Acute Rodent Tolerability, Toxicity, and Radiation Dosimetry Estimates of the S1P1-Specific Radioligand [¹¹C]CS1P1

Abstract

Purpose:

In preclinical studies with rodent models of inflammatory diseases, [¹¹C]CS1P1 has been identified as a promising imaging agent targeting sphingosine-1-phosphate receptor 1 (S1P1) in central nervous system and other tissue. In preparation for USA Food and Drug Administration (FDA) approval of [¹¹C]CS1P1 for human use, an acute biodistribution study in mice and an acute tolerability and toxicity evaluation in rats were conducted.

Procedures:

Acute organ biodistribution and excretion data was obtained using male and female Swiss Webster mice intravenously (IV) injected with 4.8-10 MBq of [¹¹C]CS1P1. The organ residence times for each harvested organ were calculated using the animal biodistribution data, and were entered in the program OLINDA/EXM for C-11 to obtain human radiation dosimetry estimates. Acute tolerability and toxicity studies were conducted in male and female Sprague Dawley rats. Rats were administered an IV bolus of either the vehicle control or 0.3 mg/kg CS1P1. Blood samples were collected and a gross post-mortem examination was conducted at day 2 or day 15 post injection.

Results:

The extrapolated human radiation dose estimates revealed that the highest organ dose was received by liver with 24.05 μGy/MBq in males and 32.70 μGy/MBq in females. The Effective Dose (ED) estimates of [¹¹C]CS1P1 were calculated at 3.5 μSv/MBq in males and 5.9 μSv/MBq in females. The acute tolerability and toxicity study identified 0.3 mg/kg as a no observable adverse effect level (NOAEL) dose, which is a ~300-fold dose multiple of the human equivalent dose of the mass to be injected for positron emission tomography (PET) imaging studies in humans as a no-observable effect limit.

Conclusions:

The toxicity study in rats suggested that injection dose of radiotracer [¹¹C]CS1P1 with mass amount < 10 μg is safe for performing a human PET study. The Dosimetry data supported an injection of 0.74 GBq (20 mCi) dose for human studies would be acceptable.

Introduction

S1P is a bioactive lysophospholipid metabolite that exerts pathophysiological functions through binding to the G protein-coupled receptors S1P1-5 [1-2]. S1P1 is among the most abundant receptors in this family. Dysregulation of S1P/S1P1 signaling is associated with various inflammatory diseases [3]. S1P1 is extensively expressed on lymphocytes and endothelial cells, and it participates in the neuroinflammatory process by regulating immune cell trafficking in the brain [1]. In the central nervous system (CNS), S1P1 is expressed in neurons and glial cells including microglia and astrocytes [3-4]. Recently the S1P1 modulator fingolimod (FTY720) was approved by FDA to treat relapsing-remitting multiple sclerosis (RR-MS), which is a chronic autoimmune, inflammatory, demyelinating neurodegenerative disease [5]. S1P1 has been involved in the multiple pathophysiologic events of MS, including regulating lymphocyte egress into the circulation, activating glial cells and potentially disrupting blood-brain barrier [6-7]. For example, S1P/S1P1 signaling directly activates the Jak-STAT3 signal-transduction pathway via IL-6, whereas impaired S1P1 phosphorylation enhances TH17 polarization and exacerbates neuroinflammation [8]. S1P1 signaling has also been linked to pathophysiology and progression of cancers. High S1P1 expression in tumors is associated with shorter disease-specific survival of estrogen receptor positive (ER+) breast cancer patients [9]. S1P1 is also involved in regulating the neovascularization of tumors. FTY720 decreased tumor-associated angiogenesis and suppressed tumor cell proliferation, thus inhibited tumor growth in a mouse melanoma model [10]. In addition, S1P/S1P1 is also implicated in modulating the expression of hypoxia-inducible factor 2 alpha (HIF2α), which can drive aggressive cancer [11].

