INDs for PET Molecular Imaging Probes—Approach by an Academic Institution

We have developed an efficient, streamlined, cost-effective approach to obtain Investigational New Drug (IND) approvals from the Food and Drug Administration (FDA) for positron emission tomography (PET) imaging probes (while the FDA uses the terminology PET drugs, we are using “PET imaging probes,” “PET probes,” or “probes” as the descriptive terms). The required application and supporting data for the INDs were collected in a collaborative effort involving appropriate scientific disciplines. This path to INDs was successfully used to translate three [18 F]fluoro-arabinofuranosylcytosine (FAC) analog PET probes to phase 1 clinical trials. In doing this, a mechanism has been established to fulfill the FDA regulatory requirements for translating promising PET imaging probes from preclinical research into human clinical trials in an efficient and cost-effective manner. Electronic supplementary material The online version of this article (doi:10.1007/s11307-014-0735-2) contains supplementary material, which is available to authorized users.

Rats were provided ad libitum RO (Reverse Osmosis) water from individual bottles attached to the cages throughout their stay in the Testing Facility. UCLA maintains AAALAC accredited animal facilities and all work was conducted under authorization by the UCLA Animal Research Committee (IACUC equivalent).

Test substance
Non-radioactive FAC compounds were purchased from Moravek Biochemicals and Radiochemicals (Brea, CA) and dissolved in 1% ethanol in saline. The technician administering the test substance was blinded as to the identity of the animals. A single 100µl volume dose of either the control (only the carrier solution of 1% ethanol in saline) or 27.5µg of non-radioactive FAC per kilogram of body weight was administered intravenously to the rats. This is equivalent to 0 or 100X the maximum tracer mass of FAC administered to human subjects, respectively.

Physiological assessments
Acute arterial blood pressure measurements were obtained via femoral or carotid artery catheterizations to determine the hemodynamic status of the isoflurane anesthetized rats. The Electrocardiogram (ECG) was simultaneously recorded via needle electrodes inserted under the skin in the lead II configuration. These pressure and ECG data were acquired via Grass amplifiers, then digitized, displayed and analyzed in real time with HEM V4.0 software (Notocord Systems, France) on a PC. Heart rates were determined from the R-R interval of the ECG. The oxygen saturation was monitored simultaneously with a MouseOx system and displayed with WinDaq software on the same PC. Baseline data was recorded continuously for at least 15-30 minutes to ensure that physiological levels of pressures, oxygen saturation and heart rates were achieved.

Blood collection
Approximately 1.0-1.5 ml of whole blood were collected by caudal lateral vein bleed under isoflurane anesthesia from rats in groups 1-4 to determine baseline values at the end of the acclimation period (day -1). Subsequent blood samples were collected terminally before pentobarbital overdose euthanasia at day +1 (groups 1-4) and day +14 (groups 5-8) just prior to euthanasia and necropsy, as shown in figure S1. The whole blood samples were divided as follows: at least 350 µL into an EDTA-containing tube (submitted for hematology [CBC, complete blood count]) and 0.5-1.0 mL into a serum-separator tube for blood chemistries.
Samples in serum-separator tubes were centrifuged at 2000 x g for 15 minutes after clotting.
The serum was evaluated for hemolysis according to colorimetric scale. Hemolysis was acceptable in the range of 20-50, according to the scale. Samples collected for CBC were assessed before running the test for clots. Smears were prepared in the diagnostic lab and read by a pathologist if the CBC equipment (HemoVet) flagged the results.

Euthanasia and necropsy
At scheduled times; blood was collected under general anesthesia using a 24G catheter placed in the lateral caudal vein. Using the same intravenous catheter, animals were euthanized with 100mg/kg pentobarbital. Immediately after euthanasia necropsies were conducted by a pathologist.

