Use of Small Animal PET/MRI for Internal Radiation Dose Assessment

Use of small animal PET/MRI for internal radiation dose assessment

Dissertation zur Erlangung des akademischen Grades Dr. rer. med. an der Medizinischen Fakult¨at der Universit¨at Leipzig

eingereicht von: M.Sc. Mathias Kranz Geburtsdatum/Geburtsort: 21.08.1986/Bad Salzungen angefertigt am/in: Universit¨atsklinikum Leipzig (UKL), Klinik und Poliklinik fur¨ Nuklearmedizin sowie Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Forschungsstelle Leipzig, Abteilung Neuroradiopharmaka

Betreuer: UKL: Prof. Dr. -Ing. Bernhard Sattler HZDR: Prof. Dr. Peter Brust

Beschluss uber¨ die Verleihung des Doktorgrades vom:

Table of Content

1 Introduction ...... 1 1.1 Radiation dosimetry ...... 2 1.1.1 Quantities and units ...... 3 1.1.2 Dose calculation ...... 5 1.1.3 Dose limits ...... 6 1.2 Preclinical dosimetry ...... 7 1.2.1 Small animal imaging ...... 8 1.2.2 Small animal PET/MRI for image based dosimetry ...... 9 1.2.3 Correction methods of preclinical data used for human dose assessment...... 11 1.3 Objective of the study ...... 12

2 Publications ...... 15 2.1 Study 1: Internal Dose Assessment of (-)-[18F]flubatine, Comparing Animal Model Datasets of Mice and Piglets with First-in-Human Results ...... 15 2.2 Study 2: Radiation Dosimetry of the α4β2 Nicotinic Receptor Ligand (+)-[18F]flubatine, Comparing Preclinical PET/MRI and PET/CT to First-in-Human PET/CT Results ...... 24 2.3 Study 3: Evaluation of the Enantiomer Specific Biokinetics and Radiation Doses of [18F]fluspidine - A New Tracer in Clinical Translation for Imaging of σ1 Receptors ...... 42

3 Summary of the dissertation ...... 57 3.1 Summary and conclusions ...... 57

References ...... 63

4 Appendix ...... 71 4.1 Erklärungen uber¨ den wissenschaftlichen Beitrag zu den Publikationen 71 4.2 Erklärung uber¨ die eigenständige Abfassung der Arbeit ...... 75 4.3 Lebenslauf ...... 76 4.4 Publikationsverzeichnis ...... 79 4.5 Danksagung...... 87

Introduction

The health risk of biological specimens exposed to ionizing radiation became appa- rent shortly after the discovery of ionizing radiation (X-rays) in 1895 by Wilhelm Conrad Roentgen [1, 2, 3, 4, 5]. High radiation doses1 which exceed a certain threshold level damage living cells which finally results in cell death (deterministic effects). However, cell information might be modified even by lower doses (stochastic effects2). This modification is usually repaired by intrinsic cell mechanisms such as DNA repair or the elimination of damaged cells [8]. If this process is incorrect, the modification (mutation) will be transmitted to other cells and might cause cancer. Notably, the world population is constantly exposed to natural- (terrestrial, solar etc.) and man-made ionizing radiation of different kind and sources. The latter includes nuclear bomb tests, nuclear power plants (and accidents) and the use of radiation for clinical applications. The medical use and application of ionizing radiation (i.e. α-, β- or γ) includes machine-generated radiation such as from accelerated particles and from radioactive isotopes that have been artificially produced. In diagnostic applications, γ rays are of major interest as they consist of high-energy photons with a mass of zero resulting in less interactions with the target material (e.g. tissue), and therefore high penetration through the whole body. While cell death in tumors or inflamed tissue is desired in therapeutic nuclear medicine [9, 10], in diagnostic nuclear medicine and diagnostic radiology the dose should be kept to a minimum to prevent these effects. Hence for therapeutic and diagnostic application of radiation in humans, radiation dosimetry is necessary to control the

1 Hereinafter referred to as ”dose”. 2 In the scientific literature there is controversy concerning the correct model which accounts for the stochastic effects at low doses [6]. Most regulatory bodies rely on the linear-no-threshold (LNT) model which assumes that even the smallest doses increase cancer risk. However, the LNT model lacks information at low doses where a linear fit function was created from higher dose rate data (collected from nuclear bomb survivors). Hence, the extrapolation was done beyond the limits of observed data.[7, 8] 2 chapter 1. Introduction dose (see 1.3) absorbed by tissues, organs and systems of organs with respect to the extend of a desired effect or the probability of a (stochastic) effect, respectively. This thesis is based on the diagnostic application of radioligands (radiation emit- ting substances bound to an active biomolecule) after intravenous injection. These compounds are called radiotracers. This concept is based on the pioneering work of George Hevesy [11, 12]. It describes a radiotracer as a chemical compound in which one or more atoms have been replaced by one of its radioactive isotopes. The injected amount of radiotracer will not interfere with the processes which are studied, as the concentrations used are extremely low as compared to the concentrations that would cause pharmacological effects [11, 13]. Thus, in vivo measurement including imaging of several different targets improves the early diagnosis for example of neurodegenerative diseases [14, 15], schizophrenia [16] and cancer [17]. Furthermore, it may support the development of new drugs by target evaluation, drug efficiency studies and contribute to the understanding of complex biochemical processes [18, 13]. At the University Hospital Leipzig, brain disorders are routinely investigated with positron emission tomography (PET). Neuropathological studies indicated that alterations of physiological metabolic processes in the brain may set on before the age of 30 years in patients who develop neurodegenerative disease [15, 19, 20]. Hence, different tracers e.g. targeting proteins or specific receptors were developed aiming at an early diagnosis of those diseases [15, 21, 22, 23, 24, 25]. At the research site Leipzig of the Helmholtz-Zentrum Dresden-Rossendorf two promising radiotracers 18 were developed, [ F]flubatine [26, 27, 28] for PET-imaging of α4β2 nicotinic acetyl- 18 choline receptors and [ F]fluspidine [29] for the imaging of σ1 receptors in several brain disease such as depression and neurodegeneration. The preclinical and clinical radiation dosimetry of the four radiotracers (-)-[18F]flubatine, (+)-[18F]flubatine, (S)-(-)-[18F]fluspidine and (R)-(+)-[18F]fluspidine are presented and discussed in this thesis. The summary in chapter 3 is based on the three publications Sattler3 et al. (2014)[30], Kranz et al. (2016a)[31] and Kranz et al. (2016b)[32].

1.1 Radiation dosimetry

One part of radiation physics deals with the investigation of the mean absorbed energy from the interaction of ionizing radiation with matter while radiation dosimetry is the quantitative determination of that energy [33]. There are two types of voluntary or involuntary exposure of humans to ionizing radiation, external or internal, which require particular dosimetric considerations. In

3 Bernhard Sattler and Mathias Kranz are equally contributing ﬁrst authors. 1.1. Radiation dosimetry 3 medical applications of ionizing radiation in humans, both approaches are used: (i) external dosimetry in X-ray applications like computed tomography (CT), brachy- therapy applications or external beam radiation therapy and (ii) internal dosimetry in radionuclide therapy or diagnostic imaging with radionuclides in nuclear medicine for instance planar/emission computed tomography -scintigraphic applications. External dosimetry describes the dose in humans if the radiation source is outside of the body. Hence, with types of radiation or accelerated particles that penetrate the skin (beta, photon or neutron radiation). In turn, internal dosimetry is the measurement of the dose caused by the ionizing radiation from radioactive substances that have entered the body (e.g. via ingestion, inhalation or injection).[34, 35]

Internal and external dosimetry observes the mean absorbed dose (statistically averaged) in the respective volume disregarding the inherent random fluctuations. Hence, the macroscopic effects are objective for this type of investigation. Another field of dosimetry is referred to as microdosimetry. It observes the energy deposition in cellular and sub-cellular structures where fluctuations are known to have large effects to these small volumes.[36] This thesis is based on internal (macroscopic) dosimetry of voluntary intravenous application of radioligands for visualization of brain functions with PET. To meet the regulatory requirements of the competent German authority (section 1.1.3) for newly developed radioligands applied first time in human, the radiation safety has to be proven in preclinical studies. Therefore, different steps need to be followed to achieve dosimetry results in animals that yield to represent a dose estimate for humans, like (i) choosing the right investigational protocol so it fits the subsequent human study, (ii) quantitatively exact PET measurement, (iii) data reconstruction and image registration, (iv) collection of the time-dependent radioactivity concentration data (biodistribution), (v) conversion and extrapolation of the data to the respective human scales, (vi) calculation of the number of disintegration per organ and (vii) calculation of the organ doses (OD) and the effective dose (ED). These steps will be described in the following subsections.

1.1.1 Quantities and units

The quantity used to express the amount of radiation, which equals the energy absorbed per unit mass of a material is called the absorbed dose:

dE J D = (1 = 1 Gy (gray)) (1.1) dm kg 4 chapter 1. Introduction

While equation 1.1 is used to calculate a point source, DT,R is used for three- dimensional objects e.g. tissues and organs:

ER DT,R = (Gy) (1.2) mT

which describes the ratio of the released energy ER of a radiation R in a tissue T

divided by the tissue weight mT and is called the organ energy dose. Subsequently, the organ dose is calculated:

J H = w × D ( = 1 Sv (sievert)) (1.3) T R T,R kg with wR being the radiation weighting factor which depends on the type and energy of the radiation species (per deﬁnition 1.0 for photons, electrons and positrons).

When multiplying DT,R with the radiation weighing factor a new unit, the Sievert, is introduced for the result.[37] Regarding the overall radiation risk, the effective dose (equation 1.4) plays an important role in dose calculation. It involves the biological effects based on differences in the radiation sensitivity of different tissues. These are defined by the tissue weighting factor wT .

E = wT × HT (Sv) (1.4)

The values for the radiation weighting factors and the tissue weighting factors were published by the International Commission on Radiological Protection (ICRP, Publication 60 and 103)4 and can be found in [40, 39]. They are based on epidemio- logical studies on cancer induction in different organs and risk assessment of genetic defects in populations exposed to ionizing radiation. In general, the data originates from a follow up study [42] from 1958 to 1998 of Japanese A-bomb survivors. The ICRP used four components for the evaluation of the so called ”detriment”, the induction of fatal cancer, non-fatal cancer, hereditary effects and loss of life time [43]. Each weighting factor represents the fraction of health risk to a specific tissue. However, they are averaged over age and gender and are suitable to represent the radiation risk to a population but not to an individual.[39]

4 The radiation weighting factor considers the relative biological effectiveness taking different biological effects of various types and energies of radiation into account.[38] Although new radiation weighting factors were published by the ICRP 103 [39], for Germany the values of ICRP 60 [40] are valid and settled by the StrlSchV Annex 6, section C, part 1 [41] (refer to subsection 1.1.3). 1.1. Radiation dosimetry 5

1.1.2 Dose calculation

There are several methods described in the literature to calculate the internal radiation dose. That is, the Marinelli/Quimby method [44, 45], the ICRP method in publication 71 [46] and the MIRD (Medical InternalRadiation Dose system) method [47]. An advanced version of the MIRD method, the RADAR (Radiation Dose Assessment Resource) method [48], was used for the dose estimation calculation in this thesis. It is widely used and accepted for the assessment of the radiation exposure to humans [49, 50, 51, 52, 53] and animals [54, 55, 56, 57] from applications of radioactive substances. In addition, it is available as software implementation in OLINDA/EXM (Organ Level Internal Dose Assessment/Exponential Modeling)[58]. The calculation is based on the fundamental equation for the organ dose D:

D = N × DF (1.5) with N being the total number of disintegrations that occurred in a source organ5 during the respective observation period. It is the time integral (from the respective organ time-activity curve) starting from the injection up to the last available time frame from an investigation of the biodistribution and -kinetics in the whole body.

DF is the dose factor with yi being the yield of photons or particles per nuclear decay with the energy, E, and φ, the fraction of absorbed energy in the target organ with the weight m, multiplied with the radiation weighting factor wRi . It expresses the absorbed dose fraction in a target region per nuclear disintegration in a source region [59].

k yiEiφiwRi DF = Pi (1.6) mT The software OLINDA v. 1.1 contains a hermaphroditic 3D model of an adult with the DFs as published by Cristy and Eckerman [60]. These are calculated based on mathematical phantoms of Snyder et al. [61] consisting of diﬀerent densities and chemical compositions for lung, bone and soft tissue [62]. The calculations were performed with a Monte Carlo radiation transport program [63] which simulates the transport of radiation in the respective mathematical phantom while the source of radiation is assumed to be uniformly distributed [62].

5 The MIRD system is based on a combination of ”source” and ”target” organs. Usually the radioactive decay occurs in a source organ (e.g. liver), exposing the surrounding organs (stomach, spleen, pancreas etc.) with ionizing radiation. In addition, a source organ is also a target organ of the radioactivity in the proximity.[58] 6 chapter 1. Introduction

The OLINDA calculation results in the organ doses (see 1.3) for 25 organs. Con- sequently, the eﬀective dose is calculated by multiplying the organ doses with the radiation weighting factors (equation 1.4).

1.1.3 Dose limits

The main principle of radiation protection states that all doses based on medical exposure must be kept as low as reasonably achievable while the required diagnostic information has to be obtained from this data [64]. Furthermore, the justification of using ionizing radiation in humans must be given and deemed beneficial (§93, §94 StrlSchV [65]). Generally, in Germany the public is protected under the Radiation Protection (§46 StrlSchV) and X-ray Ordinances (§2b, §32 RöV) defining a limit of 1 mSv effective dose per person and year when exposed to ionizing radiation6 originating from civil use. However, the German public is exposed to several naturally and man-made radiation sources7 which altogether cumulate to an effective dose of about 4 mSv per year. For research purposes, healthy subjects may receive up to 20 mSv (§24(3) StrlSchV). However, excluded from the application are healthy volunteers in which radioactive materials or ionizing radiation was used for research or treatment purposes over the last decade, if an effective dose of more than 10 mSv is to be expected through reapplication in medical research (§88(2) StrlSchV). There is no actual limit for patients if they could potentially benefit from the application of radioactive materials or ionizing radiation in the research project, respectively. If this is not the case, the Federal Office for Radiation Protection/Bundesamt fur¨ Strahlenschutz usually ap- plies the 20 mSv rule too but without the 10 mSv exclusion criterion as mentioned above. There are different international organizations which set regulatory effective dose limits for scientific purposes (e.g. first-in-man trials). In Europe several countries follow the ICRP recommendation of 10 mSv [67] which is the strictest known from the literature. Furthermore, the ICRP Publication 60 [68] stated that “the limits are designed to prevent deterministic effects and to reduce stochastic effects to an acceptable level” [9]. However, the U.S. Food and Drug Administration (FDA) per-

6 For example: A commercial ﬂight exposes the passengers to cosmic radiation with an eﬀective dose rate of 3 to 7 µSv/h [66]. 7 While there exists natural sources such as cosmic radiation (14C,3 H), terrestrial (40K,222 Rn) and exposures based on incorporation (228T h,40 K) which cumulates to 1.75 mSv/year, there are other radiation sources based on human civilization (medical radiation sources, atom bomb tests, nuclear waste) which cumulates to 0.6 mSv/year. 1.2. Preclinical dosimetry 7 mits the research in radioactive drug development when the compound meets the requirements of the “Radioactive Drug Research Committee” [69]. For an adult volunteer (a single study or summed up to one year) the dose limits are:

For whole body, blood-forming organs, eye lens, gonads • single dose of 30 mSv • annual and total dose of 50 mSv Remaining organs • single dose of 50 mSv • annual and total dose of 150 mSv As there exist no dose limits for patients in Germany (§81 section 4 StrlSchV), for clinical routine diagnostic reference levels (DRL) were established (§81 section 2 StrlSchV). DRLs are used to monitor the injected activities during routine PET scans. As described in the ICRP publication 103 [39] the DRLs should be a dimen- sion which can be easily measured and quantiﬁed. Following the ICRP recommendations, the European Commission drafted a directive (2013/59/EURATOM [70]) which binds all member states to include that concept in their radiation protection program. The DRLs should not be used as upper reference levels. Rather they are optimal values for deriving a satisfying image quality in relation to the injected activity. Furthermore, they are deﬁned for standard patients and should be in compliance with the mean value out of ten unselected patients. For PET investigations the reference values for a [18F]FDG brain PET scan are between 200 and 250 MBq and for whole body tumor detection between 350 and 380 MBq.[71]

1.2 Preclinical dosimetry

A preclinical biodistribution and dose assessment is required in accordance with the radiation protection law prior to first-in-man application of a new radiotracer (StrlSchV §24 (1)6) in order to ensure radiation safety in humans. Hence, the dose estimation needs to be calculated from biokinetic data using animal models. Typi- cally mice or rats are used. Although monkeys are more closely related to humans, results from dosimetry studies based on primates show poor dose estimates as the effective dose to humans is overestimated [72]. Preclinical dose estimates remain preliminary and need to be confirmed in subsequent human studies. Small animal based dosimetry can identify critical organs, the route of excretion and yield estimates of the effective dose but should always be 8 chapter 1. Introduction

considered with regard to their methodical limitations.[73] Zanotti-Fregonara et al. [74] suggested a new pathway of tracer safety evaluation without using preclinical data. They compared 54 clinical dosimetry studies resulting in an average ED of 20.6 µSv/MBq which is well within the range of the limits presented earlier. Hence, a patient should receive an injection of 74 MBq of a new tracer followed by a PET scan ﬁrst. Subsequently, the data is evaluated for unex- pected tracer accumulation in a radiosensitive organ (gonads, ovaries, red marrow etc.). If no limitation is observed and the ligand looks promising, a whole-body dosimetry should be performed in a larger number of volunteers.[74]

To perform dosimetric calculations, the course of radioactivity concentration and accumulation in the relevant organs is required. This information can be achieved by two different approaches known as the dissection- (harvesting-) method and the imaging method (refer to 1.2.2 for image based dosimetry) [75]. Both methods were carried out for this thesis, the results compared to each other and discussed in section 3. A comparison of both methods can be found in table 1.2.2. The harvesting method (animals conscious during tracer injection and accumulation) is performed by sacrificing the animal subjects at certain desired times post i.v. injection of the radiotracer. Therefore, several animals per time point (up to 10 time points) need to be used in order to achieve statistical power. At each time point an animal is sacrificed, all relevant organs are collected and the radioactive content is determined in a γ counter. The results are normalized to the injected activity and expressed as %ID8. There are several disadvantages of this method that can be bypassed by performing image based dosimetry:

• An ethical reason is the large number of up to 30 animals that is needed for one dosimetry study • Time consuming preparation and handling of up to 30 animals • Multiple tracer productions • Nonuniform tracer distribution in an organ cannot be detected

1.2.1 Small animal imaging

Due to the availability of semiconducting photo-multiplying-tubes, the construction of high resolution small animal PET scanners is possible [76, 77, 78, 79, 80]. Since the introduction of small animal PET scanners diﬀerent models are commercially available e.g. Siemens microPET Focus [81] and microPET Inveon [82], Philips

8 Organ percentage of total injected dose (i.e. amount of activity) 1.2. Preclinical dosimetry 9

Mosaic HP [83, 84], GE Healthcare LabPET [85], Raytest ClearPET [86] and Medi- so nanoScan R PET/CT [87] or PET/MRI [88]. While most systems are combined with a CT or radioactive sources for transmission scans, the soft tissue contrast is poor. Therefore, a combined sequential PET/MRI system was chosen for this thesis in order to achieve optimal soft tissue contrast. As a result, the organ delineation is facilitated and the accuracy increased for the determination of the accumulated organ activity to create time-activity curves for dosimetry calculations. The collection of preclinical data was performed with the first commercially available combined PET/MRI system (Mediso nanoScan R PM PET/MRI) [88]. The data is collected by a continuous scan and stored in list-mode format9 while energy, position and time stamp are recorded to reconstruct any user defined time frame.[92] During the image acquisition the animals are immobilized and kept under isoflurane anaesthesia (induction: 4 % , maintenance: 1.8 % in 60 % oxygen/40 % pressured air) positioned prone on a heated (37 ◦C) platform. The imaging characteristics were investigated by Nagy et al. [88] following the guidelines of NEMA NU 4-2008 standard [93]. The results show a spatial resolution of 0.91 mm (FWHM, axial 5 mm off center) which is one of the best compared to current commercially available scanner models [94, 95]. Furthermore, the absolute system sensitivity was calculated to be 52.8 cps/kBq which corresponds to 5.83 % relative sensitivity (mouse-sized phantom). For morphological image information, a MRI subunit based on a 1 T permanent magnet with high homogeneity (<5 ppm, at center) and small fringe field (<13 mT, at magnet surface) was used. The gradient coils deliver 450 mT/m pulses (ramp time: 250 µs). For animal imaging different body coil types are available while the dedicated mouse body coil was used for the preclinical studies.