Given the importance of S1P1 in inflammatory diseases, cancer, and other diseases, positron emission tomography (PET) with a specific S1P1 radioligand would provide a unique imaging tool to quantify S1P1 expression in vivo, and to evaluate neuroinflammatory response and therapeutic effects. Our group reported an S1P1-targeted PET radiotracer, [¹¹C]CS1P1, named [¹¹C]TZ3321 (IC₅₀ = 2.13 ± 1.63 nM for S1P1, > 1000 nM for S1PR2-5, Fig. 1), which showed great promise for quantifying S1P1 expression in rodent models of inflammation [12-15]. The increase of [¹¹C]CS1P1 uptake is correlated to the higher expression of S1P1 at sites of inflammation in three animal models including the rat experimental autoimmune encephalomyelitis (EAE) model of MS [14]; the ApoE^−/− mouse femoral artery wire-injury model of neointimal hyperplasia [12]; and the rat carotid injury model of vascular inflammation [13]. The increase of S1P1 expression was also observed in human and murine atherosclerotic plaques [15].

Fig. 1.

Chemical structure of [¹¹C]CS1P1.

Based on the breadth of our promising results with this tracer in different rodent models of inflammatory diseases and human atherosclerotic plaques, and PET brain imaging studies in macaques which suggested that [¹¹C]CS1P1 has favorable pharmacokinetic properties and good metabolic stability (unpublished data), we conducted additional studies to evaluate the safety of CS1P1 based on FDA regulatory of PET radiopharmaceuticals for human use. Human radiation dosimetry estimates were calculated from acute biodistribution and excretion studies of [¹¹C]CS1P1 in adult male and female Swiss Webster mice. The single-dose acute tolerability and toxicity study was carried out in adult male and female Sprague Dawley (SD) rats by the contract research organization (CRO), Seventh Wave, LLC, following the recommendation of the FDA’s August 2018 Microdose Radiopharmaceutical Diagnostic Drugs: Nonclinical Study Recommendations, Guidance for Industry. The August 2018 recommendations are intended to reduce the time and resources expended in microdose radiopharmaceutical drug development without compromising patients.

Materials and Methods

All applicable institutional and/or national guidelines for the care and use of animals were followed in the acute biodistribution study in mice and the acute tolerability and toxicity evaluation in rats.

Human dosimetry estimate based on acute biodistribiton of [¹¹C]CS1P1 in mice

The acute biodistribution of [¹¹C]CS1P1 was carried out using standard ex vivo organ dissection methodology in groups of male and female Swiss Webster mice. Studies were carried out over two days. Residence times were computed and absorbed doses were then derived using the standard Medical Internal Radiation Dose (MIRD) methodology [16]. The human residence times were extrapolated from mice using simplifying assumptions on species differences. Human radiation dosimetry dose estimates were then calculated by the OLINDA/EXM dosimetry software.

Radiochemistry

Radiosynthesis of [¹¹C]CS1P1 was carried out according to our previously published procedure [12]. The radiochemical yield was ~10% (decay corrected to end of bombardment) with a radiochemical purity of > 99%, chemical purity of > 95%, and molar activity > 145 GBq/μmol (decay corrected to end of bombardment).

Biodistribution in Healthy Mice

Acute organ biodistribution and excretion data was obtained using male and female Swiss Webster mice (Charles River Laboratories, Inc., Wilmington, MA) intravenously (IV) injected with 4.8-10 MBq of [¹¹C]CS1P1 in 100 μl 10% ethanol/saline. The average mouse weight was 31.0 g for males and 24.7 g for females and the age was between 8 - 10 weeks. Animals were euthanized under anesthesia at 5 min, 15 min, 30min, 60 min and 90 min post injection (n = 5 for each time point each sex). Samples of blood, lungs, liver, gall bladder, spleen, kidneys, bladder, muscle, heart, brain, fat, bones, red marrow, testes, adrenals, thyroid, pancreas, uterus, ovaries, stomach, small intestine, upper and lower large intestines were collected, weighed and counted in a gamma counter. In addition, the last group of mice was kept in metabolic cages, and samples of urine and feces excretion were also weighted and counted in the gamma counter.

Residence Times

The residence times (in min) for each organ were obtained by numerical integration of the mouse biodistribution data, which is in the unit of percentage injected dose per gram. We assumed that there was no biological excretion occur after 90 min post injection of tracer and that the decrease of radioactivity was fully attributed to physical decay. Then the human organ residence times were calculated using the “relative organ mass scaling” method for most organs [17]. For the gallbladder, stomach, small intestine, upper and large intestines, the residence times were calculated using the percent injected dose in the organ.

