Biodistribution and Radiation Dosimetry of ¹⁸F-CP-18, a Potential Apoptosis Imaging Agent, as determined from PET/CT Scans in Healthy Volunteers

Abstract

¹⁸F-CP-18 or (18S,21S,24S,27S,30S)-27-(2-carboxyethyl)-21-(carboxymethyl)-30-((2S,3R,4R,5R,6S)-6-((2-(4-(3-[F18]fluoropropyl)-1H-1,2,3-triazol-1-yl)acetamido)methyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxamido)-24-isopropyl-18-methyl-17,20,23,26,29-pentaoxo-4,7,10,13-tetraoxa-16,19,22,25,28-pentaazadotriacontane-1,32-dioic acid is being evaluated as a tissue apoptosis marker for positron emission tomography (PET) imaging. The purpose of this study was to determine the biodistribution and estimate the normal-organ radiation absorbed doses and effective dose from ¹⁸F-CP-18.

Methods:

Successive whole-body PET/CT scans were performed at approximately 7, 45, 90, 130, and 170 minutes after intravenous injection of ¹⁸F-CP-18 in seven healthy human volunteers. Blood samples and urine were collected between the PET/CT scans, and the biostability of ¹⁸F-CP-18 was assessed using high-performance liquid chromatography. The PET scans were analyzed to determine the radiotracer uptake in different organs. OLINDA/EXM software was used to calculate human radiation doses based on the biodistribution of the tracer.

Results:

¹⁸F-CP-18 was 54% intact in human blood at 135 minutes after injection. The tracer cleared rapidly from the blood pool with a half-life of ~30 minutes. Relatively high ¹⁸F-CP-18 uptake was observed in the kidneys and bladder, with diffuse uptake in the liver and heart. The mean standardized uptake values (SUVs) in the bladder, kidneys, heart and liver at ~50 minutes after injection were approximately 65, 6, 1.5, and 1.5, respectively. The calculated effective dose was 38±4 μSv/MBq with the urinary bladder wall having the highest absorbed dose at 536±61 μGy/MBq using a 4.8-hr bladder voiding interval for the male phantom. For a 1-hr voiding interval, these doses were reduced to 15±2 μSv/MBq and 142±15 μGy/MBq, respectively. For a typical injected activity of 555 MBq, the effective dose would be 21.1±2.2 mSv for the 4.8-hr interval, reduced to 8.3±1.1 mSv for the 1-hr interval.

Conclusion:

¹⁸F-CP-18 cleared rapidly through the renal system. The urinary bladder wall received the highest radiation dose and was deemed the critical organ. Both the effective dose and the bladder dose can be reduced by frequent voiding. From the radiation dosimetry perspective, the apoptosis imaging agent ¹⁸F-CP-18 is suitable for human use.

Programmed cell death, known as apoptosis, is a regulated cellular mechanism that maintains cell population homeostasis and prevents the proliferation of unwanted or damaged cells (1). When apoptosis becomes unregulated, biological dysfunction ensues and is a contributing factor in the onset of autoimmune diseases, ischemic heart failure and even transplant rejection. A cell’s apoptotic potential can also be reduced or even halted, a common trait of cancer cells as they progress into malignant phenotypes (2, 3).

The apoptotic process invokes distinct biochemical alterations triggered by either extrinsic signaling via the death receptor or through intrinsic pathways via mitochondrial signaling. Regardless of how apoptosis is initiated, the signaling pathways ultimately converge at the execution phase which commits the cell to undergo apoptosis and eventually die. Interestingly, apoptosis directed via the mitochondria initiates the release of pro-apoptotic proteins which activates a family of cysteinyl aspartate proteases (caspases) as critical mediators of the intrinsic apoptosis pathway. While caspase proteins are involved in the initiation of apoptosis (caspase −2, −8, −9, −10), the executioners (caspase-3, −6, −7) cleave multiple intracellular substrates to provoke the apoptotic cascade. Caspase-3, as a downstream apoptosis effector, cleaves key proteins involved in DNA repair (PARP), signaling proteins (Akt, Ras) and cell cycle regulators (p27Kip1). The detection of activated caspase-3 could therefore be a valuable and specific tool for identifying apoptotic cells prior to the morphological tissue changes.