1.2.2 Small animal PET/MRI for image based dosimetry

Being the gold standard for preclinical mouse based dosimetry, the dissection method was applied first in this thesis prior to the small animal PET/MRI based approach. However, as mentioned in section 1.2, the image based dosimetry has some considerable advantages. While for the dissection method some animals need to be sacrificed at every individual time point, with the imaging based method a whole body scan over the investigation period will deliver time-dependent data required for dose calculations. 9 Using an online file storage of sinograms is very inefficient in terms of high data storage (in very short times) which would be required [89]. This would result in a lower time resolution [90]. Therefore, to reduce data storage and processing time, only the coordinates of the coincident events are saved. A rebinning of the data set to the desired time-frame will be necessary prior to reconstruction.[91] 10 chapter 1. Introduction

The whole-body PET scan was rebinned and reconstructed into 10 time frames with increasing duration to keep the number of γ-counts per frame constant and, thus, maintain the image quality. An advantage of the Mediso nanoScan PET/MRI system compared to other small animal PET systems is the combination with a MRI subunit. Thus, a clear soft tissue delineation is possible and even small organs like the gallbladder can be identified. However, the publications of Constantinescu et al. [54, 55] show the feasibility of preclinical dosimetry and biodistribution studies of new radiotracers using a small animal PET/CT while the delineation of the inner organs is very challenging. When using small animal systems, the correction for the partial volume effect (PVE) plays an important role in collecting quantifiable activity concentration data. That effect occurs if the region of interest (ROI) is smaller than the spatial resolution of the detector system resulting in an underestimation of the radioactive content in that area (due to the “spill-out” of radioactivity) [96]. There are correction methods available to eliminate these deviations. As described in the publications in section 2 the software ROVER (ABX, Radeberg, Germany) was used to create the ROI’s as well as to correct for the PVE10 [98].

10 The image resolution of PET scanners is usually reported as “full width at half maximum” (FWHM) which is the width of the region where the pixel values reach the half of the maximum of a point source response [97]. Consequently, it is the distance between two reconstructed PET point sources where the peaks can be clearly distinguished. The code automatically defines within the ROI data a “spill-out” region (with the distance d from the ROI contour) depends on the scanner resolution. It is defined as the set of all voxels with the distance smaller than the FWHM from the ROI contours. Furthermore, the ”general” background voxels (d) are defined to FWHM < d < 2.5 FWHM. Finally, the local background of each single voxel in the ’spill-out’ region is defined as each voxel belonging to the ’general’ background and fulfilling d ≤ 1.5 FWHM to the respective voxel. With these definitions, knowing the amount of radioactivity in the “spill-out” region, the partial volume corrected ROI can be calculated which significantly contributes to quantifiable image data.[98] 1.2. Preclinical dosimetry 11

Table 1.2.2 : Comparison of the harvesting and imaging method used for preclinical dose assessment

Attribute Harvesting method Imaging method

Number of animals approx. 30 3 to 5 Tracer productions multiple 1-2 Organ activity collection organ dissection and VOI analysis γ counter (counts/min) Bq/ml Output for dosimetry %ID %ID Organ weight determination weighing VOI based in MR image Advantages no expensive imaging dynamic whole body device needed tracer information no influence of same method as for image artifacts human investigation no anaesthesia good tissue influencing metabolism separation in MR reuse of image data activity inhomogeneities within organs visible multiple use of animal Disadvantages inhomogeneities are unknown anaesthesia not detected effects blood contamination blood contamination image artifacts: partial volume, limited resolution

1.2.3 Correction methods of preclinical data used for human dose assessment

When assessing the radiation dose in humans based on animal data, an extrapolation of the biokinetic data to the human scale has do be done. This is to compensate for the faster metabolism and the anatomical diﬀerences of rodents. There are several methods described in the literature [99, 100, 101] such as using no extrapolation of the preclinical data, a relative organ mass scaling or the time scale extrapolation. An allometric scaling11 based on the combination of time and mass adaptation was used in the current studies [30, 31, 32] as it was described as the most promising method [101]. The %ID/g method was described by Kirschner et al. [104, 105] with the equation

11 This scaling approach is based on the power-law equation P = kmb [102] where P is the respective parameter to be scaled, m the relevant organ mass, b the allometric exponent and k the allometric coeﬃcient. By deﬁning k = 1 and b=1 it is assumed that the observed parameter scales with mwholebody organ mass as fraction of the total body weight mwholebody.[103] 12 chapter 1. Introduction

(%ID) (%ID) manimal(g) = · morganhuman (g) · (1.7) organ human ganimal mhuman(g) with (%ID) being the resulting % injected dose per organ in humans, (%ID) organ human ganimal the % ID per gram organ mass in animals multiplied by morganhuman the respective organ weight as deﬁned by the dosimetry model (e.g. Cristy-Eckerman model of an adult human [60]) and manimal(g) the ratio of whole-body animal to human weight. mhuman(g) This method assumes that the investigated physiological parameter (the organ % ID) scales with the organ mass as a fraction of the total body weight [103].

In addition, time scaling was applied to compensate for the faster metabolism of smaller species compared to the human species:

mhuman 0.25 thuman = tanimal( ) (1.8) manimal with thuman being the time in human scale and tanimal the corresponding time at animal scale as well as the ratio between human body weight mhuman and animal body weight manimal. The allometric exponent for scaling was found to be close to 0.25 for example when comparing blood ﬂow per cycle of mice (15 s [106]) to humans (50 s [107]).[108, 103]

1.3 Objective of the study

To date, there are no publications available using small animal PET/MRI with mice for human dose estimation. Only Constantinescu et al. and Bretin et al. showed the feasibility of preclinical dosimetry based on hybrid imaging with PET/CT or microPET12 with mice [54, 55, 56]. However, there is no comparison or validation with the same tracer to other preclinical, or clinical internal dosimetry methods. Furthermore, the image information of soft tissue derived by small animal CT for anatomical orientation is poor and the organ delineation is challenging. Another approach was shown by Bretin et al. [56] using a hybrid method by imaging mice with a microPET followed by microCT and ﬁnally sacriﬁcing the animals. The organs were collected and measured in a gamma counter for radioactive content. The result was used to calculate the ratio from the organ radioactive content in the last image frame and the gamma counter data to obtain a correction factor. Finally, all frames were corrected with that value. However, the organ delineation

12 For attenuation correction an integrated 57Co point source was used [56]. 1.3. Objective of the study 13 with these imaging devices is challenging too and the demand for an alternative method is obvious.

Hence, in this thesis the first time use of a small animal PET/MRI system for estimations of the internal exposure to radiation based on mouse data was evaluated with four different tracers. To confirm the small animal PET/MRI derived results, an additional investigation method based on animal dissection was used. In addition, a second species was chosen for preclinical dosimetry in order to clarify if the dose calculation using larger animals (piglets) results in a better ED estimate for humans compared to CD-1 mice (for (+)- and (-)-[18F]flubatine [30, 31]). Finally first-in-man investigations were performed with (+)- and (-)-[18F]flubatine as well as (S)-(-)-[18F]fluspidine and compared to the preclinical dosimetry studies [32]. In conclusion the thesis is based on the following main hypotheses which will be evaluated in detail in chapter 3:

• Hypothesis 1: The dosimetry results based on the gold standard of animal dissection combined with a gamma counter compared to PET/MRI based studies are reproducible and can be compared to each other (feasibility study).

• Hypothesis 2: When using larger species (e.g. piglets) than mice for preclinical dose estimates in humans, a better dose estimation is achieved compared to clinical results.

• Hypothesis 3: Enantiomeric tracer kinetic differences influence the effective dose.

• Hypothesis 4: Preclinical PET/MRI image based dosimetry is feasible and can be used for the estimation of the ED in humans prior to the application of ﬁrst- in-man studies.

Publications

2.1 Study 1: Internal Dose Assessment of (-)-[18F]ﬂubatine, Comparing Animal Model Datasets of Mice and Piglets with First-in-Human Results

Journal of Nuclear Medicine (2014): 55(11):1885-92. doi: 10.2967/jnumed.114.137059.

Bernhard Sattler∗, Mathias Kranz∗, Alexander Starke, Stephan Wilke, Cornelius K. Donat, Winnie Deuther-Conrad, Marianne Patt, Andreas Schildan, Jörg Patt, RenéSmits, Alexander Hoepping, Peter Schoenknecht, Jörg Steinbach, Peter Brust∗, Osama Sabri∗ ∗ Contributed equally Downloaded from jnm.snmjournals.org by Yale University Medical Library on October 25, 2016. For personal use only.

Internal Dose Assessment of (–)-18F-Flubatine, Comparing Animal Model Datasets of Mice and Piglets with First-in-Human Results

Bernhard Sattler*1, Mathias Kranz*1,2, Alexander Starke3, Stephan Wilke1, Cornelius K. Donat3, Winnie Deuther-Conrad3, Marianne Patt1, Andreas Schildan1,Jörg Patt1, René Smits4, Alexander Hoepping4, Peter Schoenknecht5,Jörg Steinbach6, Peter Brust*3, and Osama Sabri*1

1Department of Nuclear Medicine, University Hospital Leipzig, Leipzig, Germany; 2Institute of Radiopharmaceutical Cancer Research, Research Site Leipzig, Helmholtz-Zentrum Dresden-Rossendorf, Dresden/Leipzig, Germany; 3Department of Nuclear Medicine, Diaconal Hospital Henriettenstiftung Hannover, Hannover, Germany; 4ABX Advanced Biochemical Compounds Ltd., Radeberg, Germany; 5Department of Psychiatry, University Hospital Leipzig, Leipzig, Germany; and 6Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany

Key Words: radiation dosimetry; positron emission tomography; − 18 (−)-18F-flubatine is a promising tracer for neuroimaging of nicotinic ( )- F-flubatine; nicotinic receptors; α4β2 acetylcholine receptors (nAChRs), subtype α4β2, using PET. Radi- J Nucl Med 2014; 55:1885–1892 ation doses after intravenous administration of the tracer in mice DOI: 10.2967/jnumed.114.137059 and piglets were assessed to determine the organ doses (ODs) and the effective dose (ED) to humans. The results were compared with subsequent clinical investigations in human volunteers. Methods: Twenty-seven female CD1 mice (weight ± SD, 28.2 ± 2.1 g) received ± − 18 intravenous injection of 0.75 0.33 MBq of ( )- F-flubatine. Up to Neuronal nicotinic acetylcholine receptors (nAChRs) are 240 min after injection, 3 animals per time point were sacrificed and involved in memory processes and in learning and neuropsy- the organs harvested, weighed, and counted in a γ counter to determine mass and activity, respectively. Furthermore, whole-body chiatric diseases. The degeneration of nAChRs is related to PET scans of 5 female piglets (age ± SD, 44 ± 3 d; weight ± SD, brain disorders like Parkinson disease (1) and schizophrenia 13.7 ± 1.7 kg) and 3 humans (2 men and 1 woman; age ± SD, 59.6 ± (2). Furthermore, a decrease of nAChRs availability is suggested 3.9 y; weight ± SD, 74.3 ± 3.1 kg) were obtained up to 236 min to be a potential indication for patients with a progressive (piglets) and 355 min (humans) after injection of 186.6 ± 7.4 and Alzheimer dementia (3). By visualizing nAChRs in vivo, a pre- ± − 18 353.7 10.2 MBq of ( )- F-flubatine, respectively, using a PET/CT diction of the transformation from mild cognitive impairment scanner. The CT was used for delineation of the organs. Exponential to Alzheimer dementia might be possible (4). More informa- curves were fitted to the time–activity-data, and time and mass scales were adapted to the human anatomy. The ODs were calcu- tion on the role of nAChRs in PET imaging of the brain can lated using OLINDA/EXM (version 1.0); EDs were calculated with the be found in the supplemental material (available at http://jnm. tissue-weighting factors of ICRP103. Results: After the injection of snmjournals.org). (−)-18F-flubatine, there were no adverse or clinically detectable Although there are 2 existing enantiomers of 18F-flubatine, (2)- pharmacologic effects in any of the subjects. The highest activities and (1)-18F-flubatine, the (2)-derivate was investigated in this after injection were found in the kidneys, urinary bladder, and liver. study because it showed faster kinetics than those of (1)-18F- The urinary bladder receives the highest OD in all investigated spe- flubatine (5). As a regulatory requirement and to ensure the safety cies, followed by the kidneys and the liver for animals and humans, respectively. On the basis of mouse, piglet, and human kinetic data, and tolerability in the human body, a whole-body (WB) biodistri- the projected human ED of (−)-18F-flubatine was estimated to be bution and dosimetry study was performed. Determining the esti- 12.5 μSv/MBq in mice, 14.7 ± 0.7 μSv/MBq in piglets, and 23.4 ± mated internal radiation dosimetry to humans using animal models 0.4 μSv/MBq in humans. Conclusion: As has been demonstrated for is essential before permission for clinical testing can be granted (6). other PET radiotracers, preclinical (i.e., animal-derived) dosimetry under- Therefore, the study was divided into a preclinical (nonhuman − 18 estimates the ED to humans, in the current case of ( )- F-flubatine biologic systems, i.e., mouse, piglet) and an early clinical part by 34%–44%. including the first-in-human data collection. The preclinical part includes the WB dosimetry of 27 mice as well as the first-time (to our knowledge) use of 5 piglets. The clinical part includes 3 healthy volunteers. The aim of this study was to perform a com- Received Jan. 6, 2014; revision accepted Aug. 7, 2014. For correspondence or reprints contact: Bernhard Sattler, University parison of biodistribution and internal radiation dosimetry by Hospital Leipzig, Department of Nuclear Medicine, Liebigstrasse 18, 04103 extrapolating animal data to humans and to validate the results Leipzig, Germany. E-mail: [email protected] by additional investigation in human volunteers. Furthermore, we *Contributed equally to this work. propose the general conclusion to correct for the underestimation Published online Oct. 6, 2014. 18 COPYRIGHT © 2014 by the Society of Nuclear Medicine and Molecular in the translation of preclinical dose estimates for F-labeled Imaging, Inc. ligands to humans.

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FIGURE 2. Representative PET image (100 min after injection) of human (man), with (top row) and without (bottom row) VOIs highlighted. Different views are shown as coronal (A), sagittal (B), transversal (liver, kidneys) (C), and maximum-intensity projection (D) of 1 representative dataset. FIGURE 1. Representative PET image (80 min after injection) of piglet, MATERIALS AND METHODS with (top row) and without (bottom row) VOIs superimposed. The ﬁgure shows a summary of all investigated organs in piglets from different Synthesis of (−)-18F-Flubatine views: coronal (A), sagittal (B), transversal (liver) (C), and maximum- A detailed description of the synthesis was published by Deuther- intensity projection (D) of 1 representative dataset. Conrad et al. (7) and Patt et al. (8), and an improved 2-step strategy was published by Fischer et al. (4). Including the 3 healthy volunteers

FIGURE 3. Time–activity curve (A) and micturition diagram (B) of uri- FIGURE 4. Series of human PET scans from injection time until 414 min nary bladder (human subject 3). after injection (maximum-intensity projection).

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Piglets. Five female piglets (age 6 SD, 44 6 3.0 d; weight 6 SD, 13.7 6 1.7 kg) were anesthetized using ketamine (20 mg/kg), azaperone (2 mg/kg), and 0.5% isoflurane in 70% N2O/30% O2, after immobilization with pancuronium bromide (0.2 mg/[kg h] intrave- nously). All incision sites were infiltrated with 1% lidocaine. The animals were sequentially PET-imaged up to 5 h after intravenous (vena jugularis) injection of 186.6 6 7.4 MBq of (2)-18F-flubatine. The artificial respiration, after surgical tracheotomy, was controlled by a ventilator (Ventilator 710; Siemens-Elema) whereas the existing vein access was used for volume substitution (lactated Ringer solution, 5 mL/[kg h]). The ventilation was set to normoxic and normocapnic blood gas values. To control the arterial blood gas pressure and the acid-base parameters, blood samples from the arteria femoralis with a polyurethane catheter to the aorta abdominalis were manually taken and measured (ABL 50; Radiometer). The body temperature was controlled by an in-ear thermometer and kept at 38°C using an electric blanket. After all preparations for the scan were finished, the concentration

of anesthetic gas was reduced to 0.25% isoﬂurane, 65% N2O, and 35% O2.

TABLE 1 ODs and EDs of Mice for (−)-18F-Flubatine

ED contribution Target organ OD (mSv/MBq) (mSv/MBq)

Adrenals 9.74E−03 8.38E−05 Brain 1.07E−02 1.07E−04 Breasts 5.34E−03 6.41E−04 Gallbladder wall 9.28E−03 7.98E−05 Lower large intestine wall 1.26E−02 7.56E−04 Small intestine 1.30E−02 1.12E−04 Stomach wall 8.51E−03 1.02E−03 − 18 – FIGURE 5. Representative ( )- F-flubatine time activity curves (pig- Upper large intestine wall 1.07E−02 6.42E−04 let). To determine number of disintegrations, we used exponential least- − − mean squared fit. Fit function is shown with the respective curve. Heart wall 7.86E 03 6.76E 05 Kidneys 2.42E−02 2.08E−04 as described in this work, we have injected more than 40 preparations Liver 1.40E−02 5.60E−04 of (2)-18F-flubatine in human subjects and had no clinically detectable Lungs 8.78E−03 1.05E−03 side effects from the radiopharmaceutical. At the time of injection, the − − radiopharmaceutical had an injected mass of 0.1 6 0.08 mg(maxi- Muscle 7.07E 03 6.08E 05 mum, 0.4 mg). The use of (–)-18F-flubatine was authorized by the re- Ovaries 1.03E−02 8.24E−04 sponsible authorities in Germany, the Federal Institute for Drugs and Pancreas 1.40E−02 1.20E−04 Medical Devices (Bundesamt für Arzneimittel und Medizinprodukte, Red marrow 1.09E−02 1.31E−03 BfArM) and the federal Office for Radiation Protection (Bundesamt − − für Strahlenschutz, BfS), as well as by the institutional review board Osteogenic cells 1.18E 02 1.18E 04 (ethics committee). Skin 4.99E−03 4.99E−05 Spleen 1.15E−02 9.89E−05 Preclinical Studies − 1 All experiments involving animals were approved by the respective Testes 7.84E 03 0.00E 00 Institutional Animal Care and Use Committee and are in accordance Thymus 7.95E−03 6.84E−05 with national regulations for animal research and laboratory animal Thyroid 6.46E−03 2.58E−04 care. Urinary bladder wall 1.04E−01 4.16E−03 Mice. The animals were housed under a 12-h–12-h light–dark − − cycle and had 2 d for acclimation before the experiments. Twenty- Uterus 1.41E 02 1.21E 04 four hours before the investigation, they were fasted while water was Total body 8.32E−03 0.00E100 still available. Twenty-seven female CD1 mice (weight 6 SD, 28.2 6 ED (mSv/MBq) 1.25E−02 2.1 g) received an intravenous injection of 0.75 6 0.334 MBq of (2)-18F-flubatine with a specific activity greater than 100 GBq/mmol through the tail vein (vena caudata lateralis). At fixed time points, Mean over 27 subjects; ODs calculated for adult male model the animals were sacrificed and dissected, and the organs and tissues (73.7 kg, implemented in OLINDA) based on mouse biodistribution and organ geometry data that were scaled to human circum- were harvested and counted in a g counter to evaluate the activity in stances. each organ.