The remainder of the body residence time was calculated from the maximum theoretical residence time minus the excreted residence time minus the sum of all residence times measured in the organ above at the exception of blood and fat. This resulted in a residence time associated to the remainder of the body of 10.80 min in males and 10.20 min in females. Activity in bone was assumed to be uniformly distributed to the entire bone volume and divided in equal part to trabecular and cortical bones. The blood was assumed to be 7% of the human body mass. The errors bars on the measured residence times were determined from the standard deviation of the biodistribution data points. Minimal excretion was observed in the urine and feces and thus no excretion was assumed in the calculations.

Radiation Dosimetry

The residence times were entered in the program OLINDA/EXM 1.1 for C-11 and using the standard MIRD adult male and female models, to obtain human radiation dosimetry estimates. The error bars on the radiation dose estimates were assumed to be in proportion of the organ residence time uncertainties.

Acute tolerability and toxicity studies of CS1P1 in rats Methods and rationale for dose preparation and dose verification

Based on our prior experience with ¹¹ C-labeled PET radiopharmaceuticals, the maximum mass of CS1P1 allowed for human PET imaging studies will be 10 μg per patient dose. This mass limit was readily achievable in our cGMP Cyclotron Facility. As noted above, proposed human PET studies with [¹¹C]CS1P1 fall under the FDA’s guidance for toxicology studies for microdose drugs and approval for first-in-human studies will be pursued through the exploratory Investigational New Drug (IND) mechanism. The FDA July 2005 guidelines for allometric scaling from human to rat were used to calculate a ~300-fold dose multiple of the human equivalent dose (HED, the rat dose is divided by 6.2 to obtain the HED). The CS1P1 was administered IV, since this is the route of administration for the radiopharmaceutical. Due to the solubility of the compound, CS1P1 could not be formulated in 10% ethanol/saline at a concentration of ~300 ppm, therefore the vehicle used for the acute toxicity study of CS1P1 was 10% PEG-300, 30% hydroxypropyl beta-cyclodextrin, and 60% sterile water for injection. All components of the vehicle for the rat study were approved for use in human IV formulations and the reagents used to prepare the dose were USP or National Formulary Grade. Following preliminary studies which included characterization of the cold standard (the HCl salt of CS1P1), validation of high performance liquid chromatography (HPLC) conditions needed to accurately determine the concentration of the injectate, and the stability of the compound after filtration and over the duration of the toxicity study, compounding and sterile filtration of the vehicle, and the drug dose verification were carried out with the assistance of the Washington University School of Medicine Cyclotron Facility. A dose of ~ 0.3 mg/kg and an injection volume of 5 ml/kg was used for the rat study. Immediately following HPLC confirmation of the concentration and purity of sterile-filtered drug, the drug and sterile filtered vehicle were delivered to the staff of the CRO. HPLC injections of an aliquot of reserved injectate were repeated ~ 5 hours later after the in vivo injections had been completed.

Protocol for study conducted by the CRO

Acute tolerability and toxicity studies were conducted by the CRO, Seventh Wave, LLC. Male and female SD rats were used and divided into two groups with different endpoints (Day 2 or Day 15 post injection). Each treatment group was comprised of 10 male and 10 female rats. Rats were administered an IV bolus of either the vehicle control or 0.3 mg/kg CS1P1 via an indwelling tail vein catheter. The injection volume was adjusted for individual body weight at 5 ml/kg dosing to ensure that each animal received the appropriate dose. Clinical observations and body weights were recorded once daily. Blood samples for the evaluation of hematology and clinical chemistry endpoints were collected on either Day 2 or Day 15 from both groups. Following blood sample collections, rats were euthanized and a gross post-mortem examination was conducted. The following tissues were collected and evaluated grossly by the CRO study pathologist: adrenal glands, brain, cecum, colon, duodenum, epididymis, eyes, femur with bone marrow, heart, ileum, injection site (IV), jejunum, kidneys, liver, lung, lymph node (mesenteric), mammary gland (both genders), ovaries, pancreas, skeletal muscle (quadriceps femoris), spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thyroid with parathyroids, urinary bladder, uterus, and vagina.