Several ¹⁸F-labeled positron emission tomography (PET) imaging agents have been developed for the in vivo detection of apoptosis. The PET-labeled agents ¹⁸F-ML10 (4–6), ¹⁸F-C2A (7), ¹⁸F-annexin-V(8–10) and ¹⁸F-FBnTP (11, 12) detect key apoptotic pathways involved in membrane imprints (13), membrane blebbing or mitochondrial membrane potential but do not specifically target members of the caspase family. A separate class of PET imaging agents based on the small-molecule isatin scaffold has been developed including ¹⁸F-AF-110(14), ¹⁸F-ICMT-11 (15, 16), ¹¹C-WC-98, ¹⁸F-WC-II-89 and ¹⁸F-WC-IV-3 (14, 17–19). The utility of these ¹⁸F-radiolabeled isatins as PET apoptosis imaging agents has been described previously (15, 16, 19–21). Several isatin-based radiotracers are reported to undergo hepatic-based metabolism and gut clearance, which may limit abdominal imaging applications (15, 16). In addition, radiolabeled isatins can potentially bind other cysteine proteases (e.g. cathepsins) which may lower the specificity of these tracers in imaging apoptosis (22).

¹⁸F-CP-18 was designed as a caspase-3 peptide substrate, similar in principle to fluorescent reporters that are used in caspase-3 activity assays. ¹⁸F-CP-18 bears the tetra-peptidic caspase-3 recognition sequence (“D-E-V-D”), which has high specificity for caspase 3 (23), and a polyethylene glycol (PEG) chain to facilitate membrane transport into cells, which is necessary as the high polar DEVD peptide sequence itself poorly diffuses across cell membranes. Even though ¹⁸F-CP-18 contains multiple peptide functional groups, the tracer is readily radiolabeled with ¹⁸F using click-chemistry, a technique that employs primary azides and terminal alkynes in the presence of Cu(I) to selectively form 1,2,3-triazoles (24–26). From a mechanistic standpoint, the cell permeating PEG moiety facilitates the internalization of ¹⁸F-CP-18 into cells undergoing cleavage in the presence of active intracellular caspase-3. This cleavage step effectively traps the polar radiolabeled fragment within the cell as a function of caspase-3 activity. As more ¹⁸F-CP-18 is cleaved inside the dying cell, more labeled fragments are accumulated resulting in signal enhancement. The localization of ¹⁸F-CP-18 in apoptotic tissues has been previously evaluated in preclinical models (27). Further exploration of its use as an apoptosis marker in patients is warranted.

The main goal of this study was to determine the biodistribution and dosimetry of ¹⁸F-CP-18 in humans using PET.

MATERIALS AND METHODS

Radiopharmaceutical Preparation

The name of the investigational product being studied in this protocol is ¹⁸F-CP-18, or (18S,21S,24S,27S,30S)-27-(2-carboxyethyl)-21-(carboxymethyl)-30-((2S,3R,4R,5R,6S)-6-((2-(4-(3-[F18]fluoropropyl)-1H-1,2,3-triazol-1-yl)acetamido)methyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxamido)-24-isopropyl-18-methyl-17,20,23,26,29-pentaoxo-4,7,10,13-tetraoxa-16,19,22,25,28-pentaazadotriacontane-1,32-dioic acid. The chemical structure of ¹⁸F-CP-18 is shown in Figure 1.

FIGURE 1.