Gross necropsy and histopathology
Each rat was weighed on a calibrated, digital scale immediately upon euthanasia and before necropsy. A complete necropsy was performed on each rat and all gross lesions were recorded.
Additionally, the following organs were weighed and recorded for comparison between the treated and non-treated groups: brain, heart, thymus, liver, spleen, right and left kidney, and testes when applicable. Samples of all of the organs were collected for histologic examination.
All tissues were immersed in 10% formalin immediately after collection. The tissues were sectioned for paraffin embedding within 24-48 hours post fixation. The UCLA-DLAM staff Page 5 of 20 veterinary pathologist performed or directly supervised all tissue sectioning. The staff veterinary pathologist performed all histopathologic evaluations for this study.
Gross necropsy included an initial physical examination of external surfaces and all orifices. A skin incision was made extending from the mandible through the anogenital region. The incision was extended through the subcutaneous tissues and muscular layers. An internal examination of tissues and organs in situ was performed. The following was examined: external and internal portions of all hollow organs; the external surfaces of the brain and spinal column, the nasal cavity and neck with associated organs and tissues; the thoracic, abdominal and pelvic cavities with associated organs and tissues; and the musculoskeletal carcass. The lungs were perfused with neutral buffered 10% formalin. The entire carcass was fixed in 10% formalin post necropsy and filed for future sectioning if needed.

Study plan
Sprague-Dawley rats were randomly assigned to eight groups of eight rats (four male and four female rats in each group) to determine the safety pharmacology and toxicity effects upon one, single, intravenous administration of non-radioactive form of each of the FAC PET probe analogs in rats ( Figure S1). The rats were monitored prior and up to 14 days following the administration of the test substance (carrier solution or FAC). The early effects of the administered agents were monitored on day (+1) post injection in groups 1-4 and the late effects of the administered agents were monitored on day (+14) post injection in groups 5-8. Rats in groups 1/5 were administered with the carrier solution control and those in groups 2/6, groups Page 6 of 20 The dose of each FAC agent used for these toxicity studies was in excess of 100x the maximum estimated dose of the FAC tracer that may be injected into a human subject, equivalent to 27.5ug per kg. The control groups received the carrier solution instead.

Safety pharmacology
The safety pharmacology parameters evaluated during the study included, body weight, body temperature, heart rate, blood pressure, blood oxygen levels, respiration rate and daily clinical observations.
Body weight: Rats were purchased from Charles River Laboratories with average weights of 200-220grams and 300-320grams for female and male rats respectively. Body weight measurements were taken on days -1, 0, +1, +7 and +14 and averaged for males and females in vehicle control (groups 1 and 5), D-FAC (groups 2 and 6), L-FAC (groups 3 and 7) and L-FMAC (groups 4 and 8) administered rats. The results demonstrated no significant body weight  (Figure S2 A, B and C).
Page 8 of 20 Page 13 of 20 Clinical observations: All the rats involved in the study were monitored daily for clinical signs.
No clinical signs linked to the administration of the FAC test substance were observed.

Toxicology studies
The early toxicity (day +1) and late toxicity (day +14) of the agents were determined by obtaining complete blood counts (CBC), clinical chemistry, necropsy and histopathology data in rats from groups 1-4 and 6-8 respectively.
CBC: The CBC results for each group under early (Table S1A) and late time points (Table S1B) were averaged and the standard deviations displayed. The standard deviations with a p<0.05 compared to the same sex controls under respective early or late time points are annotated with an asterisk and highlighted.
Serum chemistry: The serum chemistry results for each group under early (Table S2A) and late time points (Table S2B) were averaged and the standard deviations displayed. The standard deviations with a p<0.05 compared to the same sex controls under respective early or late time points are annotated with an asterisk and highlighted.
Necropsy: The necropsy data results for each group under early (Table S3A) and late time points (Table S3B) were averaged and the standard deviations displayed. The standard deviations with a p<0.05 compared to the same sex controls under respective early or late time points are annotated with an asterisk and highlighted.
A significant change was detected in the heart weight (P<0.05) of male and female D-FAC treated groups. Once the data had been presented to the FDA, we were requested to conduct a second toxicity study for the D-FAC group. The heart weight measurements from 6 male and female rats were obtained and the results are displayed in tables S3A and S3B under "Heart Wt