Results

Dosimetry studies of [¹¹C]CS1P1 in mice

The mean concentration of radioactivity in each organ is shown in Table 1 and 2. [¹¹C]CS1P1 accumulation was high in the liver, gall bladder and kidney in both male and female Swiss Webster mice. The liver uptake (% injection dose/gram, %ID/gram) increased from 15.87 (male)/18.72 (female) at 5 min post injection to 46.17 (male)/ 65.07 (female) at 90 min. The initial uptake in gallbladder was 9.72 in males and 42.77 in females at 5 min, and the values escalated to 37.91 and 126 at 90 min, respectively. The kidney uptake increased from 16.56 at 5 min to 29.47 at 90 min in males, but changed less in females (18.57 at 5 min and13.77 at 90 min). The biodistribution pattern was consistent with our previous findings in male C57/BL6 mice [12]. The organ residence times (RTs) extrapolated to human (adult) are listed in Table 3 and Fig. 2. The highest cumulative activity was found in the liver, with 8.34 ± 1.50 min in males and 8.58 ± 1.44 min in females. The RT values for small intestines were 1.80 ± 0.24 min in males and 2.70 ± 0.60 min in females. The data indicated that the tracer was mainly excreted by the hepatobiliary system, as this accounted for the excretion of > 30% activity, whereas merely <0.1% activity was found in the urinary bladder.

Table 1.

Organ activity concentration from male mice dissection expressed in percent injected dose per gram of tissue (%ID/g).

Organs	5 min	15 min	30 min	60 min	90 min
Blood	1.78 ± 0.29	1.61 ± 0.30	1.26 ± 0.60	1.04 ± 0.74	1.34 ± 0.11
Lung	11.38 ± 2.61	7.82 ± 2.73	3.87 ± 1.45	2.61 ± 0.78	3.74 ± 0.56
Liver	15.87 ± 7.96	38.53 ± 3.36	39.09 ± 7.47	40.24 ± 15.62	46.17 ± 3.66
Spleen	4.27 ± 0.78	3.04 ± 0.42	1.47 ± 0.24	1.14 ± 0.78	2.04 ± 0.26
Kidney	16.56 ± 1.95	28.53 ± 3.62	23.23 ± 3.78	28.91 ± 9.69	29.47 ± 3.86
Bladder	1.30 ± 0.33	1.83 ± 0.32	0.96 ± 0.22	0.58 ± 0.79	1.36 ± 0.71
Gallbladder	9.72 ± 6.93	17.88 ± 13.50	22.07 ± 3.85	30.06 ± 23.68	37.92 ± 16.96
Muscle	1.27 ± 0.15	1.80 ± 0.35	0.87 ± 0.19	0.37 ± 0.23	0.73 ± 0.08
Fat	0.34 ± 0.07	0.77 ± 0.21	0.38 ± 0.05	0.13 ± 0.14	0.56 ± 0.12
Heart	7.19 ± 0.87	4.67 ± 0.89	1.96 ± 0.47	1.17 ± 0.72	2.03 ± 0.16
Brain	2.08 ± 0.26	3.55 ± 0.41	2.89 ± 0.54	2.66 ± 0.15	3.89 ± 0.15
Bone	0.89 ± 0.35	1.04 ± 0.26	0.35 ± 0.17	0.03 ± 0.08	0.62 ± 0.31
Marrow	8.56 ± 3.13	3.26 ± 0.51	2.51 ± 1.78	0.00 ± 0.00	1.08 ± 1.01
Testes	0.49 ± 0.06	0.81 ± 0.12	0.55 ± 0.14	0.14 ± 0.15	0.98 ± 0.17
Prostate	1.56 ± 2.03	1.20 ± 0.37	0.33 ± 0.45	0.11 ± 0.20	0.71 ± 0.66
Adrenals	2.90 ± 1.21	2.56 ± 1.13	1.87 ± 0.85	0.96 ± 1.33	3.34 ± 2.68
Thyroid	2.49 ± 0.54	2.30 ± 0.40	1.05 ± 0.26	0.31 ± 0.45	0.62 ± 0.44
Pancreas	4.04 ± 0.63	5.29 ± 0.55	2.59 ± 0.47	1.17 ± 0.40	2.48 ± 0.16
Thymus	1.89 ± 0.12	2.21 ± 0.46	1.06 ± 0.47	0.90 ± 1.37	1.27 ± 0.43
Stomach	1.25 ± 0.26	1.79 ± 0.37	1.43 ± 0.81	0.98 ± 0.49	1.89 ± 0.68
Sm Int	4.18 ± 0.51	9.15 ± 1.20	7.01 ± 1.38	9.94 ± 4.10	13.45 ± 1.32
ULI	1.87 ± 0.27	3.46 ± 0.70	2.67 ± 0.52	3.74 ± 1.07	5.18 ± 1.71
LLI	0.93 ± 0.17	1.44 ± 0.17	0.84 ± 0.12	0.82 ± 0.10	1.38 ± 0.35
Salivary Gland	3.00 ± 0.48	3.71 ± 0.53	1.59 ± 0.22	0.75 ± 0.58	1.56 ± 0.30
Skin	0.56 ± 0.12	1.02 ± 0.10	0.69 ± 0.15	0.30 ± 0.25	0.86 ± 0.27
Tail	4.52 ± 2.60	3.85 ± 1.48	9.41 ± 10.65	5.04 ± 3.35	1.65 ± 0.69

