissues, except in the femur (up to 9.8 nmol/g) and kidneys (up to 56.6 nmol/g) (Figs. 3, 4). Clearance of Gd from the brain, cerebellum, and femur was above 78% and up to 96%, and in the kidneys, liver, and skin above 96% and up to 100% compared with 2 days after the end of the dosing period (Figs. 3, 4).

Gd Tissue Retention in Juvenile and Adult Rats

In adult and juvenile rats treated with gadopiclenol on single and repeated occasions, the highest Gd concentration after administration was detected in the kidneys, followed by the liver, femur or skin, cerebellum or brain, and plasma (Fig. 5). The day after dosing, the Gd concentrations of juvenile animals compared with adults were lower in the kidneys (approximately 2- to 6-fold), higher in the brain and cerebellum (approximately 2- to 10-fold), but similar in other tissues. After repeated dosing with and without recovery, and single dosing with recovery, these values were equivalent between juvenile and adult animals (Fig. 5).

FIGURE 5.

Mean Gd concentrations in different tissues in rats (juvenile and adult) after single or repeated administrations (every 4 days during 8 weeks) of gadopiclenol or gadodiamide (0.6 mmol/kg per occasion) with a recovery period (8 weeks) or without (the day after dosing).

After the recovery period, tissue Gd concentrations were very low and close to the LLOQ in all tissues, except in the femur and mainly the kidneys.

In juvenile rats, after single dose and recovery period, tissue Gd concentration decreased by more than 95%, compared with the day after dosing. In animals receiving repeated administrations and with a recovery period, the same decrease was observed in the kidneys, liver, plasma, and skin. The decrease was comprised between 71% and 85% in the brain and cerebellum, and up to 55% in the femur.

In adult rats, after single dose and recovery period, values decreased by more than 95%, compared with the day after dosing, except for the femur (up to 77%). In animals receiving repeated administrations, these values decreased by more than 90% in the kidneys, liver, plasma, and skin, up to 82% in the brain, up to 93% in the cerebellum, and up to 28% in the femur.

Overall, the tissue Gd concentrations were much lower after gadopiclenol treatment than gadodiamide treatment at the end of the dosing period and after an 8-week recovery period with either single or repeat dose, except in the kidneys and liver of adult rats after the end of repeated dosing with approximatively equivalent Gd mean concentrations (see Fig. 5). The difference in tissue Gd concentrations between both GBCAs was more pronounced after repeat dosing and end of the treatment-free period. The tissue Gd washout during the 8-week treatment-free period was lower after gadodiamide administration than gadopiclenol: mainly in the bone (after repeated doses a reduction up to 14% for gadodiamide vs reduction up to 28% for gadopiclenol), followed by the brain/cerebellum (after repeated doses a reduction up to 32% for gadodiamide vs reduction up to 93% for gadopiclenol), and the skin (after repeated doses a reduction up to 25% for gadodiamide vs reduction up to 94% for gadopiclenol).

DISCUSSION

The objective of safety pharmacology is to investigate the potential undesirable pharmacological effects of a substance on functions of vital organs and systems in relation to exposure in the therapeutic/diagnostic range and above.²² Those studies conducted at high-dose levels showed a satisfactory tolerance of gadopiclenol on main body systems. Some treatment-related effects were noted on the cardiovascular system in anesthetized dogs, with some slight to moderate hemodynamic changes, sometimes transient and not always dose-related. In vitro, no statistically significant effect on the hERG tail current was observed up to 2.5 mmol/L of gadopiclenol, which represents 4.6 times the C_max obtained in humans after a single IV administration at the clinical dose level of 0.05 mmol/kg.¹² At higher concentrations, the inhibition of the hERG current observed should not be attributed to a deleterious effect of gadopiclenol on cardiac repolarization but rather to a nonspecific and nonbiologically relevant effect. Similar results were described for other approved GBCAs,^23,24 and a specific effect of those agents on hERG current has always been ruled out. This effect is probably driven by hyperosmolality of these substances, as suggested elsewhere.^23,24 This effect was not confirmed by another in vitro study on rabbit Purkinje fibers where no effect on action potentials was observed up to 10 mmol/L. In conscious dogs, no modifications were seen, as well as no effect on QT/QTc interval, up to 22 times the human dose level. In addition, the absence of any treatment-related effect on the ECG was confirmed in single and repeat-dose toxicity studies in dogs up to the maximum dose tested (4 mmol/kg, ie, 26 times the HED) and after 28 daily administrations. In a clinical study assessing the effect of gadopiclenol on the QTc interval, the macrocyclic GBCA did not prolong the QT interval at clinical and supraclinical doses (up to 0.3 mmol/kg) and was well tolerated in healthy volunteers.²⁵