Chemical structure of ¹⁸F-CP-18

¹⁸F-labeling was performed in an automated synthesis module having minor hardware reconfigurations. A solution of anhydrous ¹⁸F-fluoride complexed with Kryptofix® 222 and potassium carbonate in acetonitrile was reacted with pent-4-yn-1-yl 4-methylbenzenesulfonate to generate ¹⁸F-fluoropentyne. This ¹⁸F-labeled intermediate was then reacted with the CP-18 azide precursor, sodium ascorbate, CuSO₄ and TBTA dissolved in a solution of ethanol:water:acetonitrile (See Figure 2). The crude reaction mixture was purified via semi-preparative HPLC and reformulated using a C18 SepPak cartridge to achieve a final formulation of ¹⁸F-CP-18 in a maximum of 10% ethanol:water (v/v). The solution was then processed through a 0.22 μm sterile filter into a sterile vial. The average decay-corrected radiochemical yield of ¹⁸F-CP-18 was 18.7%, and the average specific activity was 171,093 MBq/μmol (n=7).

FIGURE 2.

Synthesis of ¹⁸F-CP-18 from Precursor

Subjects

The study was approved by the Research Review Committee, Institutional Review Board and Radiation Safety Committee of Fox Chase Cancer Center. Seven healthy volunteers (5 female, 2 male) with ages 50±9 years (mean± SD, age range 31–55 years) were included in the study. Written informed consent was obtained from each subject. The subjects’ weights were 71±8 kg (range 59–82 kg). All subjects were healthy based on medical history, physical examination, EKG, urinalysis, and standard blood tests.

PET/CT Acquisition and Image Analysis

Following the intravenous injection of 604±74 MBq (range 537–699 MBq) of ¹⁸F-CP-18 to the volunteers, five successive whole-body PET/CT scans were performed on a Siemens Biograph Truepoint 16 PET/CT Scanner (Siemens Healthcare). A very low mA setting on the scanner was used to perform the CT scans for attenuation correction and organ localization. The helical CT scan acquisition parameters were: 130 kVp, 20 mA, 0.6 sec rotation, pitch =1.

The whole-body PET scans were acquired in 3-D mode and ranged from the top of the head down to mid-thigh. The five whole-body scans were conducted at approximately 7, 45, 90, 130, and 170 minutes after injection. The scan time was 1.5 minutes per bed position and each scan covered 8 or 9 bed positions. In addition, after the first whole-body scan, a PET/CT scan of mid-thigh to toes was performed. Blood pressure, body temperature, respiratory rate, pulse and EKG were monitored before the administration of ¹⁸F-CP-18 and following the first, second, and last PET/CT scans, and then at 24 hrs post-dose.

Blood and urine were collected before the radiotracer injection and in the breaks between the PET scans. Urine was also collected at the end of the last PET scan. Samples of urine were assayed in a well counter to estimate the excreted activity in urine. High-performance liquid chromatography was performed to determine the amounts of intact ¹⁸F-CP-18 and its radioactive metabolites in the plasma. Briefly, the activity of the whole blood samples was measured in a well counter as counts per minute. Of the seven blood samples submitted for analysis, one sample was not analyzed due to technical difficulties with the HPLC instrumentation. For the remaining six blood samples, the whole blood was centrifuged at 3,500 RPM for 5 minutes to separate plasma from whole cells. The plasma fraction was removed and placed in a separate, empty, pre-weighed tube to record the weight of plasma. An aliquot of plasma (400 μL) was removed followed by spiking the aliquot with non-radioactive CP-18 standard (16 μl of a 1 mg/mL aq. stock solution) and acetonitrile (100 μL). After vortexing the sample for 30 seconds and centrifuging at 13,000 RPM for 8 minutes to separate proteins from plasma, the plasma supernatant was removed, weighed and the counts per minute of pellet and extract were measured. An aliquot (50–100 μl) of the processed plasma sample was injected into the HPLC loop (Agilent 1100 HPLC system equipped with a Luna C18 analytical column (10 μm, 250× 4.5 mm), mobile phases A: Water w/ 0.05% TFA, B : Acetonitrile w/ 0.05% TFA , gradient 95% A to 50% A in 15 min, UV 200 or 206 nm, Flow rate : 1 mL/min). The radioHPLC eluent was collected in one minute fractions and individually counted and the percent of plasma-borne activity in the form of parent compound (i.e.¹⁸F-CP-18) and in the form of metabolites was extracted from the radioHPLC traces.