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TABLE 2 ODs and EDs of Piglets for (−)-18F-Flubatine

Target organ OD (mSv/MBq) SD (mSv/MBq) ED contribution (mSv/MBq) SD (mSv/MBq)

Adrenals 1.15E−02 8.07E−04 9.89E−05 6.94E−06 Brain 2.78E−02 6.40E−03 2.78E−04 6.40E−05 Breasts 1.68E−02 2.60E−02 2.02E−03 3.11E−03 Gallbladder wall 1.71E−02 2.32E−03 1.47E−04 1.99E−05 Lower large intestine wall 1.10E−02 1.40E−03 6.60E−04 1.69E−04 Small intestine 1.48E−02 2.76E−03 1.27E−04 2.37E−05 Stomach wall 1.27E−02 2.13E−03 1.52Ev03 2.56E−04 Upper large intestine wall 1.54E−02 3.08E−03 9.24E−04 1.85E−04 Heart wall 1.56E−02 2.65E−03 1.34E−04 2.28E−05 Kidneys 4.18E−02 7.29E−03 3.59E−04 6.27E−05 Liver 2.88E−02 8.09E−03 1.15E−03 3.23E−04 Lungs 1.32E−02 1.94E−03 1.58E−03 2.32E−04 Muscle 7.76E−03 7.10E−04 6.67E−05 6.11E−06 Ovaries 1.02E−02 1.17E−03 8.16E−04 9.34E−05 Pancreas 3.39E−02 2.82E−02 2.92E−04 2.42E−04 Red marrow 1.20E−02 1.51E−03 1.44E−03 1.81E−04 Osteogenic cells 1.39E−02 1.46E−03 1.39E−04 1.46E−05 Skin 5.90E−03 5.47E−04 5.90E−05 5.47E−06 Spleen 1.99E−02 4.63E−03 1.71E−04 0.00E100 Testes 7.36E−03 9.59E−04 0.00E100 8.25E−06 Thymus 1.85E−02 4.45E−03 1.59E−04 3.83E−05 Thyroid 1.56E−02 5.77E−03 6.24E−04 2.31E−04 Urinary bladder wall 4.55E−02 1.94E−02 1.82E−03 7.76E−04 Uterus 1.14E−02 1.62E−03 9.80E−05 1.39E−05 Total body 9.39E−03 5.20E−04 0.00E100 0.00E100 ED (mSv/MBq) 1.47E−02 7.25E−04

Mean over 5 subjects; ODs calculated for adult male model (73.7 kg, implemented in OLINDA) based on piglet biodistribution and organ geometry data that were scaled to human circumstances.

Clinical Study scan was preceded by a WB low-dose CT scan for attenuation cor- The institutional review board approved this study, and all subjects rection and anatomic orientation. signed a written informed consent form. Three healthy volunteers The piglets were positioned prone with legs alongside the body (2 men; age 6 SD, 59.6 6 3.9 y, weight 6 SD, 74.3 6 3.1 kg) were and the head in a custom-made head-holder. During the dynamic sequentially imaged in a PET/CT system after injection of 353.7 6 (sequential, without time period between the WB scans) part, 7 WB 10.2 MBq of (2)-18F-flubatine. At 2, 3.5, 5, and 7 h after injection, the scans were obtained up to 96 min after injection. The subsequent static subjects left the PET/CT table, and the urine was collected. The ac- part included 3 WB scans (22 min between each WB scan) up to tivity excreted in urine was calculated by volume measurement in 281 min after injection. Supplemental Table 1 shows the scan protocol combination with determination of activity in 1 mL aliquots with used for the PET (Fig. 1) and CT scans of the piglets. a g counter (Auto-Gamma-Counting System, Cobra II 5003, 7.62-cm PET imaging in humans was performed with volunteers posi- [3-in] NaI crystal; Packard). tioned supine, with their arms alongside their body. The PET frame was acquired using 9 bed positions starting from 1.5 up to 6 min PET Scan Protocol and Data Acquisition/Evaluation per bed position (Supplemental Table 2), divided into a dynamic Data in the 3 species were collected in 2 different ways. Although and static part. Each of these parts started with a CT scan, at a the harvesting method (6) was chosen for the mice, data acquisition maximum of 50 mAs and using automated tube current modulation. for piglets and humans was applied by sequential, decay-corrected CT attenuation correction based on low-dose CT transmission data PET imaging on a Siemens Biograph16 PET/CT system (imaging and iterative reconstruction using ordered-subsets expectation max- method (6)). The system is a lutetium oxyorthosilicate, 3-dimensional, imization (4 iterations and 8 subsets) were applied in both imaged 41-detector-ring system with an axial field of view of 162 mm, species. a minimal slice thickness of 2 mm, and a transaxial and axial resolu- Three mice at each time point (5, 15, 30, 45, 60, 90, 120, 180, and tion of 6.5 and 6 mm, respectively. Each sequential and late WB PET 240 min after injection) were sacrificed and dissected. All relevant

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TABLE 3 ODs and EDs of Humans for (−)-18F-Flubatine

Target organ OD (mSv/MBq) SD ED contribution (mSv/MBq) SD

Adrenals 1.41E−02 6.66E−04 1.22E−04 5.73E−06 Brain 2.76E−02 1.87E−03 2.76E−04 1.87E−05 Breast 7.27E−03 1.36E−04 8.73E−04 1.63E−05 Gallbladder wall 2.78E−02 3.52E−03 2.39E−04 3.02E−05 Lower large intestine wall 1.57E−02 1.85E−03 9.44E−04 1.11E−04 Small intestine 2.56E−02 3.27E−03 2.20E−04 2.81E−05 Stomach wall 2.42E−02 5.80E−03 2.90E−03 6.96E−04 Upper large intestine wall 2.78E−02 3.61E−03 1.67E−03 2.17E−04 Heart wall 1.69E−02 2.38E−03 1.45E−04 2.05E−05 Kidneys 3.86E−02 5.10E−03 3.32E−04 4.39E−05 Liver 4.47E−02 5.47E−03 1.79E−03 2.19E−04 Lungs 3.11E−02 5.37E−03 3.73E−03 6.44E−04 Muscle 8.79E−03 3.41E−04 7.56E−05 2.93E−06 Ovaries 1.31E−02 1.32E−03 1.05E−03 1.06E−04 Pancreas 2.66E−02 3.23E−03 2.29E−04 2.78Ev05 Red marrow 1.91E−02 1.63E−03 2.30E−03 1.95E−04 Osteogenic cells 1.76E−02 6.51Ev04 1.76E−04 6.51E−06 Skin 6.29E−03 2.52E−04 6.29E−05 2.52E−06 Spleen 3.84E−02 1.16E−02 3.30E−04 9.93E−05 Testes 1.54E−02 6.45E−03 1.23E−03 5.16E−04 Thymus 8.76E−03 1.36E−04 7.53E−05 1.17E−06 Thyroid 3.28E−02 1.19E−02 1.31E−03 4.75E−04 Urinary bladder wall 8.02E−02 3.78E−02 3.21E−03 1.51E−03 Uterus 1.49E−02 2.57E−03 1.28E−04 2.21E−05 Total body 1.13E−02 1.00E−04 0.00E100 0.00E100 ED (mSv/MBq) 2.34E−02 4.45E−04

Mean over 3 subjects; ODs calculated for adult male model (73.7 kg, implemented in OLINDA) based on human biodistribution and organ geometry data.

organs including the brain, heart, lungs, stomach, small intestine, large with ROVER, visually checked and manually corrected when needed. intestine, liver, kidneys, urinary bladder, spleen, thymus, pancreas, Relevant organs including brain, gallbladder, large intestine, small intes- adrenals, ovaries, blood, skin, muscle, and skeleton were isolated tine, stomach, heart wall, kidneys, liver, lungs, pancreas, red marrow, (harvested) and weighed, and their activities were measured in a g spleen, testes, thyroid, and urinary bladder were manually deﬁned counter. To obtain activity data for muscle and skeleton, an allometric (VOIs) as shown in Figure 1 (piglets) or Figure 2 (humans), and the scaling of the weights and volumes using tissue samples was performed respective activity values were extracted. The %ID was calculated using (9). The activity data of each organ at the respective time point were Equation 1. subsequently transformed to the percentage injected dose (%ID) using A calibration of the image data was performed as described in the the following equation: supplemental material.

AorganðtÞ · Cscan WB Dosimetry %IDorgan 5 ; Eq. 1 A0ðtÞ To make the investigations of 3 species comparable to each other, the hermaphroditic adult male model (the 73.7-kg standard man) was where AorganðtÞ is the total activity in the respective organ at the used for animal as well as human dosimetry calculations. This was corresponding time, Cscan the correction factor (supplemental mate- possible because of the scaling of animal biokinetic data (%ID values rial), and A0ðtÞ the decay-corrected total activity at time t. The resulting and time scales) from mice and piglets to human anatomy before input time–activity data are shown in Supplemental Table 3. into OLINDA/EXM. The scaling is described in detail in the supple- The software tool ROVER (ABX advanced biochemical com- mental material. pounds) was used to deﬁne the volumes of interest (VOIs)—that is, the The OLINDA/EXM program outputs the mean doses to humans to target organs—to determine the activity as well as the respective 25 target organs, and the ED was calculated using the tissue weighting volume. A rigid image registration of the PET and CT data was done factors provided in ICRP publication 103 (11).

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emptying of the urinary bladder, it is not suitable for this investigation. Instead, the trapezoidal-integration equation

2 f 1 n 1 %IDurinary bladder 5 + ð%IDi 1 %IDi 1 1Þðti 1 1 2 tiÞ; Eq. 2 2 i 5 1

was used to determine the cumulated activity in the urinary bladder f (%IDurinary bladder), where %IDi is the activity value at a time point ti, respectively. Using the image information and the counted urine, we created a graph showing the %ID and the voided volume of urine over time (Fig. 3). The relationship shown in this chart highlights that the urinary bladder is not completely emptied, so that the voiding-bladder model (12) for dose calculation is not optimal. Application of the trapezoid equation (integrate the surface under f the curve) yields the cumulative activity %IDurinary bladder and the number of disintegrations in the urinary bladder for input of the kinetic data in OLINDA.

FIGURE 6. Organ doses of all investigated species. Calculated ODs are summarized and visualized in this chart for examined species. Liver, RESULTS kidneys, and urinary bladder in all 3 species uniformly take up high After an intravenous injection of (2)-18F-flubatine, no study amount of activity. Dose values of spleen, thyroid, and skin are highly spread. LLI 5 lower large intestine; ULI 5 upper large intestine. drug–related adverse pharmacologic effects occurred during the investigation time. Urine and blood tests showed normal results. Representative (2)-18F-flubatine PET images of piglets (80 min Dosimetry of Urinary Bladder after injection) and humans (100 min after injection) are presented The dosimetry of the urinary bladder requires special consideration. in Figures 1 and 2, respectively, in coronal, sagittal, and transverse This organ accumulated the highest amounts of the radiotracer, with views. The brain (target organ), liver, kidneys, and urinary bladder the activity in the urinary bladder contents therefore contributing can clearly be identified. These figures also illustrate the delineated relatively high doses to the bladder wall as well as nearby organs. VOIs, created after coregistration with the CT images, of the re- The mice data were obtained by counting the dissected urinary spective subject in ROVER. The WB PET series shown in Fig- bladder together with the collected urine at each time point in the g ure 4 clarifies the filling and emptying of the urinary bladder as counter and used to create the time–activity curve for that organ. Be- well as the high activities in the liver during the first hour after cause there was no micturition during the imaging sessions of the piglets, injection and the subsequent clearance. The resulting activity val- we extracted the activity data by creating a VOI inside the organ at ues are presented in Supplemental Table 3. Representative time– different time points. To validate the accuracy of the measured activity, activity curves and the respective exponential fit functions are the urine was collected and counted in 1 mL samples after the piglets shown in Figure 5 for 6 organs. The organ doses based on the animal were sacrificed. To verify, the data were decay-corrected to the micturition time and compared with the urinary bladder VOI of the last data were calculated using the adult male model (73.7-kg hermaph- scan. As shown in the scan protocol (Supplemental Table 2), the human roditic standard man) as implemented in OLINDA. A comparison volunteers left the PET/CT table 4 times for collection of urine. One- of the mouse data to the human data is therefore possible as follows. milliliter samples of the collected urine were assayed in a g counter Because of the clearance circumstances of this tracer as ob- and decay-corrected to the micturition time. The total excreted activity served during the first hour after injection, a high uptake in the is obtained by multiplying the measured whole volume of voided liver and urinary bladder can be identified. As a result, the organ urine with the counted activity concentration. Dividing this result doses in all 3 species were found to be highest in the urinary with the administered activity and multiplying by 100 yields the %ID. bladder wall (104.0, 45.5 6 19.4, and 80.2 6 37.8 mSv/MBq Because the dynamic voiding-bladder model (10) assumes the complete for mice, piglets, and humans, respectively) and the kidneys

TABLE 4 Comparison of ED for Different PET Tracers Based on Preclinical and Clinical Dosimetry Studies

Tracer Target organ Clinical (μSv/MBq) Preclinical (μSv/MBq) Deviation (%) Reference

(−)-18F-ﬂubatine Brain 23.4 12.5 (mouse) 44.0 This study 14.7 (piglet) 34.0 18F-RGD-K5 Cancer imaging 31.0 22.2 (monkey) 28.4 (20) 18F-BMS747158 Myocardial imaging 19.0 15 (monkey) 21.1 (21,22) 18F-FEDAA1106 Evaluation of neuroinﬂammatory 36.0 23.5 (mouse) 34.7 (23) diseases 11C-choline Oncologic 4.4 2.8 (rat) 36.0 (24) 11C-DASB Brain 7.0 6.2 (monkey) 11.4 (25,26) 11C-telmisartan Liver 4.2 3.7 (rat) 13.1 (27)

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(24.2, 41.8 6 7.3, and 38.6 6 5.1 mSv/MBq for mice, piglets, Bottlaender et al. (18). As stated there, the contribution to the res- and humans, respectively). Tables 1–3 show the mean OD as well idence time (normalized cumulated activity) in an organ is mostly as ED values of 25 organs in the 3 investigated species. In this affected by the short half-life of 18F rather than by the different study, the calculated EDs to humans based on mouse, piglet, and biologic half-lives of the active molecules. This statement empha- human data were 12.5, 14.7 6 0.7, and 23.4 6 0.4 mSv/MBq, sizes that the preclinical underestimation of the effective dose to respectively. For an administration of 300 MBq in a brain study, humans of 40% would be applicable not only for tracers similar to the corresponding ED estimates to humans are 3.75, 4.41, and flubatine but also for other 18F-labeled substances (19). 7.02 mSv. CONCLUSION DISCUSSION As has been demonstrated for other PET radiotracers, pre- The values (%ID/organ, 15 min after injection) of the liver clinical (i.e., animal-derived) dosimetry underestimates the ED to (mice, 8.7; piglets, 15.9 6 6.9; humans, 18.9 6 2.4) and kidneys humans, in the current case of (–)-18F-flubatine by 34%–44%. (mice, 4.6; piglets, 4.2 6 2.3; humans, 4.6 6 0.7) were significantly higher than those of the remaining organs but decreased DISCLOSURE because of the clearance in these organs. At the end of the re- The costs of publication of this article were defrayed in part by spective measurement, the values decreased significantly for the the payment of page charges. Therefore, and solely to indicate this liver (mice, 0.05 6 0.02; piglets, 5.4 6 2.1; humans, 8.4 6 1.2) fact, this article is hereby marked “advertisement” in accordance and kidneys (mice, 0.01 6 0.003; piglets, 1.4 6 0.3; humans, with 18 USC section 1734. The trial was funded by the German 1.4 6 0.3). These organs are the most highly exposed in all Federal Ministry of Education and Research (project code investigated species, with organ doses comparable to those of 01EZ0820) and partially cofunded by Strahlenschutzseminar in other 18F-labeled tracers (e.g., 18F-mefway, 18F-nifene) (13,14) Thüringen (registered association, project-code F2010-10). The 11 11 15 as well as to the C-labeled C-WAY ( ). Figure 6 shows a use of (–)-18F-flubatine in humans was authorized by the respon- summary of all ODs for the 3 investigated species. Information sible authorities in Germany, the Federal Institute for Drugs and on the comparison to tracers that target similar receptors can be Medical Devices (Bundesamt für Arzneimittel und Medizinpro- found in the supplemental material. dukte) and the federal Office for Radiation Protection (Bundesamt To achieve an adequate image quality, the German Federal für Strahlenschutz), as well as by the local ethics committee. The Office for Radiation Protection (Bundesamt für Strahlenschutz) animal experiments were approved by the regional administration proposes for 18F-labeled tracers the administration of between Leipzig of the Free State of Saxony, Germany. Alexander Hoepping 200 MBq (brain imaging) and 350 MBq (WB PET imaging for and René Smits are employees of ABX advanced biochemical tumor detection) to an adult human (16). After the injection of compounds Radeberg, Germany. No other potential conflict of in- 300 MBq of (2)-18F-flubatine, for instance, this will result in an terest relevant to this article was reported. effective dose of about 3.75, 4.41, and 7.02 mSv (mouse, piglet, and human, respectively) based on the data as presented in this ACKNOWLEDGMENT work. In general, the dosimetry results and the deviation of preclinical We thank Dr. Tatjana Sattler, DVM, Large Animal Clinic for Internal Medicine, University Leipzig, Germany, for her support in from clinical phase data is within the range of those of other 18F- keeping and preparing the piglets for the imaging sessions. labeled tracers as shown in Table 4. As described in the “Methods” section, a scaling of the time and activity data to the human entity REFERENCES primarily can compensate for the faster metabolism of small animals but not for the differences in tracer distribution due to different 1. Quik M, Wonnacott S. a6b2* and a4b2* nicotinic acetylcholine receptors as drug targets for Parkinson’s disease. Pharmacol Rev. 2011;63:938–966. species. For a prediction of further preclinical studies and the re- 2. Brasiˇ c´ JR, Cascella N, Kumar A, et al. 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1892 THE JOURNAL OF NUCLEAR MEDICINE • Vol. 55 • No. 11 • November 2014 24 chapter 2. Publications

2.2 Study 2: Radiation Dosimetry of the α4β2 Nicotinic Receptor Ligand (+)-[18F]ﬂubatine, Comparing Preclinical PET/MRI and PET/CT to First-in-Human PET/CT Results

European Journal of Nuclear Medicine and Molecular Imaging, Physics (2016): 3(1):25. doi: 10.1186/s40658-016-0160-5