Gadopiclenol did not exert any relevant effect on respiratory parameters in conscious rats.

No effect on the main central and peripheral nervous system functions was attributable to gadopiclenol. A minor proconvulsant effect was observed at the highest tested dose in PTZ-treated rats (at 5 mmol/kg, ie, 16 times the HED). Seizure is a known adverse effect of the GBCA class,^26,27 even if there is no evidence suggesting that gadopiclenol directly precipitates convulsions at the human dose from the clinical trials conducted with this agent.^10,11,13,14

Even if the effects on renal function are generally well addressed during toxicology studies and are not included in the ICH S7A safety pharmacology core battery, it is of particular importance to focus on the functional effects of a contrast agent on kidneys, because it is the excretory organ for such products, and consequently a potential target organ. Furthermore, it is expected to be the most sensitive system due to the osmolality of the product. Within this context, renal function was evaluated in both toxicology and specific safety pharmacology studies in rats treated with gadopiclenol. In the dedicated safety pharmacology study, gadopiclenol exhibited only a slight dose-related antidiuretic and antinatriuretic effect up to 6 hours after the injection.

The objective of toxicology studies is to evaluate the safety of the candidate drug by a characterization of potential adverse effects with respect to target organs, dose dependence, relationship to exposure, and, when appropriate, potential reversibility. It will enable a risk benefit assessment in order to support the clinical trials and marketing authorization. The ultimate goal is to translate the animal model responses into an understanding of the risk for human subjects.

Rats and dogs were chosen as the nonclinical species based on the standard acceptance of these species in nonclinical toxicology studies and the demonstrated drug exposure in these species after IV administration and a lack of metabolism demonstrated in all species. Otherwise, concerning the choice of the nonrodent species, dogs are the most commonly used species in nonclinical studies of pharmaceuticals when not disqualified for metabolic or physiological considerations. Furthermore, dogs are a well-adapted model for IV route administration, especially to assess hemodynamic or cardiovascular effects.

In single-dose toxicity studies in mice, rats, and dogs, a single injection of gadopiclenol up to dose levels 25 to 59 times the human dose based on comparison of systemic exposure revealed a low acute toxicity and was well tolerated. No or only minor and reversible effects were observed attributable to acute circulatory disorders caused by bolus injection of hypertonic and viscous solutions: mainly minimal to mild tubular cell vacuolation observed in the kidneys, with a dose-related incidence and severity, and transient swelling of the face and/or limbs (reversible in a few hours) only observed at the highest injected doses due mainly to the injection of high volumes of a hyperosmolar solution with high viscosity. The studies are deemed to be the most relevant ones for human risk assessment of a compound given as a single dose in humans.