Sm Int = small intestine, ULI= Upper Large Intestine, LLI = Lower Large Intestine

Table 2.

Organ activity concentration from male mice dissection expressed in percent injected dose per gram of tissue (%ID/g).

Organs	5 min	15 min	30 min	60 min	90 min
Blood	2.08 ± 0.60	1.65 ± 0.25	1.14 ± 0.47	0.72 ± 0.52	1.17 ± 0.15
Lung	15.24 ± 4.06	7.12 ± 1.30	4.67 ± 0.52	1.52 ± 1.14	3.29 ± 0.63
Liver	18.72 ± 8.02	46.86 ± 11.48	56.14 ± 9.20	69.84 ± 20.24	65.07 ± 6.61
Spleen	5.30 ± 1.38	3.42 ± 0.35	1.99 ± 0.26	0.83 ± 0.52	1.97 ± 0.11
Kidney	18.57 ± 2.82	25.79 ± 2.74	20.46 ± 3.25	17.22 ± 4.88	13.77 ± 2.24
Bladder	1.86 ± 0.36	1.94 ± 0.39	1.60 ± 0.33	0.66 ± 1.47	1.12 ± 0.48
Gallbladder	42.77 ± 34.77	14.21 ± 13.70	43.27 ± 32.36	20.79 ± 24.02	126.82 ± 32.86
Muscle	1.82 ± 0.35	2.01 ± 0.34	1.22 ± 0.09	0.69 ± 0.55	0.90 ± 0.18
Fat	0.77 ± 0.23	0.89 ± 0.20	0.62 ± 0.20	0.10 ± 0.14	0.82 ± 0.45
Heart	8.54 ± 1.80	4.88 ± 0.61	2.35 ± 0.45	0.95 ± 0.86	2.03 ± 0.34
Brain	2.67 ± 0.61	4.11 ± 0.99	3.82 ± 0.23	4.01 ± 1.09	4.21 ± 0.42
Bone	1.07 ± 0.51	0.72 ± 0.23	0.49 ± 0.25	0.00 ± 0.00	0.35 ± 0.39
Marrow	11.76 ± 5.01	3.86 ± 0.91	3.74 ± 2.14	2.01 ± 4.48	2.43 ± 1.15
Testes	2.40 ± 0.28	2.31 ± 0.28	1.57 ± 0.08	1.92 ± 3.09	1.01 ± 0.44
Prostate	2.32 ± 0.77	2.60 ± 0.54	1.60 ± 0.25	0.11 ± 0.26	1.66 ± 1.04
Adrenals	7.04 ± 1.97	5.04 ± 0.60	4.36 ± 1.55	0.58 ± 0.84	2.86 ± 1.33
Thyroid	3.26 ± 0.83	2.60 ± 0.33	1.27 ± 0.11	0.08 ± 0.17	2.04 ± 0.83
Pancreas	4.89 ± 1.09	5.96 ± 0.79	3.43 ± 0.47	1.65 ± 1.05	3.05 ± 0.69
Thymus	2.18 ± 0.66	2.01 ± 0.44	1.45 ± 0.29	0.29 ± 0.36	1.43 ± 0.61
Stomach	1.40 ± 0.39	2.28 ± 0.53	1.29 ± 0.40	0.61 ± 0.15	2.51 ± 1.08
Sm Int	5.99 ± 1.81	11.30 ± 1.74	12.82 ± 1.67	22.92 ± 10.95	24.31 ± 3.80
ULI	2.42 ± 0.68	3.70 ± 0.45	3.74 ± 0.91	6.68 ± 1.16	7.43 ± 3.15
LLI	1.24 ± 0.26	1.55 ± 0.27	1.29 ± 0.15	1.39 ± 0.38	1.77 ± 0.19
Salivary Gland	3.86 ± 1.25	4.31 ± 0.49	2.39 ± 0.25	1.02 ± 0.81	2.07 ± 0.25
Skin	0.96 ± 0.18	1.21 ± 0.08	0.92 ± 0.08	0.66 ± 0.58	0.98 ± 0.31
Tail	7.57 ± 4.91	9.83 ± 6.73	4.20 ± 2.37	2.69 ± 1.99	4.37 ± 1.75