Mathias Kranz∗, Bernhard Sattler∗, Solveig Tiepolt, Stephan Wilke, Winnie Deuther- Conrad, Cornelius K. Donat, Steffen Fischer, Marianne Patt, Andreas Schildan, Jörg Patt, RenéSmits, Alexander Hoepping, Jörg Steinbach, Osama Sabri∗, Peter Brust∗ ∗ Contributed equally Kranz et al. EJNMMI Physics (2016) 3:25 DOI 10.1186/s40658-016-0160-5 EJNMMI Physics

ORIGINALRESEARCH Open Access

Radiation dosimetry of the α4β2 nicotinic receptor ligand (+)-[18F]flubatine, comparing preclinical PET/MRI and PET/CT to first-in-human PET/CT results Mathias Kranz1†, Bernhard Sattler2†, Solveig Tiepolt2, Stephan Wilke2, Winnie Deuther-Conrad1, Cornelius K. Donat1,4, Steffen Fischer1, Marianne Patt2, Andreas Schildan2, Jörg Patt2, René Smits3, Alexander Hoepping3, Jörg Steinbach1, Osama Sabri2† and Peter Brust1*†

* Correspondence: [email protected] †Equal contributors Abstract 1 Institute of Radiopharmaceutical 18 Cancer Research, Research Site Background: Both enantiomers of [ F]flubatine are new radioligands for neuroimaging Leipzig, Helmholtz-Zentrum of α4β2 nicotinic acetylcholine receptors with positron emission tomography (PET) Dresden-Rossendorf, exhibiting promising pharmacokinetics which makes them attractive for different clinical Permoserstraße 15, 04318 Leipzig, 18 Germany questions. In a previous preclinical study, the main advantage of (+)-[ F]flubatine 18 Full list of author information is compared to (−)-[ F]flubatine was its higher binding affinity suggesting that available at the end of the article 18 (+)-[ F]flubatine might be able to detect also slight reductions of α4β2 nAChRs and could be more sensitive than (−)-[18F]flubatine in early stages of Alzheimer’s disease. To support the clinical translation, we investigated a fully image-based internal dosimetry approach for (+)-[18F]flubatine, comparing mouse data collected on a preclinical PET/MRI system to piglet and first-in-human data acquired on a clinical PET/CT system. Time-activity curves (TACs) were obtained from the three species, the animal data extrapolated to human scale, exponentially fitted and the organ doses (OD), and effective dose (ED) calculated with OLINDA. Results: The excreting organs (urinary bladder, kidneys, and liver) receive the highest organ doses in all species. Hence, a renal/hepatobiliary excretion pathway can be assumed. In addition, the ED conversion factors of 12.1 μSv/MBq (mice), 14.3 μSv/MBq (piglets), and 23.0 μSv/MBq (humans) were calculated which are well within the order of magnitude as known from other 18F-labeled radiotracers. (Continued on next page)

© The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Kranz et al. EJNMMI Physics (2016) 3:25 Page 2 of 17

(Continued from previous page) Conclusions: Although both enantiomers of [18F]flubatine exhibit different binding kinetics in the brain due to the respective affinities, the effective dose revealed no enantiomer-specific differences among the investigated species. The preclinical dosimetry and biodistribution of (+)-[18F]flubatine was shown and the feasibility of a dose assessment based on image data acquired on a small animal PET/MR and a clinical PET/CT was demonstrated. Additionally, the first-in-human study confirmed the tolerability of the radiation risk of (+)-[18F]flubatine imaging which is well within the range as caused by other 18F-labeled tracers. However, as shown in previous studies, the ED in humans is underestimated by up to 50 % using preclinical imaging for internal dosimetry. This fact needs to be considered when applying for first-in-human studies based on preclinical biokinetic data scaled to human anatomy. Keywords: Image-based internal dosimetry, (+)-[18F]flubatine, Preclinical hybrid PET/MRI, Radiation safety, Nicotinic receptors, Dosimetry, OLINDA/EXM

Background

For neuroimaging of α4β2 nicotinic acetylcholine receptors (nAChRs), a new promising radioligand [18F]flubatine, formerly called [18F]NCFHEB, was recently discovered

[1]. The α4β2 receptor subtype comprises ~90 % of all nAChRs in the brain [2] and is involved in different neuronal functions and diseases like Parkinson’s disease [3], schizophrenia [4], and Alzheimer’s disease (AD) [5]. Its density is reduced in post mortem studies of patients who suffered from AD throughout the cerebral cortex [6, 7]. The possibility of detecting disease-related alterations of nAChRs might provide important information on the transformation from mild cognitive impairment to AD [8, 9]. Most of other tracers targeting nAChRs, e.g., 2-[18F]fluoro-A-85380 [10] or [18F]nifene [11] have the drawback of long acquisition times or a low specific binding. The enantiomers (−)-[18F]flubatine and (+)-[18F]flubatine have a high affinity (Ki = 0.112 nM, Ki =

0.064 nM, respectively) and selectivity [12] to α4β2 receptors as demonstrated in mice [12], piglets [13], and rhesus monkeys [14]. Although both enantiomers are suitable for clinical application, the (−) enantiomer was chosen first due to faster binding kinetics in pig brains [15] for an early clinical study [16]. As expected, (−)-[18F]flubatine showed fast kinetics in humans reaching its pseudo equilibrium within 90 min p.i. in the thalamus and within 30 min p.i. in all cortical regions [7, 15]. Metabolite analysis have shown that (−)-[18F]flubatine has a high stability after i.v. injection in patients with AD and healthy controls. More than 85 % of unchanged tracer was found at 90 min p.i. determined by dynamic blood sampling followed by radio-HPLC [17]. However, a metabolite correction of the positron emission tomography (PET) data is inevitable [17]. In the previous preclinical study, the main advantage of (+)-[18F]flubatine compared to (−)-[18F]flubatine was its higher binding affinity [13]. Therefore, the data suggest that (+)-[18F]flubatine

might be able to detect also slight reductions of α4β2 nAChRs and could be more sensitive than (−)-[18F]flubatine in early stages of AD. For the translation of newly developed radiotracers into clinical study phases, a radiation dosimetry, i.e., the calculation of the absorbed organ and effective dose in humans is mandatory. These dose assessments are mainly based on biokinetic data obtained in small animals prior to permission for first-in-human use [18]. The human Kranz et al. EJNMMI Physics (2016) 3:25 Page 3 of 17

safety and tolerability has to be shown in a whole-body biodistribution and dosimetry study. To determine the biodistribution of a tracer, terminal methods (sacrificing the animals followed by organ harvesting) or non-invasive methods using quantitative molecular imaging techniques like PET and single-photon emission computed tomography (SPECT) can be applied [19]. Imaging-based methods should be preferred in order to minimize the necessary number of laboratory animals and the duration of the investigation. Constantinescu et al. [20, 21] has shown the feasibility of a preclinical assessment of the radiation dose in humans by radiotracers using mice and rats on a small animal PET/CT. However, since the soft tissue contrast is comparatively poor in CT, the organ delineation remains difficult. In this work, we introduce our preclinical PET/MRI system to estimate the absorbed radiation dose in humans based on small animal quantitative PET data and evaluate the feasibility and suitability of the proposed protocol. Additionally, piglets are used to investigate the influence of the body size on the dosimetry result. Subsequently, we report on the first-in-human internal dosimetry using (+)-[18F]flubatine obtained in three healthy volunteers. We compare the results of the image-based approach after i.v. injection of (+)-[18F]flubatine in mice, piglets, and humans, as presented in this work, to previous dosimetry data of an ex vivo biodistribution study (organ harvesting method) in 27 mice and PET/CT imaging of 6 piglets and 3 humans after i.v. injection of (−)-[18F]flubatine [16].

Methods Synthesis of (+)-[18F]flubatine A detailed description of the synthesis was published by Deuther-Conrad et al. [1], a fully automated radiosynthesis by Patt et al. [22] and a highly efficient 18F-radiolabeling based on the latest generation of trimethylammonium precursors by Smits et al. [15]. The automated radiosynthesis under full GMP conditions as described by Patt et al. [22] was used for the dosimetry investigations in mice, piglets, and humans. Enantiomeric pure (+)-[18F]flubatine was synthesized in 40 min with a radiochemical yield of 30 %, a radiochemical purity of >97 % and a specific activity of about 3000 GBq/μmol.

Preclinical investigations All animal experiments were approved by the respective Institutional Animal Care and Use Committee and by the regional administration Leipzig of the Free State of Saxony, Germany, and are in accordance with national regulations for animal research and laboratory care (§ 8 section 1 Animal protection act) as well as with the standards set forth in the eighth edition of Guide for the Care and Use of Laboratory Animals.

Mice Three female CD1 mice (age = 12 weeks; weight = 30.1 ± 0.4 g) were housed in a temperature-controlled box with a 12:12 h light cycle at 26 °C. They underwent anesthesia (U-410, AgnTho's AB, Sweden) in a chamber using 4.0 % isoflurane in 60 %

O2 and 40 % synthetic air (Gas blender 100, MCQ Instruments, Italy) until they were fully motionless demonstrated by the lack of pedal withdrawal reflex. Afterwards, they were positioned prone on the mouse imaging chamber and the anesthesia was reduced to 1.7 % isoflurane at 250 mL/min. The animals were imaged up to 4 h after i.v. injection Kranz et al. EJNMMI Physics (2016) 3:25 Page 4 of 17

of 9.4 ± 2.4 MBq (+)-[18F]flubatine into the lateral tail vein. The imaging chamber was maintained to keep the animals at 37 °C continuously to prevent hypothermia, and their breath frequency was controlled by a pneumatic pressure sensor at the chest.

Piglets Three female piglets (age = 43 days; weight = 14 ± 1 kg) underwent anesthesia using

20 mg/kg ketamine, 2 mg/kg azaperone, 0.5 % isoflurane in 70 % N2O/30 % O2. Subsequently, they were artificially ventilated after surgical tracheotomy by a ventilator (Ventilator 710, Siemens-Elema, Sweden) followed by a 0.2 mg/kg/h pancuronium bromide i.v. injection. All incision sites were infiltrated with 1 % lidocaine. Volume substitution was supplied through a vein access (lactated Ringer’s solution, 5 mL/kg/h]). To monitor the arterial blood gas and acid-base parameters, blood samples were taken from the arteria femoralis with a polyurethane catheter to the aorta abdominalis and analyzed for relevant parameters (Radiometer ABL 50, Copenhagen, Brønshøj, Denmark). After finishing all surgical preparations, the concentration of anesthetic gas was reduced

to 0.25 % isoflurane, 65 % N2O, and 35 % O2. The body temperature was monitored by an in-ear thermometer and maintained using a heating blanket throughout the imaging session. PET scans were sequentially conducted up to 5 h after i.v. (v. jugularis) injection of 183.5 ± 9.0 MBq (+)-[18F]flubatine.

Clinical study All experiments with (+)-[18F]flubatine were authorized by the responsible authorities in Germany, the Federal Institute for Drugs and Medical Devices (Bundesamt für Arzneimittel und Medizinprodukte, BfArM), the federal Office for Radiation Protection (Bundesamt für Strahlenschutz, BfS), the local ethics committee, and the institutional review board. All study participants gave their written consent to take part in the first- in-human study, particularly, that the data obtained can be analyzed scientifically including publication. The trial was registered in the EU clinical trials database, https:// eudract.ema.europa.eu on 25/07/2012 with the trial registration number 2012-003473-26. The first subject was enrolled on 11/04/2014. Three non-smoking healthy volunteers (two males, one female; age = 58 ± 6 years; weight = 81 ± 6 kg) gave their written informed consent. During the screening procedure, the individual medical and surgical history, including medication and allergies, was documented. Furthermore, a physical examination of the major body systems (plus height and weight), including vital signs, ECG (12-lead) and blood as well as urine samples was performed. None of the three volunteers fulfilled any exclusion criteria (i.e., significant abnormal physical examination, evidence of any significant illness from history, clinical, or para-clinical findings). The subjects were sequentially imaged in a PET/CT system (Biograph16, SIEMENS, Erlangen, Germany) after i.v. injection of 285.9 ± 12.6 MBq (+)-[18F]-flubatine.

Instrumentation Preclinical PET/MRI The mouse studies were performed using a commercially available preclinical PET/ MRI system (nanoScan®, MEDISO Budapest, Hungary). The three-dimensional lutetium-yttrium-orthosilicate (LYSO) 12 PS-PMT based PET-detector system with an Kranz et al. EJNMMI Physics (2016) 3:25 Page 5 of 17

axial field of view (FOV) of 94.7 mm has a resolution of 1.5–2.0 mm (transaxial) and 1.3–1.6 mm (axial) [23]. Each PET data set was corrected for random coincidences, dead time, scatter, and attenuation correction (AC), based on a whole-body MR scan segmented into soft tissue and air. The reconstruction parameters were as follows: 3D- ordered subset expectation maximization (OSEM), four iterations, six subsets, energy window 400–600 keV.

Clinical PET/CT The piglet and human studies were performed on the clinical PET/CT system as mentioned above using a low-dose CT-AC and iterative PET reconstruction (OSEM, four iterations, eight subsets). Both systems are subjected to periodically detector normalization and activity calibration. Furthermore, all peripheral devices to be used for the investigation (dose calibrator, gamma counter) are cross calibrated in terms of timing and radioactivity adjustment.

PET scan protocol and data acquisition/evaluation Mice The animals were positioned prone in a special mouse imaging chamber (MultiCell, MEDISO Budapest, Hungary), with the head fixed to a mouth piece for the anesthetic gas supply. The PET data was collected in list-mode by a continuous whole-body (WB) scan during the entire investigation using the whole FOV at one bed position (BP). Subsequently, the list-mode-data was rebinned into sinograms of time frames (3 × 5 min, 1 × 10 min, 6 × 15 min) up to 240 min. p.i. (Fig. 1). Following the PET scan, a T1-weighted WB gradient echo sequence (TR = 20 ms; TE = 3.2 ms) was performed for anatomical orientation and segmentation of a μ-map (soft tissue and air) for AC.

Piglets The piglets were positioned prone with their legs alongside the body on a custom-made plastic trough including a special head-holder. The PET acquisition was divided in a sequential (4 × 9 min, 3 × 12 min) and a static part (1 × 24 min, 1 × 30 min,1 × 36 min) (Fig. 1) each of which was preceded by a low-dose CT for AC.

Humans The subjects were positioned supine with their arms down. The PET acquisition was similar to that for piglets but with an extended timeline for the sequential (4 × 13.5 min, 3 × 18 min) and static part (1 × 36 min, 1 × 45 min, 1 × 54 min) (Fig. 1). The subjects left the system during the examination to stretch out and for voiding the urinary bladder. The volume of all urine p.i. was measured and its activity concentration determined by sampling 3 × 0.5 ml in tubes to be measured with a gamma-counter (Cobra II 5003, PACKARD, 3-in NaI crystal). The activity concentration was decay corrected to the actual time of micturition.

Image analysis All images were analyzed with the Software package ROVER (ABX, Radeberg, Germany; v. 2.1.17) [24]. The organs that exhibited tracer uptake were identified and manually delineated using 3D volumes of interest (VOI). The MR and CT images were Kranz et al. EJNMMI Physics (2016) 3:25 Page 6 of 17

Fig. 1 Dynamic PET image series (MIP) of mice (a), piglets (b), and humans (c). Each series shows the tracer accumulation in whole brain and particularly in the striatum followed by a washout through the hepatobiliary and the renal system. The animals did not void during the course of imaging. The healthy volunteers were asked to void as indicated in the timeline of the human investigational protocol

used for anatomical orientation and for image registration with the PET data. Source organs are the brain, gallbladder, large intestine, small intestine, stomach, heart, kidneys, liver, lungs, pancreas, red marrow (backbone, pelvis, sternum), spleen, thyroid, testes, and urinary bladder. The activity data of each VOI was assigned to the individual

organ or tissue and subsequently transformed into percentage of injected dose (%IDorgan) with Eq. 1 [16], A ⋅c % organt scant % IDorgant ¼ ½ ð1Þ AtðÞ0

where Aorgant is the activity in the organ at the corresponding time t, cscant is an image calibration factor representing differences between imaged and injected activity which is calculated from the injected wholebody activity decay corrected to t and divided by the imaged body activity (whole body mask delineated by threshold so that the ROI volume equals the body weight of the animal/volunteer assuming a tissue density of

1 g/cm3) of the respective image time frame, and At(0) is the injected activity decay

corrected to t0.

Dose calculation Due to differences in weight, size, and metabolic rates between the smaller animal species and the human volunteers, it is necessary to map the preclinical (mouse and piglet) extracted biodistribution data to the human circumstances. Thus, the animal Kranz et al. EJNMMI Physics (2016) 3:25 Page 7 of 17

biokinetic data (time scale and %ID values) was adapted to the human circumstances using Eqs. 2 and 3 to fit the human weight, size, and metabolic rates [25]. The allometric metabolic rate scaling was found in physiological processes between species to be close to 1/4 for example when comparing blood flow per cycle of mice (15 s [26]) and rats (15 s [27]) to humans (50 s [28, 29]). Therefore, the following equation was chosen for time adaption to humans:

0:25 mhuman thuman ¼ tanimal ð2Þ manimal

where t is the animal timescale, t is the human time scale, and mhuman is the animal human manimal ratio between animal and human body weights. Among other mass scaling methods, the %ID/g method [30] has been widely applied:

%ID %ID m ¼ ⋅ m ⋅ animal ð3Þ organ g organ human m human animal ÀÁ human

where %ID is the fraction of the injected activity in the corresponding human organ, organhuman %ID is the fraction of injected activity per gram animal organ tissue, and morgan is ganimal human the mass of the corresponding human organ (e.g., hermaphrodite model as implemented in OLINDA). The scaled biokinetic data served as input into the EXM module in the OLINDA software [31]. The time-activity curves were fitted by exponential curves (least mean squares fit), and the area under the curve was calculated giving the total number of disintegrations (NOD) occurring in the respective organ during the time of investigation. Mice and piglets did not void urine during the investigation time, and thus, the curve fits of the urinary bladder were done using the activity data from PET imaging. The voided urine from human and therefore the decrease of activity needs to be included into the calculation in another way. As fitting as well as the voiding bladder model does not represent the time course of activity in the urinary bladder very well, the integral (=NOD) of the time-activity curve in the human urinary bladder was calculated by the trapezoidal equation [32] (Eq. 4). This gives a better approximation of the real course of activity in the human urinary bladder and, thus, the cumulated activity in it (Fig. 2).