The PET scans were reconstructed to 62 cm display field of view using the Siemens TrueX reconstruction algorithm with 21 subsets and 2 iterations and a 4 mm FWHM Gaussian post-processing filter. The reconstruction included corrections for random coincidences and scatter. Attenuation correction was applied based on the low-dose CT. The accuracy of the activity in the PET images was verified by summing the activity in the first whole-body PET scan and the PET scan from mid-thigh to toes, and comparing the total-body activity thus calculated to the decayed injected activity.

The PET images for the middle time point for each patient were displayed on an imaging workstation. For each of the following organs including brain, liver, heart, spleen, kidneys, bladder, breasts (for females), and testes (for males), volume regions of interest (ROIs) were drawn. The PET images for other time points were displayed and registered with this PET scan to transfer the respective volume ROIs and determine the total activity in each organ. Adjustments to the individual ROIs were made to ensure the inclusion of the respective organs. The percent of the injected activity in each organ was determined at each imaging time point.

Normalized Number of Disintegrations

The percent administered activity for each organ for each time point was fitted to an exponential or sum of exponentials function in OLINDA/EXM (Organ Level Internal Dose Assessment) software (28) to determine the total number of disintegrations per unit administered activity, hereafter referred to as ‘normalized number of disintegrations’. Activity in the ‘remainder of body’ was calculated for each time point as the injected activity minus the activity in all the source organs and in collected urine. The half-times for biological excretion were computed by exponential fitting of injected activity minus accumulated urine activity as a function of time. The 1.0-hr and 4.8-hr bladder voiding intervals in OLINDA/EXM were used to determine the normalized number of disintegrations in bladder. Absorbed doses to the various organs were calculated by entering the normalized number of disintegrations of all source organs for each subject into OLINDA/EXM, using the standard adult male and female models.

RESULTS

The injection of ¹⁸F-CP-18 (604±74 MBq) in the seven subjects produced no clinically significant effects on either the vital signs (blood pressure, temperature, respiratory rate, pulse, and EKG) or blood tests during the 3 hr observation period following administration and in the follow-up visit at 24 hours.

The analysis of plasma samples of 6 healthy human subjects showed that the ¹⁸F-CP-18 metabolized slowly with the percentage of plasma-borne activity in the form of ¹⁸F-CP-18 decreasing from 99% to 52% from 5 to 135 min post-injection. Based on these plasma activity measurements, ¹⁸F-CP-18-derived activity exhibited a clearance half-time of ~30 minutes. The radioHPLC trace indicated that one metabolite was present in plasma along with the parent compound. (see Supplemental Figure 1). Based on the relative retention time of the metabolite peak, the plasma metabolite is tentatively assigned as the fragment cleaved between “DE” and “VD”. This pattern of cleavage is consistent with metabolites observed in mice for which those metabolites were identified by mass spectroscopy (data not shown). Urine analysis revealed the presence of the parent tracer ranging from 37% at 35 minutes to 21% at 135 minutes (see Supplemental Figure 1). The urine samples contained predominantly two major metabolites in combined percentages of 63% and 79% after 35 and 135 minutes, respectively. The enrichment of metabolites in the urine relative to the parent compound suggests a faster clearance rate for the metabolites relative to the intact tracer. Again, based on relative retention time analysis of the metabolite signals, these fragments are tentatively assigned as the “DE” fragment and the parent tracer minus the PEG group, respectively.

Figure 3 displays PET maximum intensity projection (MIP) images for one of the subjects from the PET scans. In the first scan, the predominant uptake was seen in both the kidneys and urinary bladder with moderate uptake observed in the liver, heart, and testes. All other organs had near background levels of activity. The mean SUVs at ~50 minutes after injection were approximately 65 in the bladder, 6 in the kidneys, and 1.5 in both the heart and liver. The rest of the body had much lower SUVs. The time-activity data for the urinary bladder, liver, and kidneys are shown in Figure 4. There was large inter-subject variation in both the bladder uptake (16% to 27%) and kidney uptake (3.5% to 11%), as indicated by the large error bars. The renal system (kidneys and bladder together) had more consistent uptake, ranging from 22% to 31%. The liver had the next highest uptake, ranging from 3.2% to 5.7%. The study radiotracer was excreted primarily via the renal system. By the end of the study (~2.8 hrs) ~77% of the injected activity of 18F-CP-18 had been excreted in the urine, as determined by assaying the urine samples in a well counter.