Although GBCAs are intended to be used as single IV administration in patients, gadopiclenol was tested in repeat-dose toxicity studies in rats and dogs after daily IV administration, over a period of up to 4 weeks, as recommended by current guidelines,¹⁸ which are very drastic conditions in terms of dose levels and duration of dosing compared with the conditions for human use. Gadopiclenol was well tolerated in both species, even at very high repeated dose levels. Mortality was never observed with gadopiclenol in rats and dogs, even at high-dose levels. The most remarkable finding after single and repeated administrations was vacuolation of the renal tubular epithelial cells, with a dose-related incidence and severity and exacerbated, particularly in rats, by repeat dosing. This is a classical finding will all GBCAs^28–31 and iodinated contrast agents³² administered at high volume, which was associated with no functionally significant impairment of tubular or cellular processes and no physiological consequences on kidney function. This phenomenon was mainly caused by the presence of the contrast agent in kidneys and is considered to be without clinical relevance.

Some additional treatment-related adverse effects were also seen in rats, on body weight, food consumption, clinical signs, clinical pathology parameters, and histology (vacuolated/granular macrophages), but these changes were always partially or totally reversible. No additional major changes due to repeat dosing were seen in dogs: only minor effects such as discoloration of kidneys, kidney weight increase, or face and ear swelling were noticed.

Transient swelling of face and/or limbs (reversible in a few hours) was only observed at the highest injected doses and volumes of a hyperosmolar test compound with relatively high viscosity. Consequently, these effects are considered as nonadverse. In repeat-dose studies, the swelling was mainly observed during the first week of dosing, showing that animals were progressively adapting to the intravenous injection of the highest volumes of a hyperosmolar product. In almost all the listed studies, the higher frequency in swelling was observed in males, which is consistent with an effect related to the volume administered since males received the highest volumes (with a mean body weight 10% to 30% higher than that of females). The clinical relevance of the swelling of face and/or limbs observed after single or repeated administration with gadopiclenol in nonclinical studies is considered negligible, due to high margin of safety in respect to this finding.

Since 2006, there has been a growing safety concern regarding the association administration of GBCAs, especially linear ones, and the occurrence of NSF. The low kinetic inertness of the linear complexes (and the consequential possible release of free Gd in the body) has been recognized as a risk factor to trigger NSF symptoms.³³ Such a risk is obviously increased in patients suffering from a severe renal impairment, as the elimination of the contrast agent is much slower in these patients. Gadopiclenol is a nonionic macrocyclic GBCA with a high kinetic inertness (dissociation half-life of 20 ± 3 days at pH 1.2),⁹ suggesting a very low risk for NSF induction. In the 28-day toxicity study in rats, the histologic staining of the key marker of fibrosis TGF-β^34,35 in the dorsal lumbar skin sections of males and females at 10 mmol/kg per day was comparable to the one observed with the concurrent control. Furthermore, neither macroscopic skin lesions nor dermal fibrosis at histopathology was observed in the other single or repeated-dose toxicity studies. This is also confirmed by Fretellier et al³⁶ who demonstrated in a rat model of severe renal failure no evidence of biochemical toxicity or pathological abnormalities of the skin after a treatment of 5 consecutive intravenous injections of gadopiclenol at 2.5 mmol/kg/injection (corresponding to 8 times the HED) versus 2 widely used macrocyclic GBCAs, gadoterate meglumine and gadobutrol, and 1 linear GBCA, gadodiamide (at the same dose levels). Gadopiclenol did not induce any clinical histological or biochemical (including creatinine clearance) abnormalities, as observed with the 2 others macrocyclic GBCAs tested. On the contrary, the linear GBCA gadodiamide was associated with serious systemic toxicity (morbidity-mortality) and macroscopic and histological NSF-like skin lesions. Overall, this study on sensitized rat models of NSF did not suggest any profibrotic risk associated with the use of gadopiclenol.