1 n−1 %ID ¼ ð%IDi þ %IDi Þðti −tiÞð4Þ UB i¼1 þ1 þ1 2 X e

where %IDi, is the fraction of injected activity at time ti and %IDUB is the cumulated activity of the urinary bladder, i.e., the NOD. However, the dosef of the UB when using the International Commission on Radiological Protection (ICRP) dynamic bladder model is displayed in brackets for the human study in the “Results” and “Discussion” sections. The mean absorbed organ doses were estimated using the adult male phantom [33] based on the mapped to human animal biodistribution data or human data. To assess the overall risk in humans, the effective dose (ED) concept was used by multiplying the organ absorbed doses (OD) with the tissue weighting factors as published in the ICRP 103 [34]. However, as these weighting factors requires the ICRP 110 phantom [35] which is not available in OLINDA v.1.0, the ED results by using the tissue weighting factors published in ICRP 60 [36] can be found in the last table row (Tables 1, 2, and 3). Kranz et al. EJNMMI Physics (2016) 3:25 Page 8 of 17

Fig. 2 Exemplary time-activity curves of one human subjects and the respective exponential fit functions for different organs. For the urinary bladder, combined image and urine sample activity concentration data were used and the number of disintegrations is determined using the trapezoidal fit

Results In this study, we have investigated the preclinical dosimetry of (+)-[18F]flubatine, a

radiotracer for imaging of α4β2 nAChRs, by in vivo PET imaging of mice and piglets. The biokinetic data was extrapolated to the human scale and the ODs and ED estimated with OLINDA. In a subsequent first-in-human study in three healthy volunteers, the radiation safety was confirmed which supports the translation of this promising radioligand into further clinical phases. After i.v. injection of (+)-[18F]flubatine, no adverse effects were observed in the investigated species based on vital signs monitoring. A 60 min p.i. WB PET image of a mouse, piglet, and an adult is presented in Additional file 1: Figure S1, showing high uptake in the brain, liver, stomach, and urinary bladder. An example of the manually Kranz et al. EJNMMI Physics (2016) 3:25 Page 9 of 17

Table 1 ODs and ED for (+)-[18F]flubatine based on mice data Target organ OD SD ED contribution SD (mSv/MBq) (mSv/MBq) Adrenals 1.19E−02 2.02E−03 1.02E−04 1.74E−05 Brain 1.3E−02 5.77E−05 1.33E−04 5.77E−07 Breasts 7.24E−03 1.94E−03 8.68E−04 2.33E−04 Gallbladder wall 1.49E−02 6.43E−04 1.28E−04 5.53E−06 LLI wall 1.35E−02 1.15E−03 8.10E−04 6.92E−05 Small intestine 1.96E−02 2.81E−03 1.69E−04 2.42E−05 Stomach wall 1.47E−02 7.94E−04 1.76E−03 9.52E−05 ULI wall 2.05E−02 3.44E−03 1.23E−03 2.06E−04 Heart wall 9.75E−03 2.34E−03 8.38E−05 2.01E−05 Kidneys 4.75E−02 2.17E−02 4.08E−04 1.87E−04 Liver 2.05E−02 6.38E−03 8.21E−04 2.55E−04 Lungs 7.87E−03 1.81E−03 9.45E−04 2.17E−04 Muscle 9.02E−03 2.04E−03 7.76E−05 1.75E−05 Ovaries 1.22E−02 1.68E−03 9.76E−04 1.35E−04 Pancreas 1.41E−02 1.95E−03 1.21E−04 1.68E−05 Red marrow 9.95E−03 1.79E−03 1.19E−03 2.15E−04 Osteogenic cells 1.42E−02 3.42E−03 1.42E−04 3.42E−05 Skin 6.95E−03 1.73E−03 6.95E−05 1.73E−05 Spleen 1.41E−02 6.01E−03 1.21E−04 5.17E−05 Testes 8.84E−03 1.97E−03 –– Thymus 8.62E−03 2.27E−03 7.41E−05 1.95E−05 Thyroid 9.41E−03 1.41E−03 3.77E−04 5.65E−05 Urinary bladder wall 3.34E−02 1.68E−02 1.34E−03 6.72E−04 Uterus 1.28E−02 1.31E−03 1.10E−04 1.13E−05 Total body 9.87E−03 1.95E−03 –– ED (mSv/MBq) 1.21E−02 6.97E−04 ED (mSv/MBq) ICRP 60 1.29E−02 8.29E−04 ODs calculated for the adult male model (73.7 kg, implemented in OLINDA) based on mouse biodistribution and organ geometry data that were scaled to human circumstances OD organ dose, ED effective dose (ICRP 103), SD standard deviation mean over three animals

delineated VOIs using the MR (Additional file 1: Figure S2) and CT information is shown in Additional file 1: Figure S3 (mice, piglet, human). The WB PET series in Fig. 1 for all three species show, among others, the differences of urine excretion as described above. Furthermore, the tracer accumulation in the liver, kidneys, and brain followed by a washout can be observed. The calculated ODs and ED based on the animal data and estimated for the adult male model are presented as mean values for 25 organs in Tables 1, 2, and 3 for mice, piglets, and humans, respectively. Detailed biokinetic data expressed as %ID can be found in Additional file 1: Table S1 to S3.

Estimation of the radiation exposure in humans from preclinical imaging data The high OD absorbed by the urinary bladder, kidneys, and liver indicate the major pathways of tracer elimination through the renal and the hepatobiliary system followed Kranz et al. EJNMMI Physics (2016) 3:25 Page 10 of 17

Table 2 ODs and ED for (+)-[18F]flubatine based on piglet data Target organ OD SD ED contribution SD (mSv/MBq) (mSv/MBq) Adrenals 1.11E−02 7.64E−04 9.57E−05 6.57E−06 Brain 3.23E−02 3.24E−03 3.23E−04 3.24E−05 Breasts 6.21E−03 5.15E−04 7.45E−04 6.18E−05 Gallbladder wall 1.81E−02 1.18E−03 1.56E−04 1.02E−05 LLI wall 1.15E−02 4.04E−04 6.88E−04 2.42E−05 Small intestine 1.48E−02 1.00E−03 1.27E−04 8.61E−06 Stomach wall 1.10E−02 6.51E−04 1.32E−03 7.81E−05 ULI wall 1.52E−02 1.15E−03 9.12E−04 6.92E−05 Heart wall 1.42E−02 2.15E−03 1.22E−04 1.85E−05 Kidneys 4.51E−02 6.50E−03 3.88E−04 5.59E−05 Liver 2.69E−02 4.78E−03 1.07E−03 1.91E−04 Lungs 1.39E02 2.00E−04 1.67E−03 2.40E−05 Muscle 7.77E−03 4.03E−04 6.68E−05 3.46E−06 Ovaries 1.07E−02 2.08E−04 8.53E−04 1.67E−05 Pancreas 2.24E−02 1.78E−03 1.93E−04 1.53E−05 Red marrow 1.15E−02 1.07E−03 1.38E−03 1.28E−04 Osteogenic cells 1.35E−02 1.21E−03 1.35E−04 1.21E−05 Skin 5.87E−03 4.13E−04 5.87E−05 4.13E−06 Spleen 2.01E−02 9.07E−04 1.73E−04 7.80E−06 Testes 7.70E−03 6.51E−05 –– Thymus 1.82E−02 3.27E−03 1.56E−04 2.81E−05 Thyroid 1.77E−02 8.90E−03 7.07E−04 3.56E−04 Urinary bladder wall 7.17E−02 2.63E−02 2.87E−03 1.05E−03 Uterus 1.28E−02 1.01E−03 1.10E−04 8.73E−06 Total body 9.39E−03 7.64E−04 –– ED (mSv/MBq) 1.43E−02 2.60E−04 ED (mSv/MBq) ICRP 60 1.52E−02 5.0E−04 ODs calculated for the adult male model (73.7 kg, implemented in OLINDA) based on piglet biodistribution and organ geometry data that were scaled to human circumstances OD organ dose, ED effective dose (ICRP 103), SD standard deviation mean over three animals

by a fast washout from the remaining organs (Fig. 1). The highest ODs were observed (values in μSv/MBq for mice and piglets) in the urinary bladder (33.4 ± 16.8, 71.7 ± 26.3), the kidneys (47.5 ± 21.7, 45.1 ± 6.5), and the liver (20.5 ± 6.4, 26.9 ± 4.8). The ED calculated from biokinetic animal data mapped to humans is 12.1 ± 0.7 μSv/MBq (mice) and 14.3 ± 0.3 μSv/MBq (piglets). Exemplary time-activity curves for (+)-[18F]flubatine are presented in Additional file 1: Figure S4 to S6 for mice, piglets, and humans, respectively, compared to previous results after application of (−)-[18F]flubatine. The time-activity curves (TACs) of both enantiomers from the piglet study are similar for almost all organs. Hence, no significant difference (students t test: piglets p = 0.77) was found for the assessment of the ED. However, methodological issues between the imaging and harvesting method cause differences among the two tracers investigated in the mice studies. Kranz et al. EJNMMI Physics (2016) 3:25 Page 11 of 17

Table 3 ODs and ED for (+)-[18F]flubatine based on human data Target organ OD SD ED contribution SD (mSv/MBq) (mSv/MBq) Adrenals 1.30E−02 2.26E−03 1.12E−04 1.94E−05 Brain 2.86E−02 3.33E−03 2.86E−04 3.33E−05 Breasts 6.29E−03 3.86E−04 7.55E−04 4.63E−05 Gallbladder wall 2.34E−02 9.30E−03 2.01E−04 7.99E−05 LLI wall 1.67E−02 2.89E−03 1.00E−03 1.74E−04 Small intestine 2.94E−02 8.90E−03 2.53E−04 7.66E−05 Stomach wall 2.04E−02 3.89E−03 2.44E−03 4.67E−04 ULI wall 3.24E−02 1.02E−02 1.94E−03 6.14E−04 Heart wall 1.57E−02 1.65E−03 1.35E−04 1.42E−05 Kidneys 3.81E−02 4.52E−03 3.28E−04 3.89E−05 Liver 5.31E−02 2.98E−02 2.12E−03 1.19E−03 Lungs 2.70E−02 2.60E−03 3.24E−03 3.12E−04 Muscle 7.90E−03 1.26E−04 6.80E−05 1.08E−06 Ovaries 1.31E−02 1.22E−03 1.05E−03 9.73E−05 Pancreas 2.08E−02 4.11E−03 1.79E−04 3.53E−05 Red marrow 1.49E−02 8.50E−04 1.79E−03 1.02E−04 Osteogenic cells 1.42E−02 7.81E−04 1.42E−04 7.81E−06 Skin 5.52E−03 1.27E−04 5.52E−05 1.27E−06 Spleen 1.34E−02 4.19E−03 1.15E−04 3.60E−05 Testes 2.17E−02 1.21E−02 1.53E−03 1.33E−03 Thymus 7.48E−03 2.91E−04 6.43E−05 2.50E−06 Thyroid 2.29E−02 4.37E−03 9.17E−04 1.75E−04 Urinary bladder wall 1.02E−01 2.96E−02 4.09E−03 1.18E−03 Uterus 1.55E−02 1.71E−03 1.33E−04 1.47E−05 Total body 1.05E−02 8.42E−04 –– ED (mSv/MBq) 2.30E−02 1.91E−03 ED (mSv/MBq) ICRP 60 2.48E−02 1.18E−03 ODs calculated for the adult male model (73.7 kg, implemented in OLINDA) OD organ dose, ED effective dose (ICRP 103), SD standard deviation mean over three human subjects

First-in-human study, human internal dosimetry There were no adverse effects reported in any of the three volunteers after i.v. injection of (+)-[18F]flubatine, and no significant changes in vital signs were monitored. Repre- sentative time-activity curves were plotted and their exponential fit functions calculated and exemplarily shown for the human dosimetry in Fig. 2 for eight organs. The highest ODs values were estimated in the urinary bladder (102.4 ± 29.6 or 28.8 ± 21.5 when applying the ICRP voiding bladder model with 4 h voiding interval), kidneys 38.1 ± 4.5, and liver 53.1 ± 29.8. The ED of (+)-[18F]flubatine after i.v. injection in human volunteers was estimated to be 23.0 ± 1.9 μSv/MBq.

Discussion In this study, we have shown the radiation safety of (+)-[18F]flubatine in preclinical studies prior to a first-in-human clinical trial. Furthermore, we demonstrated the feasibility of small animal PET/MR imaging to estimate the radiation dose in humans based on mice Kranz et al. EJNMMI Physics (2016) 3:25 Page 12 of 17

biokinetic image data scaled to human anatomy. However, the presented study confirmed that preclinical internal dosimetry underestimates the ED in humans by 38–47 % as it has already been shown for (−)-[18F]flubatine [16] and other radiotracers [37–40]. The main findings of the current investigation are (i) a reproducible ED result for (+)-[18F]flubatine compared to (−)-[18F]flubatine in humans based on extrapolated biokinetic data from mice and piglets, (ii) differences in the biodistribution in mice between the two enantiomers of [18F]flubatine due to methodological issues, (iii) shortcomings regarding radiation dose estimates for humans based on extrapolated small animal data, (iv) a better dose estimation in humans when using larger species to collect biokinetic data which is extrapolated to human scale, and (v) the confirmation of the radiation safety of (+)-[18F]flubatine for clinical studies.

Preclinical PET/MRI and PET/CT for dose estimation in humans Our previous study of (−)-[18F]flubatine showed that internal dosimetry based on biokinetic data acquired by sacrificing mice, organ harvesting, and measuring organs in a gamma-counter results in an ED of 12.5 μSv/MBq. In the current study, we determined the biodistribution of (+)-[18F]flubatine with the same species (similar parameters of breed, age, weight, sex) but using image-based activity quantification with a preclinical PET/MRI system. The mice organs could be clearly identified and manually delineated by using the structural MR information as shown in Additional file 1: Figure S2. Thereby, the ED was estimated to be 12.1 μSv/MBq following i.v. injection of (+)-[18F]flubatine. This is slightly lower but not significantly different (p = 0.44, t test) than assessed by the organ harvesting method. Bretin et al. [41] found a similar deviation of the ED as determined using preclinical image based dosimetry (ED: [18F]FDOPA: 16.8 μSv/MBq, ED: [18F]FTYR: 15.0 μSv/MBq) and organ harvesting (ED: [18F]FDOPA: 16.0 μSv/MBq, ED: [18F]FTYR: 14.3 μSv/MBq). However, large differences between the two enantiomers of [18F]flubatine can be observed in mice with regard to the biokinetic data (Additional file 1: Figure S4) and the ODs (Table 1), most probably due to the following substantial methodically difference in determining organ activities by the two preclinical methods. In contrast to the organ harvesting study, a limitation of the image-based method is that the animals are under isoflurane narcosis which is known to slow down the metabolism and influences the hemodynamics [42, 43]. As a result, the tracer uptake and subsequent wash-out differs between these methods which explain partially the varying ODs. Based on the currently available data, no mechanistic explanation can be provided. The preclinical dosimetry of both enantiomers based on biokinetic PET/CT data from piglets scaled to human anatomy shows similar organ %ID values (Additional file 1: Figure S5). Consecutively, the calculated ODs are identical for most organs. This recursively confirms that the differences in the results of the two small animal-based dosimetry approaches (organ harvesting or imaging method) are really due to methodological differences as described above. In comparison to the extrapolated animal data, the radiation exposure as determined in the first-in-human trial shows a 1.6- to 1.9-fold higher ED. The reason for this underestimation when using small animal data for human dose estimation is related to the following methodological shortcomings of the extrapolation methods. First, using Kranz et al. EJNMMI Physics (2016) 3:25 Page 13 of 17

the adult male model implemented in OLINDA 1.0 assumes that the anatomical arrangement (i.e., the geometric relations of organs and tissues) of mice and piglets is identical to that in humans. Hence, the applied mass extrapolation in animals does not account for the different positions and sizes and, thus, different radiation interactions of the organs to each other as reflected by the S-values in the respective phantom. Using rodent specific models (available with OLINDA 2.0 [44]) to solve this problem remains to be assessed. Secondly, the widely applied time and mass extrapolations cancel out differences in body and organ weight but do not account for species-specific differences of the target expression in the respective organs. Hence, the extrapolated uptake values of the organs differ significantly among the species. The dosimetry data of piglets show no significant difference of the ED (students t test: piglets p = 0.77) compared to the previous study with (−)-[18F]flubatine, while using the same method (clinical PET/CT system). Additionally, using piglets for human dose estimates improves the ED results slightly while an underestimation of 38 % remains, mainly due to the limitations based on narcosis, time and mass scaling, the dosimetry phantom and species-species differences as discussed before. Although it seems that larger species can improve the human dosimetry based on animal data, Zanotti-Fregonara et al. [45] showed both under- and overestimation of the ED in humans ranging from −11 to +72 %, by using biokinetic data obtained in monkeys (weight 9.9 ± 3.6 kg). This gives further evidence to take species-specific pharmacokinetics of the radiotracers and metabolites into account as they result in significant deviations in preclinical dosimetry. Despite the obvious shortcomings introduced by the organ mass and time scaling in preclinical internal dosimetry, these results justify the use of small animal image data to roughly estimate the radiation exposure in humans. Small animal PET/MR imaging yields comparable internal dosimetry results as the state-of-the-art organ harvesting method. Obviously, the main benefit of an image-based dosimetry with mice is not only the reduction of the necessary number of laboratory animals (3 versus up to about 30 per study) but also the reduction of study duration as well as tracer production resources. Furthermore, comparability of the results to those of piglet and human studies is increased, as the determination of the biodistribution and, thus, the dose calculations are based on quantitative PET-imaging as well.

First-in-human study and dosimetry The radiation dose by (+)-[18F]-flubatine in human tissues has been estimated after injection of the radiotracer in one female and two male healthy volunteers. The TACs (presented in Fig. 2) obtained hereby confirmed the renal/hepatobiliary clearance as assumed considering the preclinical results. Within 2 h p.i., the radioligand was rapidly cleared from the kidneys, liver, lungs, and spleen. The conversion factor of the effective 18 dose by application of the α4β2 receptor ligand (+)-[ F]flubatine is 23.0 μSv/MBq and well within the range of what is known of other 18F-labeled diagnostic radiotracers. Hence, the application as PET imaging agent in humans is safe. Additional file 1: Figure S6 revealed similar biokinetics for both enantiomers of [18F]-flubatine based on %ID values. Therefore, the effective dose estimates show no significant difference (p = 0.77, 18 t test). However, (+)-[ F]flubatine showed a higher affinity to α4β2 nAChRs compared Kranz et al. EJNMMI Physics (2016) 3:25 Page 14 of 17

to (−)-[18F]flubatine and a negligible amount of metabolites in our first-in-human study (data not shown). This supports the suggestion of the preclinical data that 18 (+)-[ F]flubatine might be more sensitive detecting α4β2 nAChR reductions and that 18 (+)-[ F]flubatine allows a quantification of α4β2 nAChRs without metabolite correction. Thus, in combination with the dosimetry, the application of (+)-[18F]flubatine in

humans is safe and the further investigation as a clinical tool for PET imaging of α4β2 nAChRs is being analyzed in other parts of the aforementioned clinical trial.

Conclusions There is an underestimation of up to 47 % when using animal data for the assessment of the radiation exposure in humans although it was scaled with respect to mass and time scale differences between the species. It is of further evidence that allometric scaling can only compensate for the faster metabolism of the animals due to their size but not for actual differences in tracer uptake, species-specific target expression, and clearance in these species compared to humans. The dose conversion factor and the overall radiation risk represented by the effective dose is well within the range of what is known and well tolerated with other 18F-labeled tracers such as 19.0 μSv/MBq for [18F]FDG [46]. The deviation of preclinical dosimetry results from clinical study results suggests a systematic underestimation of the effective dose in humans below 50 % when determined using animal models.