FIGURE 3.

Decay-corrected anterior maximum-intensity projections of PET at 7, 46, 77, 144, 179 minutes (from left to right) after injection of ¹⁸F- CP-18 in a male volunteer. There was rapid clearance of activity in all the organs.

FIGURE 4.

Mean percent administered activity and SD for bladder, kidneys, and liver determined on the basis of seven ¹⁸F-CP-18 PET emission scans in human volunteers, as a function of time after injection. Rapid clearance of activity was observed in the organs.

The normalized number of disintegrations for the organs is listed in Table 1. The mean organ doses are given in Table 2. The mean (± SD) effective dose of ¹⁸F-CP-18 for the human adult male phantom was 15±2 μSv/MBq and 38±4 μSv/MBq for the 1-hr and 4.8-hr bladder voiding interval, respectively. For a typical injected dose of ¹⁸F-CP-18 (555 MBq), the effective dose for the two intervals were 8.3±1.1 mSv and 21.1±2.2 mSv, respectively. The adult female phantom doses were 20–34% higher. The two organs with the highest radiation absorbed doses were urinary bladder wall and kidneys. For the 4.8-hr bladder voiding interval, the dose to uterus was similar to the dose to the kidneys.

TABLE 1.

Normalized number of disintegrations of source organs for volunteers injected with ¹⁸F-CP-18.

Organ	Normalized number of disintegrations (MBq-h/MBq administered)
Breasts	0.015±0.0047
Brain	0.0089±0.0038
Heart Contents	0.026±0.0036
Kidneys	0.059±0.017
Liver	0.064±0.024
Spleen	0.0090±0.0024
Testes*	0.0057±0.0017
Urinary Bladder Wall (1.0-hr)	0.29±0.033
Urinary Bladder Wall (4.8-hr)	1.1±0.13
Remainder	0.82±0.14

^*-data from the 2 male subjects only.

Data are mean±SD; n = 7.

TABLE 2.

Estimated organ doses per unit administered activity for ¹⁸F-CP-18 for the human adult male phantom.

Organ	4.8-h void interval (μGy/MBq)	1-h void interval (μGy/MBq)
Adrenals	6.8±0.8	6.4±0.8
Brain	2.4±0.6	2.4±0.6
Breasts	7.7±3.6	7.7±3.6
Gallbladder Wall	7.6±0.8	6.7±0.9
Heart Wall	10±0.8	10±0.8
Kidneys	39±10	39±10
Liver	11±3.0	10±3.0
Lower Large Intestine Wall	20±1.3	8.8±0.5
Lungs	4.7±0.7	4.6±0.7
Muscle	8.1±0.4	5.3±0.6
Osteogenic Cells	7.9±0.9	6.8±1.0
Ovaries	19±1.1	8.7±0.6
Pancreas	6.9±0.8	6.5±0.8
Red Marrow	6.8±0.4	4.9±0.6
Skin	4.7±0.4	3.7±0.5
Small Intestine	11±0.5	6.8±.7
Spleen	13±2.5	13±2.6
Stomach Wall	6.1±0.7	5.5±0.8
Testes*	25±14	29±6.7
Thymus	4.8±0.7	4.8±0.7
Thyroid	4.2±0.7	4.2±0.7
Upper Large Intestine Wall	9.7±0.5	6.4±0.7
Urinary Bladder Wall	536±61	142±15
Uterus	38±3.2	14±0.6
Total Body	8.2±0.4	5.6±0.6
Effective Dose (μSv/MBq)	38±3.9	15±1.9

Data are mean±SD; n = 7.

^*-data from the 2 male subjects only.