Sm Int = small intestine, ULI= Upper Large Intestine, LLI = Lower Large Intestine

Table 3.

Human organ residence times, radiation dose, effective dose estimates for [¹¹C]CS1P1

Organ residence times	Males	Females
Total body, min	18.36	19.26
Max total body, min	29.40	29.40
Remainder, min	10.98	10.14
Radiation dose and effective dose
Total body, μGy/MBq	2.70	3.78
Effective Dose Equivalent, μSv/MBq	5.14	7.84
Effective Dose, μSv/MBq	3.51	5.95

Fig. 2.

Extrapolated human organ residence times for [¹¹C]CS1P1. Using the mouse biodistribution data, the organ residence times for each harvested organ were calculated by numerical integration of the time activity data expressed in percent injected dose per gram of tissue. The animal organ residence times for most organs were scaled to human organ weight by the “relative organ mass scaling” method. The highest cumulative activity was found in the liver, with 8.34 ± 1.50 min in males and 8.58 ± 1.44 min in females, indicating that the tracer was mainly excreted by the hepatobiliary system.

Human radiation dose estimates

Extrapolated human radiation dose estimates are shown in Table 3 and Fig. 3. The highest organ dose was received by liver (24.05 μGy/MBq in males and 32.70 μGy/MBq in females), followed by kidney (15.14 μGy/MBq in males and 14.05 μGy/MBq in females). The Effective Dose (ED) estimates of [¹¹C]CS1P1 were calculated at 3.51 μSv/MBq in males and 5.95 μSv/MBq in females.

Fig. 3.

Extrapolated human radiation dose estimates for [¹¹C]CS1P1. The residence times in Table 3 were entered in the program OLINDA/EXM for C-11 and using the standard Medical Internal Radiation Dose (MIRD) adult male and female models, to obtain human radiation dosimetry estimates. The highest organ dose was received by liver (24.05 μGy/MBq in males and 32.70 μGy/MBq in females), followed by kidney (15.14 μGy/MBq in males and 14.05 μGy/MBq in females).

Tolerability and Toxicity Studies in Rats

All rats survived until the scheduled day for euthanasia and necropsy. There were no test article-related organ weight changes and no test article-related gross pathology findings at either Day 2 or Day 15. Compared to control animals, there were no CS1P1-related effects on body weights, in hematology parameters or in clinical chemistry parameters for the duration of the study.

Any minor differences in individual or group mean hematology or clinical chemistry parameters in male and female rats administered CS1P1 compared to concurrent control rats occurred sporadically or at low incidence, which had no correlate to clinical observations or microscopic findings, and therefore, were not considered test article-related. No test article-related histopathology were observed at either Day 2 or Day 15. All findings were common spontaneous changes and/or occurred at similar incidences in treated and control groups.