In reproductive and developmental toxicity studies, the effects of gadopiclenol were assessed on all phases of reproduction, including fertility, early and late embryonic phase, fetal development phase, and perinatal and postnatal development of the offspring generation and in juvenile animals. No effect was evidenced on reproductive performance and fertility in male and female rats. Contrary to some GBCAs after repeated administrations (decrease of testes weight and degeneration of spermatogenic cells),^28,37 no effect on testes was observed with gadopiclenol. The embryofetal development studies in rats and rabbits and the prenatal/postnatal study in rats showed some signs of maternotoxicity in both species (swelling, decreased activity, lower gestation weight gain, and food consumption) at the highest dose (corresponding to 32 times the HED) but did not evidence any teratogenicity in both rats and rabbits. Gadopiclenol administrations to pregnant/lactating rats were associated with a nonadverse reduced pup (F1 offspring) body weight gain but with no influence of maternal treatment on preweaning functional development, postweaning behavior, or reproductive performance. Overall, these studies do not indicate any direct or indirect harmful effects with respect to reproductive toxicity, even if little placental transfer and low milk excretion have been evidenced in animal studies (data not shown). However, as for all other GBCAs, gadopiclenol should not be used during pregnancy unless the clinical condition of the woman requires its use and continuing or discontinuing breast feeding for a period of 24 hours after administration of gadopiclenol should be at the discretion of the doctor and lactating mother.^38,39

The safety profile of gadopiclenol in juvenile rats was satisfactory, like in adults. No new target organ was evidenced, and the NOAEL was the maximum tested dose level, corresponding to 8 times the HED. Only minor effects on the iron balance completely reversible were observed in juvenile animals. However, they were not observed in repeat-dose toxicity studies performed in adult animals at the end of treatment period or after the treatment-free period while higher dose levels were tested. Consequently, they were not considered as adverse: low magnitude, not always related to the dose, and fully reversible. This good safety profile in juvenile animals is consistent with the results of the clinical studies conducted in pediatric patients (2–17 years)⁴⁰ that did not reveal any safety issues on this population.

Gadopiclenol was not genotoxic in vitro and in vivo. A carcinogenic study with gadopiclenol was not performed because neither the substance nor the other GBCAs showed genotoxic properties or toxic effects on organs with a high cellular replication rate such as the bone marrow or gut mucosa.⁴¹ In addition, gadopiclenol will not be administered repeatedly daily to humans for diagnostic purposes (only episodically). Therefore, there is no concern about a carcinogenic potential of gadopiclenol.

The local tolerance of gadopiclenol appears acceptable after a single administration in rats (IV), dogs (IV), and rabbits (IV, IA), or after repeated IV injections in rats and dogs, but signs of intolerance/irritations were observed after a single PV and repeated IV administrations in rabbits, indicating that misadministration must be avoided like for other approved GBCAs.

Gadopiclenol did not exhibit any signs for potential to induce immediate hypersensitivity, and only minor bronchoconstriction was seen in a safety pharmacology study in guinea pigs with the highest doses tested. No diagnosis of hypersensitivity was reported during the completed clinical trials. However, as with any contrast agent, anaphylactoid/hypersensitivity reactions may occur after gadopiclenol administration.

Initially demonstrated by Kanda et al⁷ in 2014 and further confirmed in the literature by numerous clinical^42,43 and preclinical studies,^44–46 small quantities of Gd were observed in tissues including the brain (with no blood-brain barrier disruption) of subjects with or without normal renal function who received cumulative doses of GBCAs (especially linear). Although it remains unclear whether these findings have any clinical relevance^47–49 (no enough evidence to demonstrate that dechelated Gd is present in any tissue long enough to manifest a toxicologic event), the retention of Gd in healthy patients has evoked safety concerns surrounding the application of GBCAs.⁵⁰ The American College of Radiology proposed recently to use the new term SAGE (symptoms associated with gadolinium exposure) to describe symptoms related to Gd exposure. This refers to symptoms that may occur irrespective of kidney function and are unrelated to established early onset (such as acute hypersensitivity) and late onset (such as NSF). Symptoms are numerous and nonspecific, ranging from headache to bone and joint pain to peripheral neuropathic pain. However, the FDA and other regulatory agencies or professional societies have concluded that there is no convincing causal evidence establishing a direct link between Gd tissue retention and any other GBCA-specific etiology with these SAGE complaints.⁵¹

That is why Gd tissue presence was investigated in 3 studies during the regulatory nonclinical program of gadopiclenol: (a) juvenile toxicity study in rats, (b) Gd tissue retention in juvenile and adult rats, and (c) prenatal/postnatal development study in rats. These studies were the last studies performed during the nonclinical program. This information is useful to better understand the Gd distribution in the body after single or repeated administration and in different age categories. The selected tissues (brain/cerebellum, skin, kidney, femur, liver) correspond to the target organs for Gd retention.