Additional file

Additional file 1: Figures S1 to S6 and Tables S1 to S3. Figure S1. PET images (maximum intensity projection, MIP) 1 h p.i. of a (A) mouse (29.7 g), (B) piglet (14.0 kg), and (C) human (77 kg). The accumulation in the brain, liver, urinary bladder, red marrow, and the intestines can be clearly identified in all three species. Compared with humans, there is an additional tracer uptake in the salivary gland in mice and in the eyes in piglets. Figure S2. T1 weighted gradient echo sequence (TR = 20 ms; TE = 3.2 ms; MIP) of a mouse. The high soft tissue contrast in the MRI allows for clear organ delineation. The heart, stomach, large intestines, small intestines, liver, and the coronary vessels can be clearly identified. Furthermore, a map of linear attenuation coefficients was segmented (soft tissue and air) from this image for scatter and attenuation correction. Figure S3. Exemplary PET images (MIP) of a mouse (A), piglet (B), and a female human (C) with VOIs highlighted. Figure S4. Organ by organ comparison of the mice imaging ((+)-[18F]flubatine) vs. organ harvesting ((−)-[18F]flubatine) method (mean %ID). Figure S5. Organ by organ comparison of the piglet imaging method after application of (−)- and (+)-[18F]flubatine (mean %ID). Figure S6. Organ by organ comparison of the human imaging method after application of (−)- and (+)-[18F]flubatine (mean %ID). Table S1. Mouse biokinetic small animal PET/MR based data and extrapolation to human circumstances in %ID per organ (mean %ID). Table S2. Piglet biokinetic PET/CT-based data and extrapolation to human circumstances in %ID per organ (mean %ID). Table S3. Human biokinetic PET/CT based data (mean %ID). (PDF 878 kb)

Abbreviations AC: Attenuation correction; BP: Bed position; ECG: Electrocardiography; ED: Effective dose; FOV: Field of view; HPLC: High-performance liquid chromatography; ICRP: International Commission on Radiological Protection; ID: Injected dose; MRT: Magnet resonance tomography; nAChRs: Nicotinic acetylcholine receptors; NCFHEB: Norchloro- fluoro-homoepibatidine; NOD: Number of disintegrations; OD: Organ dose; OSEM: Ordered subset expectation maximization; PET: Positron emission tomography; PS-PMT: Position-sensitive photomultiplier tube; ROI: Region of interest; SPECT: Single-photon emission computed tomography; VOI: Volume of interest; WB: Whole-body

Acknowledgements We thank Dr. Tatjana Sattler, DVM, Large Animal Clinic for Internal Medicine, University Leipzig, Germany, for her support in keeping and preparing the piglets for the imaging sessions.

Funding The trial was funded by the Helmholtz Validation Fonds (HVF-0012) and partially co-funded by Strahlenschutzseminar in Thüringen (F2010-10).

Availability of data and materials Supporting data can be found in the supplemental material. Kranz et al. EJNMMI Physics (2016) 3:25 Page 15 of 17

Authors’ contributions MK performed most of the experiments, analyzed the data, and wrote the manuscript. BS designed the trial, performed the experiments, analyzed and revised the data, and wrote and revised the manuscript. ST as a study physician took care for the safety of healthy volunteers during recruitment and the actual imaging investigations, performed the tracer application, and revised the manuscript. SW was the responsible physician for human studies and co-performed the PET/CT experiments. WD-C performed the mice and piglet studies (surgery) and revised the manuscript. CKD performed the piglet experiments (surgery) and data acquisition. SF performed the radiochemistry and tracer delivery for preclinical experiments. MP performed the radiochemistry and tracer delivery for human studies and revised the manuscript. AS and JP performed the radiochemistry and tracer delivery for human studies. RS and AH performed the precursor synthesis and revised the manuscript. JS performed the experimental study design and revised the manuscript. OS is the human study responsible principal investigator and volunteer recruitment for first-in-man study and revised the manuscript. PB designed and performed the piglet experiments and wrote and revised the manuscript. All authors read and approved the final manuscript.

Competing interests Alexander Hoepping and René Smits are employees of ABX advanced biochemical compounds, Radeberg, Germany. All other co-authors have no potential conflicts of interest to report.

Consent for publication All study participants gave their written informed consent to take part in the study and to the scientific analysis of the results including publication.

Ethics approval and consent to participate All animal experiments as well as the first-in-human use of (+)-[18F]flubatine were authorized by the responsible authorities in Germany the Federal Institute for Drugs and Medical Devices (Bundesamt für Arzneimittel und Medizinprodukte, BfArM), the federal Office for Radiation Protection (Bundesamt für Strahlenschutz, BfS), the local ethics committee, and the institutional review board. All animal experiments were approved by the respective Institutional Animal Care and Use Committee and by the regional administration Leipzig of the Free State of Saxony, Germany, and are in accordance with national regulations for animal research and laboratory care (§ 8 section 1 Animal protection act).

Author details 1Institute of Radiopharmaceutical Cancer Research, Research Site Leipzig, Helmholtz-Zentrum Dresden-Rossendorf, Permoserstraße 15, 04318 Leipzig, Germany. 2Department of Nuclear Medicine, University Hospital Leipzig, Leipzig, Germany. 3ABX advanced biochemical compounds Ltd., Radeberg, Germany. 4Division of Brain Sciences, Department of Medicine, Hammersmith Hospital Campus, Imperial College London, London, UK.

Received: 11 August 2016 Accepted: 9 October 2016

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2.3 Study 3: Evaluation of the Enantiomer Speciﬁc Biokinetics and Radiation Doses of [18F]ﬂuspidine - A New Tracer in Clinical Translation for Imaging of σ1 Receptors

Molecules (2016): 21(9):1164. doi: 10.3390/molecules21091164.

Mathias Kranz∗, Bernhard Sattler∗, Nathanael Wust,¨ Winnie Deuther-Conrad, Ma- rianne Patt, Philipp M. Meyer, Steﬀen Fischer, Cornelius K. Donat, Bernhard Wunsch,¨ Swen Hesse, J¨org Steinbach, Peter Brust∗, Osama Sabri∗ ∗ Contributed equally molecules

Article Evaluation of the Enantiomer Speciﬁc Biokinetics and Radiation Doses of [18F]Fluspidine—A New Tracer in Clinical Translation for Imaging of σ1 Receptors

Mathias Kranz 1,†, Bernhard Sattler 2,†, Nathanael Wüst 2, Winnie Deuther-Conrad 1, Marianne Patt 2, Philipp M. Meyer 2, Steffen Fischer 1, Cornelius K. Donat 1,3, Bernhard Wünsch 4, Swen Hesse 2,5, Jörg Steinbach 1, Peter Brust 1,†,* and Osama Sabri 2,†

1 Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Department of Neuroradiopharmaceuticals, Leipzig 04318, Germany; [email protected] (M.K.); [email protected] (W.D.-C.); s.ﬁ[email protected] (S.F.); [email protected] (C.K.D.); [email protected] (J.S.) 2 Department of Nuclear Medicine, University Hospital Leipzig, Leipzig 04103, Germany; [email protected] (B.S.); [email protected] (N.W.); [email protected] (M.P.); [email protected] (P.M.M.); [email protected] (S.H); [email protected] (O.S.) 3 Division of Brain Sciences, Department of Medicine, Hammersmith Hospital Campus, Imperial College London, London SW7 2AZ, UK 4 Pharmaceutical and Medicinal Chemistry, University Münster, Münster 48149, Germany; [email protected] 5 Integrated Research and Treatment Center (IFB) Adiposity Diseases, University Hospital Leipzig, Leipzig 04103, Germany * Correspondence: [email protected]; Tel.: +49-341-234-179-4610 † These authors contributed equally to this work.

Academic Editor: Zhen Cheng Received: 28 July 2016; Accepted: 26 August 2016; Published: 1 September 2016

18 Abstract: The enantiomers of [ F]fluspidine, recently developed for imaging of σ1 receptors, possess distinct pharmacokinetics facilitating their use in different clinical settings. To support their translational potential, we estimated the human radiation dose of (S)-(−)-[18F]fluspidine and (R)-(+)-[18F]fluspidine from ex vivo biodistribution and PET/MRI data in mice after extrapolation to the human scale. In addition, we validated the preclinical results by performing a first-in-human PET/CT study using (S)-(−)-[18F]fluspidine. Based on the respective time-activity curves, we calculated using OLINDA the particular organ doses (ODs) and effective doses (EDs). The ED values of (S)-(−)-[18F]fluspidine and (R)-(+)-[18F]fluspidine differed significantly with image-derived values obtained in mice with 12.9 µSv/MBq and 14.0 µSv/MBq (p < 0.025), respectively. A comparable ratio was estimated from the biodistribution data. In the human study, the ED of (S)-(−)-[18F]fluspidine was calculated as 21.0 µSv/MBq. Altogether, the ED values for both [18F]fluspidine enantiomers determined from the preclinical studies are comparable with other 18F-labeled PET imaging agents. In addition, the first-in-human study confirmed that the radiation risk of (S)-(−)-[18F]fluspidine imaging is within acceptable limits. However, as already shown for other PET tracers, the actual ED of (S)-(−)-[18F]fluspidine in humans was underestimated by preclinical imaging which needs to be considered in other first-in-human studies.

Keywords: image based internal dosimetry; [18F]ﬂuspidine; preclinical hybrid PET/MRI; radiation safety; σ1 receptors

Molecules 2016, 21, 1164; doi:10.3390/molecules21091164 www.mdpi.com/journal/molecules Molecules 2016, 21, 1164 2 of 14

1. Introduction The existence of various tissues of the sigma opioid receptor (σ) was postulated first by Martin et al. in 1976 [1]; nowadays it has been proven to be a non-opioid receptor (Sigma Non-Opioid Intracellular Receptor 1; σ1 receptor). This receptor plays an important role in the cellular functions associated with the endocrine, immune, and nervous systems; however, the physiological function of the σ1 receptor is not yet fully understood [2]. Furthermore, this protein interacts with a variety of psychotomimetic drugs, including cocaine and amphetamines. Various diseases like neuropsychiatric and vascular diseases as well as cancer seem to be related to dysfunctions of the σ1 receptor [3–5]. Therefore, studying this protein with positron emission tomography (PET) could contribute to a better understanding and further evaluation of the pathophysiological role of σ1 receptors in diseases [6]. For imaging of σ1 receptors several radioligands were developed and used in human such as [18F]FPS [7] and [18F]FM-SA4503 [6]. The latter study showed that the σ1 receptor density is decreased in different brain structures in patients with early Alzheimer’s and Parkinson’s disease. Recently, our group developed and tested the chiral σ1 receptor ligand [18F]fluspidine in preclinical studies in mice and piglets [8], which revealed high brain uptake of the two enantiomers (R)-(+)-[18F]fluspidine and (S)-(−)-[18F]fluspidine along with marked enantioselectivity with regard to their biokinetics. As a consequence, the binding potential (BPnd) 18 18 of (R)-(+)-[ F]fluspidine is 5- to 10-fold higher in comparison to (S)-(−)-[ F]fluspidine in σ1-rich areas of the porcine brain [9], most probably due to differences in their affinity towards σ1 receptors 18 18 ((R)-(+)-[ F]fluspidine: Ki = 0.57 nM;(S)-(−)-[ F]fluspidine: Ki = 2.3 nM; [10]). These preclinical data indicated a suitability of both enantiomers of [18F]fluspidine for different clinical issues. For the 18 first-in-human investigation of σ1 receptors in brain we have chosen (S)-(−)-[ F]fluspidine as the enantiomer with the faster pharmacokinetics for reasons of feasibility in clinical routine (German clinical trial register ID: DRKS00008321). A radiation dose assessment, i.e., calculations of the absorbed and effective doses per unit activity administered is mandatory for the translation of novel radiotracers from preclinical to clinical study phases. These calculations are mainly based on biokinetic models using data obtained in biodistribution or imaging studies in animals. Usually rodents [11–15] or monkeys [16–19] are used and require the application of computational phantoms [20–23]. With rodents, both the organ harvesting method and the dynamic hybrid imaging method are feasible to collect biokinetic data which is later extrapolated to human anatomy (concerning organ mass and time scaling) [20]. By the organ harvesting method, the tissue activity concentration is quantified by gamma-counting and converted into percent of injected activity accumulated per organ (%ID) after dissection of the animals at different points post injection of a radiotracer. With the imaging method, the biokinetics of the radiotracer is investigated using clinical or small-animal PET/CT or PET/MRI systems. The activity in the organs as well as the weight is extracted after delineation with the help of the anatomical CT or MR images, and the organ-specific %ID values are calculated. Eventually, interspecies extrapolation of the respective animal data has to be performed to calculate the human effective dose. However, the standard procedure of these established models may lead to underestimation of radiation risk in humans as we could recently show with (−)-[18F]flubatine [24] and (+)-[18F]flubatine [25]. The preclinical dosimetry in mice revealed an underestimation of the effective dose in humans of up to 50% which could be improved only slightly when using piglets as larger species (underestimation ~38%). In this work, we report on the dosimetry and biodistribution of both enantiomers of the σ1 receptor ligand [18F]fluspidine based on in vivo and ex vivo data from mice which we obtained by the dynamic hybrid PET/MR imaging method as well as by an organ harvesting study. Subsequently, we report on the first-in-human internal dosimetry using (S)-(−)-[18F]fluspidine obtained in four healthy volunteers. This direct comparison of preclinical with clinical data is assumed to advance the use of small animal PET/MRI for the assessment of the radiation risk of novel PET imaging agents in humans. The preclinical dosimetry reveals the ED for (S)-(−)-[18F]fluspidine and (R)-(+)-[18F]fluspidine comparable with other 18F-labeled PET imaging agents, despite significant differences of the EDs due Molecules 2016, 21, 1164 3 of 14 to enantiomer specific tracer kinetics. The ED estimate from the first-in-human study confirmed that the radiation risk of (S)-(−)-[18F]fluspidine imaging is within accepted limits. However, as shown in previous studies, the ED in humans is underestimated by up to 50% by using preclinical− imaging for internal dosimetry. This fact needs to be considered when applying for first-in-human studies based on preclinical biokinetic data scaled to human anatomy.

2. Results

In this study, we have investigated the preclinical dosimetry of both enantiomers of the σ1 receptor ligand [18F]fluspidine based on in vivo and ex vivo data from CD-1 mice after i.v. injection. σ The biokinetic data was obtained either by dynamic hybrid small animal PET/MR imaging or by an organ harvesting approach in mice followed by extrapolation to the human scale. Subsequently, the ODs were estimated with OLINDA and the ED calculated using tissue weighting factors published by ICRP 60 [26] and ICRP 103 [27]. Finally, we performed a first-in-human dosimetry study of (S)-(−)-[18F]fluspidine in four healthy volunteers confirming the radiation safety of that promising− radioligand.

2.1. Human Dosimetry Estimation from Small Animal PET/MRI and Biodistribution Studies Representative dynamic PET images in mice obtained at different times p.i. of (S)-(−)-[18F]fluspidine and (R)-(+)-[18F]fluspidine are shown in Figure 1. A high initial uptake of activity− in liver, small intestines, and gallbladder wall as well as a fast clearance during the investigation time was observed. Exemplary time-activity curves (TACs) with fitting functions to calculate the numbers of disintegration (please see Section 4.4) for (S)-(−)-[18F]fluspidine and (R)-(+)-[18F]fluspidine are presented in Figure S1. − The corresponding mean uptake values (in terms of % ID at a particular time p.i.; Tables S3 and S4) reflect lower values of the S-enantiomer in comparison to the R-enantiomer.

Figure 1. Representative time series (MIP) of mice (a), (b), and a volunteer (c) after i.v. injection of − (S)-(−)-[18F]ﬂuspidine (a), (c) and (R)-(+)-[18F]ﬂuspidine (b). Furthermore, the diagram shows the scan protocol for humans clarifying the dynamic and static PET part as well as the urine voiding intervals. Molecules 2016, 21, 1164 4 of 14

The biodistribution study confirmed the enantiomer-specific performance (Figure S2). The decrease of the %ID values of (S)-(−)-[18F]fluspidine during the course of the study (Table S1) is contrasted by a stagnation of the washout of activity after administration of (R)-(+)-[18F]fluspidine (Table S2), which is most obvious in brain, spleen, kidneys, and lung. Accordingly, animal PET and biodistribution data revealed higher ODs and EDs for (R)-(+)-[18F]fluspidine compared to (S)-(−)-[18F]fluspidine (Tables 1 and 2). We estimated the highest OD values for (S)-(−)-[18F]fluspidine and (R)-(+)-[18F]fluspidine from animal PET/MRI in urinary bladder, kidneys, spleen, gallbladder wall, and liver (Table 1). From animal organ harvesting derived biodistribution, the highest values were estimated in kidneys, upper large intestine, small intestine, and lungs (Table 2). For (S)-(−)-[18F]fluspidine we estimated the ED in humans from animal PET/MRI and organ harvesting derived biodistribution to be 12.9 ± 0.4 µSv/MBq and 14.0 ± 0.5 µSv/MBq, respectively, and for (R)-(+)-[18F]fluspidine to be 16.7 µSv/MBq and 18.4 µSv/MBq, respectively Accordingly, for (R)-(+)-[18F]fluspidine the ED is higher than for (S)-(−)-[18F]fluspidine in both experimental conditions; however, statistical significance could be calculated only for the imaging-derived data (p = 0.025, students t test, n = 3/group). For the organ harvesting study, a t test is not applicable due to methodical reasons. Detailed biokinetic data expressed as mean %ID of the mice organ harvesting or imaging method can be found in the supplemental material (Tables S1–S4).

Table 1. OD and ED in µSv/MBq based on the imaging method with a small animal PET/MRI. ODs calculated for the adult male model (73.7 kg, implemented in OLINDA) based on mouse biodistribution and organ geometry data that were scaled proportionately to human circumstances.

(S)-(−)-[18F]Fluspidine (R)-(+)-[18F]Fluspidine Target Organ OD SD ED Contr. SD OD SD ED Contr. SD Adrenals 10.50 0.74 0.09 0.01 11.00 1.55 0.09 0.01 Brain 10.10 2.34 0.10 0.02 13.20 1.19 0.13 0.01 Breasts 5.93 0.10 0.71 0.01 6.19 1.77 0.74 0.21 Gallbladder Wall 25.60 9.57 0.22 0.08 30.10 11.90 0.26 0.10 LLI Wall 14.00 1.48 0.84 0.09 13.80 1.39 0.83 0.08 Small Intestine 23.10 3.22 0.20 0.03 22.60 1.92 0.20 0.02 Stomach Wall 10.50 0.60 1.26 0.07 12.70 1.10 1.52 0.13 ULI Wall 20.50 4.96 1.23 0.30 25.60 2.19 1.54 0.13 Heart Wall 9.85 0.60 0.08 0.01 10.50 1.31 0.09 0.01 Kidneys 37.60 14.80 0.32 0.13 26.90 2.74 0.23 0.02 Liver 25.00 3.23 1.00 0.13 26.10 4.65 1.04 0.19 Lungs 10.40 2.30 1.25 0.28 10.80 0.89 1.30 0.11 Muscle 7.57 0.07 0.07 0.00 7.86 1.96 0.07 0.02 Ovaries 11.50 0.82 0.92 0.07 11.90 1.95 0.95 0.16 Pancreas 10.90 0.69 0.09 0.01 24.80 1.79 0.21 0.02 Red Marrow 10.80 0.37 1.30 0.04 12.80 1.27 1.53 0.15 Osteogenic Cells 12.70 0.13 0.13 0.00 14.00 3.18 0.14 0.03 Skin 5.61 0.02 0.06 0.00 5.82 1.73 0.06 0.02 Spleen 26.10 7.29 0.22 0.06 31.80 20.00 0.27 0.17 Testes 7.46 0.39 0.00 0.00 7.63 2.04 0.00 0.00 Thymus 7.19 0.11 0.06 0.00 7.52 2.21 0.06 0.02 Thyroid 7.61 1.09 0.30 0.04 10.10 0.35 0.41 0.01 Urinary Bladder Wall 58.00 15.90 2.32 0.64 55.70 19.30 2.23 0.77 Uterus 12.80 1.28 0.11 0.01 13.00 1.49 0.11 0.01 Total Body 8.68 0.14 0.00 0.00 9.13 1.67 0.00 0.00 ED 12.9 0.4 14.0 0.5 ED ICRP 60 14.8 1.7 15.2 1.9 OD = organ dose; ED contr. = effective dose contribution; SD = standard deviation, mean over 3 animals; LLI = large lower intestine; ULI = upper large intestine. Molecules 2016, 21, 1164 5 of 14

Table 2. OD and ED in µSv/MBq based on mouse organ harvesting after dissection and organ counting in a gamma-counter. The organ geometry data were scaled proportionately to human circumstances. ODs calculated for the adult male model (73.7 kg, implemented in OLINDA).