Discussion

[¹¹C]CS1P1 has been reported as a promising imaging agent targeting S1P1 in preclinical models. In order to evaluate its suitability as a PET radiopharmaceutical for human use, it is necessary to estimate the organ radiation dose burden and examine the tolerability and toxicity in rodents before human PET studies are undertaken.

Biodistribution and dosimetry studies were conducted in order to estimate the internal radiation dose absorbed by each human organ. This allowed an assessment of radiation safety of the radiotracer and the number of studies that can be safely conducted annually. Recently small animal microPET imaging has been utilized in biodistribution and dosimetry studies as an alternative to organ harvesting [18-19]. The imaging-based methods could provide high time resolution of the dynamic data, reduce the number of animals used for the dosimetry study, and simplify the laborious sampling procedure. However, significant deviation of activity in organs from organ harvesting has been observed when using PET imaging, particularly for rodents; the large discrepancies may be largely attributed to the severe partial volume effect (PVE) or the spillover effect, due to small organ sizes comparative to the limited spatial resolution of the PET camera [19]. Thus in the current study, the traditional and widely accepted ex vivo organ harvesting method was used.

The current study revealed that the effective dose (ED) of [¹¹C]CS1P1 projected from the mouse biodistribution data (3.5 and 5.9 μSv/MBq for males and females respectively) was within the similar range as other C-11 labeled PET radiotracers (3.0-16.0 μSv/MBq). The dose limiting organ for [¹¹C]CS1P1 administration is the liver. High absorbed doses were also registered in the kidneys. According to the federal regulations specified in Title 21 CFR 361.1 (https://www.gpo.gov/fdsys/granule/CFR-2012-title21-vol5/CFR-2012-title21-vol5-sec361-1), the annual limit of the radiation doses to the whole body, gonads, active blood-forming organs, and lens of the eye should not exceed 50 mSv annually and 30 mSv for a single study. The dose absorbed by all other organs should not exceed 150 mSv annually and 50 mSv for a single study. The liver is the limiting (or critical) organ for [¹¹C]CS1P1 according to the present study and therefore dictates the allowable maximum injection dose per PET scan. Based on estimation from the rodent study, a PET study with [¹¹C]CS1P1 can be performed in human subjects using a maximum injection dose of 2.08 GBq (56.18 mCi) for male and 1.53 GBq (41.32 mCi) for female, based on the dose limit (50 mGy) to the liver. When the maximum ED of 10 mSv is taken into account [20], the maximum administered dose would be 2.85 GBq (76.92 mCi) for male and 1.68 GBq (45.45 mCi) for female. Thus a single injection dose of 0.74 GBq (20 mCi) should be easily allowed for PET studies in humans. These results support the proposed exploratory IND submission for first-in-human studies of [¹¹C]CS1P1.

In the tolerability and toxicity studies, based on the absence of abnormal clinical observations coupled with no definitive CS1P1–related effects on body weights, clinical pathology parameters, organ weight values, and anatomic findings (macroscopic or microscopic), a single IV injection of 0.3 mg/kg CS1P1 was well tolerated in male and female Sprague Dawley rats.

The 300 ppm sterile-filtered dose of CS1P1 compounded in 10% polyethylene glycol-300 (PEG-300), 30% hydroxypropyl beta-cyclodextrin, and 60% sterile water for injection was stable over the 5 hour interval from initial HPLC analysis to completion of the in-life injections by the CRO. Based on the proposed mass limit of 10 μg CS1P1 per 60 kg human subject in PET imaging studies, the rat no observable effect level (NOEL) of 0.3 mg/kg is a 300-fold multiple of the HED. Under the conditions of the acute toxicology study, this was both a NOEL and a no observable adverse effect level (NOAEL) in rats following a single IV bolus administration.

Conclusions

In conclusion, radiation dosimetry studies in mice and toxicity studies in rats suggested that [¹¹C]CS1P1 is safe for use in human PET imaging studies to assess the inflammatory response by quantifying the S1P1 expression in related binding sites. An injection of 0.74 GBq (20 mCi) dose for human PET studies would be acceptable and is safe for radiation safety consideration.