Whatever the study where tissue Gd presence was investigated (up to 9 weeks after dosing), the highest Gd content was in the kidneys, followed by the bone or liver, skin, and brain/cerebellum. Higher Gd presence was observed after repeated versus single administration in some tissues. A massive Gd washout after single and repeat dosing, except in bone, was observed during the treatment-free period. At the end of the recovery period, only traces of Gd were quantifiable almost exclusively in the kidneys (excretory organ) and to a lower level in the bone. Tissue Gd presence was also globally similar between juvenile and adult animals. Only a small difference was observed after single administration with no recovery. These are likely due to the immaturity of the kidneys and brain at this age, the first administration occurring at PND 10. In fact, renal function is immature at birth, and glomerular filtration rate continues to increase up to approximatively PND 20.⁵² Similarly, the blood-brain barrier is established only at PND 1–3 in rats,⁵³ but some others indicate functionality at approximately PND 10, and functionality is only fully achieved at PND 33–40.⁵⁴ Certain transport mechanisms are unique to the developing brain and may lead to increased drug entry into the brain.⁵⁵ In the present studies, Gd presence in the brain had no impact on neurodevelopment as assessed by validated neurobehavioral tests and histopathology (including brain) and prenatal/postnatal development. This was consistent with literature data with other macrocyclic GBCAs.^56–59 These different studies confirmed that, despite of Gd retention in different tissues after administration of gadopiclenol (single or repeated administrations), no clinical effect was associated with this finding.

Interestingly, in the study including a group of adult rats receiving gadodiamide as a comparator in the assessment of potential tissue Gd presence, the Gd concentrations were much lower in tissues after gadopiclenol treatment than gadodiamide treatment at the end of the dosing period and after an 8-week recovery period with either single or repeat dose, except in the kidneys and liver of adult rats after the end of repeated dosing with approximatively equivalent Gd mean concentrations. The difference in tissue Gd concentrations between both GBCAs was more pronounced after the repeat dosing and end of treatment-free period. This is related to the tissue Gd increased accumulation observed after repeated administrations of gadodiamide and the lower tissue Gd washout during the 8-week treatment-free period with this GBCA (mainly in the bone, followed by the brain/cerebellum and the skin).

Juvenile toxicity studies with similar design at similar dose levels were also performed with other GBCAs (gadoterate meglumine/Dotarem⁵⁸ and gadobenate dimeglumine/MultiHance⁵⁶). Similar Gd concentrations were found after administration of gadopiclenol and gadoterate meglumine,⁵⁸ except in the kidneys and femur, where lower Gd concentrations were measured for gadoterate meglumine, only after repeated administrations (femur) and recovery period (kidneys), indicating a possible faster Gd elimination with gadoterate meglumine in these tissues. A higher Gd washout was evidenced after the 9-week treatment-free period for gadopiclenol compared with gadobenate dimeglumine,⁵⁶ except in the kidneys after repeat dosing. However, 50% of gadobenate dimeglumine is excreted by the liver⁶⁰ in rats unlike other GBCAs; this may explain the lower kidney Gd concentrations. The Gd concentrations analyzed in the brain/cerebellum and bone after repeated administration of gadopiclenol with recovery are much lower than those of gadobenate dimeglumine (for example at high-dose level, in the brain 4.7/6.6-fold lower for males/females, respectively, and in bone 4.5/2-fold lower for males/females, respectively).