(S)-(−)-[18F]Fluspidine (R)-(+)-[18F]Fluspidine Target Organ OD ED Contr. OD ED Contr. Adrenals 36.0 0.3 18.6 0.2 Brain 12.4 0.1 12.6 0.1 Breasts 11.2 1.3 11.3 1.4 Gallbladder Wall 15.5 0.1 14.0 0.1 LLI Wall 19.0 1.1 16.4 1.0 Small Intestine 31.9 0.3 25.1 0.2 Stomach Wall 14.8 1.8 14.3 1.7 ULI Wall 33.3 2.0 25.6 1.5 Heart Wall 17.9 0.2 22.3 0.2 Kidneys 35.6 0.3 27.6 0.2 Liver 12.5 0.5 10.3 0.4 Lungs 30.5 3.7 45.5 5.5 Muscle 7.2 0.1 7.1 0.1 Ovaries 17.0 1.4 24.9 2.0 Pancreas 26.2 0.2 21.7 0.2 Red Marrow 13.6 1.6 13.5 1.6 Osteogenic Cells 19.6 0.2 19.1 0.2 Skin 9.1 0.1 8.7 0.1 Spleen 17.6 0.2 17.2 0.1 Testes 11.2 - 10.8 - Thymus 12.0 0.1 19.3 0.2 Thyroid 11.7 0.5 11.5 0.5 Urinary Bladder Wall 13.9 0.6 20.2 0.8 Uterus 15.9 0.1 14.9 0.1 Total Body 12.5 - 12.2 - ED 16.7 18.4 ED ICRP 60 17.3 20.1 OD = organ dose; ED contr. = effective dose contribution; LLI = large lower intestine; ULI = upper large intestine.

2.2. Human Dosimetry from the First-in-Human Study There were no adverse effects reported in any of the four volunteers after i.v. injection of (S)-(−)-[18F]fluspidine, and no significant changes in vital signs were monitored. Typical TACs and fitted curves are shown in Figure S3. The results of the dose assessment are presented in Table 3. Detailed biokinetic data expressed as mean %ID of the clinical study can be found in the supplemental material (Table S5). The highest OD values for (S)-(−)-[18F]fluspidine were estimated in gallbladder wall, small intestine, stomach, and kidneys. The effective dose of (S)-(−)-[18F]fluspidine for humans was estimated to be 21.0 ± 1.3 µSv/MBq. A summary of the ED estimates for both enantiomers of [18F]fluspidine, the different methods and species can be found in Table 4. The toxicity results (please see supplemental methods and results) of the pathologic examination in Wistar rats indicated that (S)-(−)-fluspidine after single intravenous administration did not cause toxicological changes in pathological and histopathological parameters on day 2 and day 15. The no observed effect level (NOEL) of (S)-(−)-fluspidine after single intravenous administration in this study for both day 2 and day 15 was determined to be 620 µg/kg (highest tested dose). Molecules 2016, 21, 1164 6 of 14

Table 3. First-in-human data, OD and ED in µSv/MBq. The ODs were calculated for the adult male model (73.7 kg, implemented in OLINDA) based on human biodistribution and organ geometry data.

(S)-(−)-[18F]Fluspidine Target Organ OD SD ED Contr. SD Adrenals 15.3 1.1 0.1 0.0 Brain 22.6 4.2 0.2 0.1 Breasts 6.5 0.5 0.8 0.1 Gallbladder Wall 60.7 10.6 0.5 0.1 LLI Wall 16.6 5.1 1.0 0.3 Small Intestine 56.9 10.6 0.5 0.1 Stomach Wall 31.5 3.3 3.8 0.4 ULI Wall 24.3 5.2 1.5 0.3 Heart Wall 17.7 1.3 0.2 0.0 Kidneys 31.1 5.2 0.3 0.0 Liver 76.0 17.7 3.0 0.4 Lungs 28.2 2.9 3.4 0.3 Muscle 7.8 0.5 0.1 0.0 Ovaries 13.8 1.0 1.0 0.5 Pancreas 15.9 0.7 0.1 0.0 Red Marrow 23.2 2.2 2.8 0.1 Osteogenic Cells 18.0 1.6 0.2 0.0 Skin 5.3 0.5 0.1 0.0 Spleen 24.0 4.2 0.2 0.0 Testes 8.0 2.6 0.8 0.4 Thymus 7.5 0.7 0.1 0.0 Thyroid 8.4 1.4 0.3 0.1 Urinary Bladder Wall 24.7 3.4 1.0 0.1 Uterus 13.0 0.7 0.1 0.1 Total Body 11.4 0.3 0.0 0.0 ED 21.0 1.3 ED ICRP 60 22.1 0.8 OD = organ dose; ED contr. = effective dose contribution; SD = standard deviation, mean over four volunteers; LLI = large lower intestine; ULI = upper large intestine.

Table 4. Comparison of dosimetry results (ED) for different PET tracers including the current study with [18F]ﬂuspidine.

Tracer Target Organ Clinical (µSv/MBq) Preclinical (µSv/MBq) Reference 12.9 (mouse, imaging) (S)-(−)-[18F]fluspidine brain, tumor 21.0 this study 16.7 (mouse, harvesting) 14.0 (mouse, imaging) (R)-(+)-[18F]fluspidine brain, tumor n.a. this study 18.4 (mouse, harvesting) (−)-[18F]flubatine 12.5 (mouse) brain 23.4 [24] (formerly [18F]NCFHEB) 14.7 (piglet, imaging) 12.1 (mouse, imaging) (+)-[18F]flubatine brain 23.0 [25] 14.3 (piglets, imaging) 21.0 (male mouse) [18F]FEDAA1106 brain 36 [28] 26.0 (female mouse) [18F]FET brain tumor 16.5 9.0 [29,30] 2-[18F]F-A85380 brain 19.4 n.a. [31] [18F]FDG multiple 19.0 n.a. [32]

3. Discussion With this study, we support the clinical translation of the novel radiotracer [18F]fluspidine for imaging of σ1 receptors by preclinical and clinical radiation dosimetry studies. We have derived Molecules 2016, 21, 1164 7 of 14 internal radiation dosimetry of the enantiomers (S)-(−)-[18F]fluspidine and (R)-(+)-[18F]fluspidine by organ harvesting and dynamic small animal PET/MR imaging in mice and compared the results of both methods with each other. Finally, we performed a clinical study to calculate radiation doses for humans following intravenous injection of (S)-(−)-[18F]fluspidine and to validate the results achieved by the animal dose assessment. The main findings are (i) methodical issues regarding radiation estimates for humans extrapolated from small animals; (ii) radiation dose differences between the two enantiomers (S)-(−)-[18F]fluspidine and (R)-(+)-[18F]fluspidine; and (iii) confirmation of the radiation safety of (S)-(−)-[18F]fluspidine for clinical studies. We would like to point out that both the preclinical as well as the clinical studies have shown 18 that the novel σ1 receptor imaging agent (S)-(−)-[ F]fluspidine fulfils the requirements regarding radiation dose in human clinical trials, although in comparison to the extrapolated animal data a 1.6-fold higher value (p < 0.001, students t test, n = 3/group) of the actual ED has been calculated from the human study. The main reasons for this discrepancy are assumed to be related to several methodological shortcomings of the extrapolation procedures. One deficiency is the assumption in the adult male model implemented in OLINDA 1.0, that the anatomical organ arrangement between mice and humans is identical. However, a simple mass extrapolation in animals and using a human phantom that does not take into account the spatial interactions of the organs in comparison to mouse (reflected by the S-values), is insufficient. A novel approach using the implementation of rodent specific dosimetry models in OLINDA 2.0 [33] remains to be assessed. Another limitation belongs to the extrapolation methods used to adapt the animal time scale and uptake scale. The currently most qualified methods [34] cancel out at least partially species differences in metabolism as well as body and organ weight. However, a compensation for species-specific differences in the tracer uptake, i.e., differences in the expression of the target in the respective organ, is not possible. Furthermore, the aspect of the effect of significant size differences between the species on dose estimations has been recently addressed by our group during the clinical translation of a radioligand for imaging of nicotinic acetylcholine receptors by directly comparing dosimetry in piglets (~15 kg) and humans [24,25]. However, an underestimation of the radiation dose in humans of about 40% remained. Hence, a simple size-dependent effect is not likely, as reflected by the findings of Zanotti-Fregonara et al. [16]. In this study, both under- and overestimations of the effective dose in humans, ranging from −11% to +72%, by using biokinetic data for nine PET tracers obtained in monkeys are reported (baboons and rhesus monkeys, weight: 9.9 ± 3.6 kg). Altogether, these findings clearly indicate the need to take species-specific pharmacokinetics into account of both the radiotracer and radiometabolites as they potentially result in significant deviations in the dosimetry of the radiotracer under investigation. The direct comparison between the two preclinical methods of dose estimation via organ harvesting and dynamic small animal PET imaging reveals negligible differences regarding ED values of the respective [18F]fluspidine enantiomer under investigation. However, for both radiotracers, slightly lower organ doses were detected in the imaging than in the organ harvesting approach. This outcome is most likely related to anesthesia-mediated effects on hemodynamics and metabolism [35,36], although based on the currently available data no mechanistic explanation can be provided. The attractive approach reported by Bretin et al. [14] to compensate for deviations between these two preclinical methods by correcting the image derived TACs according to the activity values measured ex vivo by gamma-counting after scanning is not applicable here, because in contrast to our study they used anesthetized animals for the organ harvesting method as well. Another interesting finding in our preclinical study is that although both enantiomers accumulate specifically in σ1 receptor rich regions in the brain [9], they exhibit pronounced differences in their ED values. This is most probably related to marked differences in their pharmacokinetics and pharmacology [9]. The TACs of (S)-(−)-[18F]fluspidine and (R)-(+)-[18F]fluspidine in mice, obtained by either organ harvesting or PET imaging, are different in terms of maximal uptake value (in %ID) and the shape of the curve. Hence, slower elimination rates, up to 1.3-fold higher OD values and Molecules 2016, 21, 1164 8 of 14 subsequently higher ED values (p = 0.025, students t test; PET imaging approach with n = 3/group) were observed for the (R)-(+)-enantiomer. Following an initial washout, detected for both enantiomers, the elimination of activity stagnates in nearly all organs after administration of (R)-(+)-[18F]fluspidine. This corresponds to the enantioselective tracer kinetics already observed in most regions of the 18 pig brain and the significantly higher BPnd values of (R)-(+)-[ F]fluspidine [9]. Assuming such enantioselective pharmacokinetics for other tissues as well due to the expression of σ1 receptors in almost all tissues [3,37], the slower washout of (R)-(+)-[18F]fluspidine from the organ tissues was to be expected. Statistical significance in terms of ED was attained solely with the imaging-derived data because only with this approach a complete set of biokinetic data of one animal and hence individual OD and ED values are available. By contrast, no individual time-activity data can be obtained from ex vivo biodistribution studies because each animal contributes to only a single OD value. The strong correlation between pharmacokinetics and ED values is demonstrated also by a comparison of the herein investigated enantiomers of [18F]fluspidine with the enantiomers of [18F]flubatine, a ligand for α4β2 nicotinic acetylcholine receptors [24,25]. Our preclinical and clinical dosimetry studies of (+)-[18F]flubatine and (−)-[18F]flubatine, both possessing very similar biokinetics in different species up to humans, revealed no significant differences in the ED between the two enantiomers. No significant differences were observed also regarding the ED values of the enantiomers of [11C]mirtazapine, although the enantioselectivity of the OD values estimated for brain corresponds with the enantioselectivity of the brain kinetics [38]. Altogether, findings on either different or comparable ED values of enantiomers of chiral compounds used as PET imaging agents strongly reflect the influence of enantioselective processes during their interaction with the chiral compounds in biological systems such as receptor proteins or metabolizing enzymes [39]. Although the ED values of both enantiomers of [18F]fluspidine show a 1.6 fold difference, the excretion route of 18F is similar. A renal/hepatobiliary clearance can be assumed from the two preclinical models due to a high uptake of activity in the intestinal and hepatobiliary as well as renal tract, which results in comparatively high OD values in the liver, gallbladder wall, small intestine, kidneys, and urinary bladder. Furthermore, in fully awake animals used in the organ harvesting distribution study the urinary bladder is less exposed to radiation than in anesthetized mice due to urinary retention under isoflurane narcosis [40–43]. Based on the preclinical biokinetic data shown herein as well as in our recent PET study using piglets [9], different clinical applications came into consideration for the two enantiomers of [18F]fluspidine. The relatively slow kinetics and nearly constant activity accumulation of (R)-(+)-[18F]fluspidine in the observed organs and tissues which might translate into high signal-to-noise ratios in σ1 expressing tumors and metastases makes this enantiomer interesting for cancer imaging. By contrast, the (S)-(−)-enantiomer provided favorable properties for neuroimaging and data analysis with a special regard to kinetic modeling due to the high initial brain uptake and fast washout and was therefore selected for a first-in-human study. The radiation dose of (S)-(−)-[18F]fluspidine in human tissues has been estimated after injection of the radiotracer in two female and two male healthy volunteers. The hereby obtained TACs (presented in Figure S3) confirmed the assumed renal/hepatobiliary clearance. The radioligand was rapidly removed from brain, stomach, liver, and spleen within one hour post injection, while a slower clearance from red marrow, already observed in earlier σ1 receptor ligand studies [44,45], reflects the high expression of σ1 receptors in rapidly dividing tissues. Hence, it was proposed that σ1 receptor ligands may also be used as 18 proliferation markers [46]. The effective dose of the σ1 receptor ligand (S)-(−)-[ F]fluspidine is 21.0 µSv/MBq, well within the range of other 18F-labeled diagnostic radiotracers (Table 4). Thus, in combination with a NOEL of at least ~600 µg/kg, the application of (S)-(−)-[18F]fluspidine as PET imaging agent in humans is safe. Molecules 2016, 21, 1164 9 of 14

4. Materials and Methods The time-dependent radioactive data for the animal and human studies was acquired with three different techniques. (i) The mice were scanned in a preclinical small animal PET/MRI while the (ii) human study was performed on a clinical PET/CT system. In addition the (iii) ex vivo biodistribution study in mice was performed by post mortem organ dissection followed by counting for radioactivity in a gamma counter.

4.1. Synthesis of [18F]Fluspidine The synthesis of (S)-(−)-[18F]fluspidine for the human application was performed as described by Fischer et al. [8] with minor modifications. Briefly, the tracer was produced by phase transfer catalyst assisted nucleophilic substitution (100 ◦C, 15 min) using a precursor molecule with a tosyl-leaving group (2 mg in 1 mL dry CH3CN). Purification and formulation was achieved by semipreparative HPLC and solid phase extraction, respectively. Overall synthesis time was 50 min, radiochemical purity exceeded 97% and specific activity was determined to be 230 ± 160 GBq/µmol (n = 16 syntheses). For the animal studies, enantiomerically pure (S)-(−)-[18F]fluspidine and (R)-(+)-[18F]fluspidine was prepared on a TRACERlab FX F-N synthesizer (GE Healthcare) as described in previous publications [9,47]. The radiochemical purity of (R)-(+) or (S)-(−)-[18F]fluspidine was >99%, and the specific activity at the end of the synthesis was 650 and 870 GBq/µmol, respectively [48].

4.2. Preclinical Dosimetry Studies All animal experiments were approved by the responsible institutional and federal state authorities (Landesdirektion Leipzig; TVV 08/13). A toxicological study was conﬁrmed and can be found in the supplemental material.

4.2.1. Ex Vivo Biodistribution Study (Organ Harvesting Method) Female CD-1 mice (age: 12 weeks) received an intravenous injection of (R)-(+)-[18F]fluspidine (0.35 ± 0.08 MBq; weight: 29.8 ± 2.2 g; n = 28) or (S)-(−)-[18F]fluspidine (0.39 ± 0.05 MBq; weight: 29.3 ± 1.9 g; n = 22). Two to three animals per time point were sacrificed by cervical dislocation at 5, 15, 30, 45, 60, 90, 120, 180, and 240 min. p.i. The brain, heart, lung, stomach, small intestine (SI), large intestine (LI), liver, kidneys, urinary bladder (UB), spleen, thymus, pancreas, adrenals, and ovaries were dissected, weighed, and the accumulated activity quantified in a gamma-counter (WIZARD Automatic Gamma Counter, PerkinElmer, Waltham, MA, USA) to determine the percentage injected activity (dose) per gram of tissue (%ID/g). In addition, the sampling time p.i. and %ID/g values were scaled proportionately to human magnitude (please see Equations (1) and (2) in Section 4.4) prior to dose estimation with OLINDA/EXM (Vanderbilt University, Nashville, TN, USA, version 1.0).

4.2.2. In Vivo Imaging Based Study (Imaging Method) The animals were initially anesthetized with 4% of isoflurane and were positioned prone in a small-animal PET/MRI system (nanoScan® PET/MRI, MEDISO, Budapest, Hungary) equipped with respiratory monitoring, heated mouse bed (37 ◦C), and inhalation anesthesia (1.8% isoflurane in a 60% oxygen/40% air gas mixture at 250 mL/min airflow; Anaesthesia Unit U-410, agntho’s, Lidingö, Sweden; Gas blender 100 series, MCQ Instruments, Rome, Italy). Prior to the PET scan, a scout image MR sequence was done to outline the animal dimensions. Female CD-1 mice (age: 12 weeks, weight: 30.9 ± 1.3 g) were injected via the tail vein with (S)-(−)-[18F]fluspidine (13.2 ± 3.0 MBq; n = 3) or (R)-(+)-[18F]fluspidine (12.6 ± 1.4 MBq; n = 3) in a volume of 200 µL saline. The injected dose was calculated by the difference of the radioactivity in the syringe before and after the injection. A dynamic whole body animal PET scan of 105 min length (Figure 1) was started simultaneously. This scanning time was chosen to represent the protocol of the human study (after time extrapolation according to Molecules 2016, 21, 1164 10 of 14

Equation (2)) based on a priori biokinetic information from the ex vivo investigation. Following the PET scan, a 20 min T1-weighted whole body MR scan (gradient echo sequence, TR = 20 ms; TE = 3.2 ms) was performed for anatomical orientation after co-registration and attenuation correction at the reconstruction step.

4.3. First-in-Human Dosimetry Study (Imaging Method) The first-in-human use of (S)-(−)-[18F]fluspidine was authorized by the competent authorities in Germany, the Federal Institute for Drugs and Medical Devices (Bundesamt für Arzneimittel und Medizinprodukte, BfArM) and the Federal Office for Radiation Protection (Bundesamt für Strahlenschutz, BfS) as well as by the local ethics committee and was conducted in accordance with the Declaration of Helsinki. Informed consent was obtained from four healthy volunteers (2 f, 2 m; age: 23 ± 3 years; weight: 56 ± 4 kg). The volunteers were positioned supine with the arms down in a clinical PET/CT system (Biograph 16, Siemens, Erlangen, Germany) and received an intravenous injection of 255 ± 9 MBq (S)-(−)-[18F]fluspidine. Simultaneously, the PET scan was started. It was divided into a dynamic part up to 2 h p.i. (7 frames) and a static part up to 7 h p.i. (3 frames) with increasing time per bed position (from 1.5 up to 6 min) as shown in Figure 1. The volunteers left the investigation table four times to stretch out. All urine was collected, weighed, and the activity was determined in a gamma-counter (Packard Cobra II 5003 Auto Gamma Counting System, GMI, Ramsey, MN, USA) cross calibrated to the PET/CT system.