DISCUSSION

¹⁸F-CP-18, which is currently under evaluation for detecting tumor apoptosis (27), was investigated in this dosimetry study in healthy volunteers. In this investigation ¹⁸F-CP-18 was shown to have a reasonable biodistribution and dosimetry profiles in human subjects and suggests this agent may have promise in PET imaging. This study provides information on the background uptake levels in normal organs. The data also addresses the potential radiation exposure in humans through whole-body PET imaging.

¹⁸F-CP-18 displayed a biodistribution profile dominated by activity uptake in both the bladder and kidneys indicative of a rapid renal clearance. Accordingly, 77% of injected activity was excreted within 2.8 hour after injection of the tracer. The liver and heart showed diffuse uptake and there was no evidence of the tracer above background levels in the large intestines during the 2.8 hour imaging period, consistent with the notion that the tracer is cleared predominantly via the renal system. The rapid clearance and relatively low non-specific uptake of the tracer as compared to other ¹⁸F-labeled apoptosis imaging agents suggests that a large imaging window is possible for detecting increased caspase-3 expression in tumors throughout the torso, including the lungs, breast and the head and neck regions.

Among all the organs observed, the urinary bladder wall received the highest dose with the 4.8-hr bladder voiding interval as a result of the tracer’s clearance profile. The mean urinary bladder wall doses were 142±15 and 536±61 μGy/MBq for the 1-hr and 4.8-hr bladder voiding intervals, respectively. Kidneys had the next highest dose, 39±10 μGy/MBq, followed by the uterus at 38±3 μGy/MBq for the 4.8-hr bladder voiding interval. The testes had doses of 29 and 25 μGy/MBq for the two intervals, respectively. The remaining organs had lower doses, in the range of 2 to 20 μGy/MBq. The average values of effective dose for ¹⁸F-CP-18 were 15±2 μSv/MBq and 38±4 μSv/MBq, respectively, for the two intervals. For a typical 555 MBq injected dose of ¹⁸F-CP-18, the effective dose for the 4.8-hr interval is 21.1±2.2 mSv. The effective dose is lower than the 30 mSv dose limit specified by the FDA for research subjects (29). By more frequent bladder voiding the doses can be reduced. For the 1-hr bladder voiding interval, the effective dose was reduced to 8.3±1.1 mSv.

¹⁸F-CP-18 was found to be initially stable in human plasma and about 54% intact at 135 minutes post injection. The urine analysis suggests that the metabolites and parent tracer are predominantly excreted via the urine. The metabolites formed are tentatively assigned as the expected cleavage products of the parent “D-E-V-D” sequence, which were previously observed during the preclinical evaluation of the tracer in rodents (27) (data not shown). The majority of metabolite activity cleared quickly through urine via the kidneys though a small amount was detectable in plasma. The combination of the clearance and biostability profile suggests that this tracer is suitable for imaging tissue apoptosis in humans.

Table 3 compares the doses to individual organs for ¹⁸F-CP-18 with ¹⁸F-FDG for the bladder voiding interval of 4.8 h (30). The absorbed doses in the brain, heart, pancreas, lungs, spleen, thymus, thyroid, stomach wall were much lower for ¹⁸F-CP-18 than those for ¹⁸F-FDG. ¹⁸F-CP-18 did not appreciably cross the blood-brain barrier in our imaging studies. The absorbed doses in urinary bladder, kidneys, testes, and uterus were higher for ¹⁸F-CP-18 than for ¹⁸F-FDG. A large fraction of ¹⁸F-CP-18 (~77% in 2.8 hrs) was excreted through the urinary system. The urinary bladder wall dose for ¹⁸F-CP-18 (540 μGy/MBq) was much higher than that for ¹⁸F-FDG (190 μGy/MBq) (with 4-hr bladder voiding interval). However, if patients void frequently, the bladder dose is reduced to ~142 μGy/MBq using a 1-hr bladder voiding interval.

Table 3.