Recently, an exploratory study has also demonstrated a difference in the Gd distribution and washout in the brain of healthy adult rats after long-term exposure (over 1 year) between macrocyclic GBCAs including gadopiclenol and the linear GBCA gadodiamide.^44,61 For both macrocyclic agents tested (gadopiclenol and gadobutrol), similar in vivo Gd distribution and washout in the brain were observed: no intrinsic signal hyperintensity on T1-weighted images in deep cerebellar nuclei, continuous washout over time, approximately 80% of Gd washout between 1 month and 12 months during the treatment-free period, and no binding to macromolecules. In contrast, gadodiamide, the linear GBCA tested, led to significantly higher Gd concentrations after 1 month in the cerebellum (at least 7-fold higher) combined with only 15% washout after 12 months and with evidence of Gd bound to macromolecules. Consistent with these results, a difference in brain distribution of gadopiclenol versus another linear GBCA, gadobenate dimeglumine, was previously shown 1 month after repeated administrations in healthy rats, and Gd retention was also minimalized in the case of gadopiclenol compared with gadobenate dimeglumine, resulting in no T1 hypersignal in the deep cerebellar nuclei.⁶¹ Furthermore, the body Gd exposure over a period of 5 months in healthy rats after a single injection of gadopiclenol or gadobutrol, both administered at the HED, has been evaluated in another rat study, showing a similar distribution profile. In all tissues except bone, Gd concentrations at half dose of gadopiclenol were lower than compared with gadobutrol at clinical standard dose during the first month after injection. A strong washout for both contrast agents is observed in all tissues during the period of follow-up. Therefore, for the same diagnostic efficacy, the Gd exposure after gadopiclenol is lower than that of the macrocyclic GBCA gadobutrol, thanks to the half-injected dose.⁶²

In these different studies, for all the tested GBCAs, Gd seems to have a particular affinity for bone (high Gd accumulation and slower Gd clearance than in other tissues). It is well known that bone is a deep compartment for Gd retention.⁶³ Higher Gd accumulation and retention was evidenced after administration of linear GBCAs than macrocyclic GBCAs, as observed in our study including a linear GBCA as comparator (gadodiamide). In their rat study, Schlatt et al⁶⁴ have shown that linear GBCAs accumulated significantly more Gd than macrocyclic GBCAs, but they have also observed, for the first time, that the Gd found in bone samples was mostly present as dechelated Gd or otherwise bound to endogenous components, but not complexed to its initial ligand, which is not the case in other tissues, even for macrocyclic GBCAs. The retention of unchelated Gd ion may be important clinically, because Gd is not a naturally occurring biological constituent, and once within the tissues, it persists for long periods with almost no washout, as observed in preclinical studies^45,65 but also in patients.⁶⁶ Despite the long-term retention of Gd observed in this tissue, no toxic effects were attributed to this specificity.

No difference in bone Gd concentrations was evidenced between juvenile and adult rats in our study, which is consistent with the results found with gadoterate meglumine, a macrocyclic GBCA, whereas higher Gd distribution in bone was found in juvenile than in adult rats with gadodiamide.⁵⁷

In conclusion, the toxicity profile of gadopiclenol was well characterized in a complete program of nonclinical studies. These studies have demonstrated the good tolerance of gadopiclenol, even at dose levels much higher than the clinical dose (the intended dosage being half the conventional dose). The safety margin is considerable. Nephrogenic systemic fibrosis has been effectively controlled by restrictions on GBCA use in patients with renal failure, and although investigated thoroughly, no toxicological consequence of Gd retention in the brain or other tissue has been found yet. The good clinical and biochemical safety profile of gadopiclenol in this substantial program is consistent with the good safety data obtained in humans during clinical trials,^10–14 including patients with mild to severe renal impairment,⁶⁷ and in the pediatric population.⁴⁰

Supplementary Material

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