4.4. Image Reconstruction and Analysis of the Preclinical and Clinical Data The PET images were iteratively (ordered-subsets expectation maximization, OSEM) reconstructed (preclinical: 4 iterations, 6 subsets; clinical: 4 iterations, 8 subsets) and corrected for decay, randoms, scatter, and dead time, µ-maps for attenuation correction of PET-emission data were derived from the CT or MR [49] structural data, respectively. The PET data were re-binned into 10 time frames (preclinical: 4 × 5 min, 1 × 10 min, and 5 × 15 min; clinical: 4 × 12 min, 3 × 16 min, 1 × 32 min, 1 × 40 min, and 1 × 48 min), and the reconstructed PET/MRI and PET/CT images were co-registered manually with ROVER (ABX, advanced chemical compounds, Radeberg, Germany, version 2.1.17). Quantitative evaluation was performed by drawing volumes of interest (VOI) for brain, gallbladder, large intestine, small intestine, stomach, heart, kidneys, liver, lungs, pancreas, red marrow (backbone, pelvis, sternum), spleen, thyroid, testes, and urinary bladder (Figure S4). The PET derived biokinetic data is expressed as percentage of injected activity (dose) per cubic centimeter (%ID/cm3). For human dosimetry estimation from animal biodistribution and PET/MR imaging, animal organ masses and time scale was extrapolated to human magnitudes [20,30]. At ﬁrst, the organ-speciﬁc animal %ID data were extrapolated to the human scale with the equation

%ID %ID m · · mouse = morganhuman (1) organhuman gmouse mhuman with the fraction of the injected activity in the corresponding human organ = %ID , the fraction of organhuman injected activity per gram animal organ tissue = %ID and m the mass of the corresponding gmouse organhuman human organ [50]. At second, a time scale extrapolation is needed due to differences in the metabolic rate using the equation 0.25 mhuman thuman = tmouse (2) mmouse including the human time scale = thuman, the animal time scale = tmouse and the ratio of animal and human body weights = mmouse . The allometric coefﬁcient of 0.25 generally describes the differences mhuman between the two species regarding physiological processes such as biological half-life [20,50,51]. Hence, using this time extrapolation approach with an exponent of 1/4, a 105 min PET scan in mice is sufﬁcient to represent 10 h in humans (Figure S1). Molecules 2016, 21, 1164 11 of 14

The human dosimetry estimation was performed with the data extrapolated as well as the genuine human data using OLINDA/EXM software [33]. The time-activity curves were estimated by exponential ﬁtting and calculating the time integral, which equals the number of disintegrations (NODs) per organ during the observation period normalized to 1 Becquerel administered activity dose. Due to narcosis, mice did not void urine during the imaging session. Therefore, activity data of the urinary bladder was derived from the image for each time point. In contrast, for humans the activity concentration data of the urinary bladder was obtained in a more direct approach. At ﬁrst, the activity and volume of urine was determined in the last frame of the PET scan before each micturition. Afterwards, the voided urine was collected, weighed, and the activity of three aliquots (assuming 1 mL = 1 g) determined with a gamma counter, and the activity of the whole sample estimated. The difference between imaged and sampled urine activity is equal to the residue of radioactive urine in the urinary bladder. To calculate the NOD in the human urinary bladder, the time-activity curve is integrated using a trapezoidal equation

1 n−1 %IDUB = ∑ (%IDi + %IDi+1)(ti+1 − ti) (3) 2 i=1 f with the fraction of injected activity %IDi at the time ti and the cumulated activity of the urinary bladder i.e., the NOD %IDUB. Furthermore, the NODsf of the gastric system were calculated following the ICRP GI model (ICRP 30) as implemented in OLINDA 1.0. The NODs obtained either from the EXM module or the trapezoidal equation were transferred to OLINDA. The OD for the chosen hermaphroditic adult male phantom is estimated following the MIRD scheme [52]. The S values [53,54] are pre-calculated and implemented for the respective phantom in OLINDA. Subsequently, the ED contribution from each organ is calculated by multiplying the ODs with the respective tissue weighting factors as published by the International Commission on Radiological Protection (ICRP 103 [27]) for each organ. As these weighting factors require the ICRP 110 phantom [55] which is not available in OLINDA version 1.0, the ED results by using the tissue weighting factors published by ICRP 60 [26] were estimated in addition (Tables 1–3).

5. Conclusions The results achieved from this study support the potential of (S)-(−)-[18F]fluspidine as a clinically applicable PET imaging agent for the investigation of σ1 receptors. As shown before, the extrapolation of preclinical data obtained by dosimetry studies in small animals by either organ harvesting or PET imaging results in an underestimation of the human ED values most due to limitations in allometric scaling and species-specific target expression. However, the imaging approach excels in comparison to the organ harvesting method for obtaining extensive whole body kinetic information using a significantly reduced number of animals. Thus, small animal image based dosimetry is recommended as the preferable method for preclinical dose estimates prior to the application for first-in-human studies. However, preclinical dose estimates remain preliminary and need to be confirmed in human studies. While we are presently evaluating the utility of (S)-(−)-[18F]fluspidine for quantification of pathological changes in the expression of σ1 receptors in major depressive disorder, the entire potential of the enantioselective pharmacokinetics of (S)-(−)-[18F]fluspidine and (R)-(+)-[18F]fluspidine for imaging of σ1 receptors in neuropsychiatric, neuro-oncological, and oncological diseases remains to be further investigated.

Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/ 9/1164/s1. Acknowledgments: The study has been funded by the German Research Foundation (DFG). Molecules 2016, 21, 1164 12 of 14

Author Contributions: W.D.-C., B.S., M.K., P.B., J.S. and O.S. conceived and designed the experiments; M.K., W.D.-C., N.W., C.K.D. and B.S. performed the experiments; M.K. and N.W. analyzed the data; M.K., W.D.C., B.S. and P.B. wrote the paper; O.S., P.M.M. and S.H. designed and performed the clinical study; B.W. performed the organic chemistry; M.P. and S.F. performed the radiosynthesis. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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Sample Availability: Samples of the compounds are available from the authors.

© 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/). 3

Zusammenfassung der Arbeit

Dissertation zur Erlangung des akademischen Grades Dr. rer. med. Use of small animal PET/MRI for internal radiation dose assessment eingereicht von: Mathias Kranz angefertigt am/in: Universit¨atsklinikum Leipzig (UKL), Klinik und Poliklinik fur¨ Nuklearmedizin sowie Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Forschungsstelle Leipzig, Abteilung Neuroradiopharmaka betreut von: UKL: Prof. Dr. -Ing. Bernhard Sattler HZDR: Prof. Dr. Peter Brust Dezember 2016

3.1 Summary and conclusions

The thesis is based on three publications investigating newly developed radiotracers in different animal models. The radiation safety and biodistribution has to be proven prior to the application of first-in-man studies. Resultantly, based on the preclinical dosimetry presented herein, a clinical trial was approved by the competent authorities of Germany for (-)-[18F]flubatine, (+)-[18F]flubatine and (S)-(-)-[18F]fluspidine. Although the radiation safety was proven for (R)-(+)-[18F]fluspidine too, so far it is used in preclinical studies only. A summary of the studies is shown in table 3.1. 1. As a preclinical dose estimate is mandatory for the approval of a first-in-man study it was investigated in this thesis if a new small animal imaging device (PET/MRI) is suitable for human dose estimation using CD-1 mice. Therefore, different approaches and animal models were used and compared to a subsequent clinical internal dosimetry trial. Following i.v. tracer application, time-activity curves (TAC) were calculated (image- or dissection-based) for the organs and the animal data extrapolated to the human scale. The time integration of the TACs equals the total number of disintegrations (from radioactive decay) per organ 58 chapter 3. Summary of the dissertation

and investigation time normalized to 1 MBq. Finally the organ doses (OD) were estimated with the software tool OLINDA and the effective dose (ED) calculated by multiplication with the tissue weighting factors of ICRP 103. 2. Starting with the gold standard; organ harvesting (dissection) method; a dose estimate was calculated for (-)-[18F]flubatine in mice after i.v. application and compared to first-in-man results. Therefore, 27 CD-1 mice received an i.v. injection of (-)-[18F]flubatine and were sacrificed at discrete time points, the organs dissected, weighed, the organ activity determined using a gamma counter and the OD calculated with OLINDA. The small animal based dosimetry (ED= 12.5 µSv/MBq) revealed an underestimation of the ED of about 47 % compared to humans (ED= 23.4 µSv/MBq) due to limitations of time and mass scaling as well as differences in tracer distribution and metabolism between the two species. Therefore, a larger animal model (piglet) was chosen to investigate size and metabolism related deviations in dosimetry with the imaging method. 3. For the dosimetry of (-)-[18F]flubatine based on piglet image-derived biokinetic data scaled to human entity, a clinical PET/CT system was used. The ED (14.7 µSv/MBq) was calculated to be 37 % lower than in humans. In conclusion this result shows that there is increased accuracy of the ED estimate when using larger species for dose assessment in humans. For both animal models the ED is well within the range of other 18F-labelled substances (Table 3.1). 4. For (+)-[18F]flubatine an image based dosimetry was calculated with piglets as was done for (-)-[18F]flubatine too in order to receive permission for the first-in- man trial. The calculated ED was 14.3 µSv/MBq and not significantly different (t test, p=0.77) from the results of the (-)-enantiomer. Therefore, it was assumed that the dosimetry of both enantiomers of [18F]flubatine is essentially equal and allows for a methodological comparison of small animal PET/MRI and organ harvesting based dosimetry in mice. 5. The dosimetry based on small animal PET/MRI resulted in an ED of 12.1 µSv/MBq for (+)-[18F]flubatine which is almost identical to the result found for the (-)-enantiomer with the organ harvesting method (ED= 12.5 µSv/MBq). 6. Based on mouse and piglet data, a first-in-man dosimetry study was performed after i.v. injection of (+)-[18F]flubatine. The ED was calculated to be 23.0 µSv/MBq revealing an underestimation of the ED based on mouse PET/MRI and piglet PET/CT data of 47 % and 37 %, respectively. The result is identical to that in humans for the (-)-enantiomer (t test, p=0.72). In addition, as shown 3.1. Summary and conclusions 59

for the piglet studies too, there is statistically no difference of the ED in humans between the two enantiomers. Finally, it could be shown that the calculated ED based on mouse biokinetic data from a small animal PET/MRI is identical to what was found for the assessment based on organ harvesting. 7. While this result was validated for two tracers targeting the same system of receptors a third radioligand, [18F]fluspidine (σ1-receptor ligand), was chosen to investigate the reproducibility of these findings. There are two enantiomers available: (S)-(-)- and (R)-(+)-[18F]fluspidine with distinctive in vivo kinetics [109]. In accordance with the previous studies an organ harvesting and small animal PET/MRI based dosimetry approach with CD-1 mice was applied for both enantiomers of [18F]fluspidine in order to assess the ED in humans. 8. In contrast to [18F]flubatine the preclinical PET/MRI based investigation of [18F]fluspidine showed a significant difference (t test, p=0.025) of the ED (12.9 µSv/MBq, 14.0 µSv/MBq (S)-(-)- or (R)-(+)-[18F]fluspidine, resp.) between the enantiomers. As shown by Brust et al. [109], the in vivo kinetics of the tracers are distinctly different. Hence, the higher ED of (R)-(+)-[18F]fluspidine is

most probably due to it’s higher specific binding value (Ki: 0.57 nM, 2.3 nM, R- or S-enantiomer resp.) and assuming that this fact is applicable for tissues other than the brain too as σ1 receptors are found all over the body [110]. In addition, the difference of the ED between the enantiomers was confirmed with the organ harvesting method (16.7 µSv/MBq, 18.4 µSv/MBq for (S)-(-)- or (R)-(+)-[18F]fluspidine, resp.). 9. Similar TAC patterns as shown by Brust et al. [109] for the brain could be found for different organs too after injection of [18F]fluspidine. While the initial uptake is similar between the enantiomers, brain, liver, spleen, kidneys and lungs show a wash out for (S)-(-)- but not for (R)-(+)-[18F]fluspidine. This affects the area under the TACs which equals the number of disintegrations per organ. As the R- enantiomer expresses slower kinetics than (S)-(-)-[18F]fluspidine, the area under the curve increases, resulting in higher ODs and ED. 10. Based on the promising preclinical results of (S)-(-)-[18F]fluspidine a first-in-man study was approved. The organs with the highest OD (liver, gallbladder, small intestines, stomach and kidneys) as found with the small animal PET/MRI study were confirmed for humans. The ED in humans was calculated to be 21.0 µSv/MBq so that the small animal PET/MRI study underestimates the ED by about 39 % and the organ harvesting study by about 20 %. The calculated ED 60 chapter 3. Summary of the dissertation

from the human study is well within the range of other 18F-labelled radioligands and conﬁrms the radiation safety. 11. In conclusion, the use of small animal PET/MRI to assess the ED in human leads to an underestimation between 39 % and 47 % as was found for other tracers too (Table 3.1). However, preclinical dose estimates remain preliminary and debatable assessments and need to be conﬁrmed in human studies. Small animal based dosimetry can identify critical organs, the route of excretion and estimates of the ED but should always be considered with regard to their methodological limitations.[73]

Consideration of the hypotheses

1. The results from the harvesting method showed comparable dose estimates to the image based method with mice after the injection of [18F]flubatine. It can be assumed that there are no biokinetic differences between the two enantiomers affecting the dosimetry as confirmed by the piglet studies. Hence, the ED was calculated to be 12.5 µSv/MBq and 12.1 µSv/MBq for (-)- or (+)-[18F]flubatine respectively. The reproducibility of the dosimetry data estimated with the dissection method suggests the feasibility of using small animal PET/MRI with mice for estimations of the radiation exposure in humans. However, an underestimation of the ED in humans of up to 47 % has to be considered in both of the small animal based approaches. 2. Using a larger species than mice for dosimetry results in better estimates of the ED in humans (piglets: 14.7 µSv/MBq, 14.3 µSv/MBq, (-) or (+)-[18F]flubatine, resp.) compared to first-in-man trials (humans: 23.4 µSv/MBq, 23.0 µSv/MBq (-) or (+)-[18F]flubatine, resp.). However, an underestimation of up to 38 % remains. This is due to the limitations of the animal-to-human scaling and differences of tracer metabolism among the species. 3. As shown by the investigations with [18F]fluspidine in mice enantiomeric differences affect the dosimetry result significantly (t test: p<0.05) and expectedly, the distinctive in vivo kinetics (Brust et al. [109]) confirm the dosimetry findings of both enantiomers. In contrast, there are no enantiomeric differences in dosimetry found for [18F]flubatine as shown in the preclinical and clinical studies (t test, piglets: p=0.8, humans: p=0.7). 4. Preclinical PET/MRI based dosimetry with mice is feasible to assess the radiation risk in humans prior to first-in-man studies. However, one need to be aware of the limitations that lead to underestimations of the radiation exposure 3.1. Summary and conclusions 61

in humans. This is true for the dissection based method in mice and the imaging approach in piglets too, concluding that preclinical dosimetry underestimates the ED in humans by up to 50 %.

Outlook The results presented in this thesis proof the radiation safety of the tracers under investigation. None of them causes unexpectedly high radiation exposures in the first-in-man studies. This is well in line with what has been shown for other 18F- labelled PET tracers. The meta-analysis by Zanotti-Fregonara et al. [72, 74] also shows, the radiation risk caused by tracers labelled with positron emitters (i.e. [18F] or [11C]) which are showing a comparative biodistribution, is similarly low and in the same order of magnitude. Usual amounts of radioactivity as applied for one PET- investigation maintain a level close to the variance of the natural annual exposure to ionizing radiation. The results presented for the tracers in this thesis show the same behaviour: underestimation of human exposure by preclinical investigations and comparative dosimetry results in human studies. Hence, if the studied tracer shows similar kinetics in preclinical studies (i.e the toxicology) as known and approved tracers too, the suggestion is strongly supported to include first-in-man trials with reduced injected activity (compared to diagnostic reference levels of approved tracers) in the evaluation of a new tracer without performing preclinical dosimetry studies. However, if in such a trial the first-in-man scan shows unexpectedly different tracer kinetics and biodistribution (i.e. high uptake in dose limiting organs like gonads etc.), the first-in-man trial should be terminated. Subsequently, a whole body internal dosimetry study in an animal model as presented in this thesis should be performed to determine the maximal injectable activity to humans.

Table 3.1: Comparison of the ED for diﬀerent PET tracers based on preclinical and clinical image based dosimetry studies. ∗piglets, †harvesting method

Tracer Target organ Clinical Preclinical (mice) Reference (µSv/MBq) (µSv/MBq) (-)-[18F]flubatine brain 23.4 12.5 (14.7∗) [30] (+)-[18F]flubatine brain 23.0 12.1 ( 14.3∗) [31] (S)-(-)-[18F]fluspidine brain, tumor 21.0 12. 9 (16.7† ) [32] (R)-(+)-[18F]fluspidine brain, tumor n.a. 14.0 (18.4† ) [32] [18F]FET brain, tumor 16.5 9.0 [111, 112] [18F]FEDAA1106 brain 36.0 21.0 (m), 26.0 (f) [113] [18F]flutemetamol brain 33.8 6.65 (rats) [51, 114] [18F]FBPA tumor 23.9 18.7 [115] [18F]FACT brain 18.6 14.8 [116] [18F]-5-FU multiple tumor 12.1 5.8 [117]

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Appendix

4.1 Erkl¨arungen uber¨ den wissenschaftlichen Beitrag zu den Publikationen

4.2. Erklarung¨ uber¨ die eigenstandige¨ Abfassung der Arbeit 75

4.2 Erkl¨arung uber¨ die eigenst¨andige Abfassung der Arbeit

Hiermit erkläre ich, dass ich die vorliegende Arbeit selbstständig und ohne unzuläs- sige Hilfe oder Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Ich versichere, dass Dritte von mir weder unmittelbar noch mittelbar eine Vergu-¨ tung oder geldwerte Leistungen fur¨ Arbeiten erhalten haben, die im Zusammenhang mit dem Inhalt der vorgelegten Dissertation stehen, und dass die vorgelegte Arbeit weder im Inland noch im Ausland in gleicher oder ähnlicher Form einer anderen Prufungsbeh¨ örde zum Zweck einer Promotion oder eines anderen Prufungsverfah-¨ rens vorgelegt wurde. Alles aus anderen Quellen und von anderen Personen uber-¨ nommene Material, das in der Arbeit verwendet wurde oder auf das direkt Bezug genommen wird, wurde als solches kenntlich gemacht. Insbesondere wurden alle Personen genannt, die direkt an der Entstehung der vorliegenden Arbeit beteiligt waren. Die aktuellen gesetzlichen Vorgaben in Bezug auf die Zulassung der klini- schen Studien, die Bestimmungen des Tierschutzgesetzes, die Bestimmungen des Gentechnikgesetzes und die allgemeinen Datenschutzbestimmungen wurden eingehalten. Ich versichere, dass ich die Regelungen der Satzung der Universität Leipzig zur Sicherung guter wissenschaftlicher Praxis kenne und eingehalten habe.

New Haven, 7.12.2016 Unterschrift