Organ doses for ¹⁸F-CP-18 and ¹⁸F-FDG for 4.8-h bladder voiding interval

	18F-CP-18 (μGy/MBq)	18F-FDG (μGy/MBq)
Adrenals	6.8	13
Brain	2.4	19
Breasts	7.7	9.2
Gallbladder Wall	7.6	14
Lower Large Intestine Wall	20	17
Small Intestine	11	14
Stomach Wall	6.1	13
Upper Large Intestine Wall	10	13
Heart Wall	10	60
Kidneys	39	20
Liver	11	16
Lungs	4.7	17
Muscle	8.1	11
Ovaries	19	17
Pancreas	6.9	26
Red Marrow	6.8	13
Osteogenic Cells	7.9	12
Skin	4.7	8.4
Spleen	13	37
Testes	25	13
Thymus	4.8	12
Thyroid	4.2	10
Urinary Bladder Wall	540	190
Uterus	38	23

A successful apoptosis imaging agent will localize within apoptotic tissue with sufficient contrast between apoptotic and non-apoptotic tissues. Within normal tissues, elevated levels of caspase-3 exist for tissue maintenance, especially within cartilage and in mammary glands (31). Using these tissues as internal controls, the PET images in several subjects reveal elevated uptake of ¹⁸F-CP-18 in both shoulder cartilage and mammary glands relative to non-caspase expressing tissue (e.g. muscle), consistent with differences in caspase-3 expression levels observed across such tissues (31). As a second confirmation that ¹⁸F-CP-18 is targeting tissue apoptosis, the SPECT apoptosis imaging agent ^99mTc EC Annexin V also appeared to exhibit uptake in shoulder cartilage similar to ¹⁸F-CP18, suggesting that both tracers target an overlapping pathology (32). The biodistribution and radiation dosimetry of ¹⁸F-CP-18 are likely compatible with its use as a PET apoptosis imaging agent. While the current imaging doses and scan times allowed for visualization of these caspase-expressing tissues, further imaging optimization studies will be required to determine the optimal scan time and dose levels for imaging apoptosis, or apoptotic fractions thereof, especially within tumors. From the data obtained in this study, PET imaging at 30 minutes after injection appears to be optimal, in comparison to the 60 minute delay that is typically used for other ¹⁸F-based imaging compounds such as ¹⁸F-FDG.

As a potential therapy target, apoptosis presents a unique opportunity for enabling a personalized medicine approach. For example, apoptosis imaging has the potential to identify treatment resistant tumors in need of alternate therapies by monitoring changes in ¹⁸F-CP18 uptake during the course of therapy, especially in instances where metabolic ¹⁸F-FDG PET imaging data is confounded by tissue inflammation. Given that all tumors undergo apoptosis to varying degrees, a change in apoptosis post-treatment is likely to be more meaningful than a single baseline apoptosis scan. Serial tumor uptake measurements are relatively easily performed using non-invasive PET imaging as a semi-quantitative tool which is a distinct advantage over the use of multiple biopsies.

PET apoptosis imaging may also enable the development of novel apoptosis-inducing therapies. Conventional chemotherapeutics are known to induce apoptosis, but do so non-specifically by targeting multiple cellular processes which can lead to both therapy resistance and a narrowing of the therapeutic window. Promoting apoptosis as a strategy for cancer drug discovery enables therapeutic agents to selectively induce apoptosis in cancer cells while improving therapeutic outcomes (33–36). In such a scenario, PET apoptosis imaging can be used to monitor the effect of apoptosis inducing/promoting drug treatments with the aim of predicting responders to such therapies perhaps earlier than metabolic ¹⁸F-FDG PET imaging. For such a strategy to be successful, the PET imaging data must be correlated with clinical, surgical, pathological, and patient outcomes. PET imaging of caspase-3 could therefore be established as a pharmacodynamic biomarker to measure the increase in apoptotic fraction after treatment, allowing clinicians to tailor patient treatments or optimize dosing regimens. Further studies are needed to evaluate the efficacy of this promising PET imaging agent.

The injection of ¹⁸F-CP-18 did not cause clinically significant changes in vital signs or blood tests suggesting that the administration of ¹⁸F-CP-18 is both safe from a dosimetry perspective and compatible with PET imaging in humans.