CHEMISTRY, MANUFACTURING, AND CONTROLS

Exploratory IND Application: Evaluation of [11C]PBR28

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PET Imaging of Peripheral Benzodiazepine Receptors Using [11C]PBR28 I. Introductory Statement of Purpose and General Plan................................................. 2 II. Study Protocol: PET Imaging Using [11C]PBR28. ..................................................... 3 III. Chemistry, Manufacture and Control. .................................................................... 3 IV. Pharmacology and Toxicology. .............................................................................. 3

A. General Description. ............................................................................................... 3 B. In Vitro Receptor Binding. ..................................................................................... 5 C. Acute 7-Day Intravenous Toxicity Study of DAA1106 in Mice............................ 5 D. In Vitro Experiments............................................................................................... 6 E. Animal Studies........................................................................................................ 6 F. Human Studies. ....................................................................................................... 6

V. Animal Experimentation............................................................................................. 7 A. Metabolism and Clearance of PBR28..................................................................... 7 B. PET Imaging of Nonhuman Primates ..................................................................... 8 C. Pharmacological Effects in Nonhuman Primates ................................................. 11 D. Estimated Receptor Occupancy ............................................................................ 11 E. Radiation Dosimetry Estimation from Nonhuman Primates. ............................... 11

VI. Human Experience................................................................................................ 12 VII. Environmental Assessment................................................................................... 12 VIII. Case Report Form. ................................................................................................ 12 IX. References............................................................................................................. 12 X. APPENDICES. ......................................................................................................... 17

A. Investigator C.V.................................................................................................... 17 B. Study Protocol....................................................................................................... 18 C. IRB Protocol Approval. ........................................................................................ 19 D. Case Report Form. ................................................................................................ 20 E. Dosimetry Report from Nonhuman Primates ....................................................... 21 F. List of Cited Pharmacology / Toxicology Studies................................................ 22

CHEMISTRY, MANUFACTURING, AND CONTROLS Document No. Section

1 CMC

2 Master Batch Record

3 QC Form

4 Radiopharmacy Form

5 SOP: Standard Operating Procedures

6 Annual Testing for Radionuclidic Identity

7 Certificate of Analysis for starting Materials

8 Precursor Acceptance Testing and Form

9 Data Form for Generating Calibration Curve

10 Validation Runs


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I. Introductory Statement of Purpose and General Plan. Since their discovery in the late 1950’s, benzodiazepines have been widely used

as anxiolytics, anticonvulsants and sedative medications (Sternbach 1983). In the last decades, two pharmacologically distinct subclasses of benzodiazepine binding site have been demonstrated. One class, the central benzodiazepine receptor (CBR) is mainly localized on the extracellular domain of the γ-aminobutyric acid (GABA)A receptor and regulates the chloride channel of GABAA receptors in the central nervous system (CNS) (Tallman et al 1978).

The second class of benzodiazepine receptor was initially identified in peripheral tissue and was called the peripheral benzodiazepine receptor (PBR). PBR is located on the mitochondrial outer membrane in several organs including the kidney, nasal epithelium, lung, heart and endocrine organs such as the adrenal gland, testis and pituitary gland (Anholt et al 1985; Anholt et al 1986; Braestrup et al 1977; Gavish et al 1999). Contrary to the original nomenclature of “peripheral” benzodiazepine receptors, later studies demonstrated the presence of PBR in the CNS (Schoemaker et al 1981; Weissman et al 1984; Zisterer and Williams 1997). In mitochondria, PBR belongs to the mitochondrial permeability transition pore (Anholt et al 1985; Bernassau et al 1993; McEnery et al 1992), where it is intimately associated in trimeric complex with adenosine nucleotide translocase and the voltage-dependent anion channel. This complex is a megachannel located on the inner and outer mitochondrial membrane contact sites. Although the physiological role of PBR in still unclear, PBR has been implicated in various functions such as neurosteroid synthesis (Culty et al 1999; Papadopoulos et al 1997), immunomodulation (Zavala 1997), cell proliferation (Okuyama et al 1999; Schlichter et al 2000; Verma et al 1998) and apoptosis (Bono et al 1999). In the normal brain, PBRs are mainly found in glial cells and are especially highly localized to the ependyma lining of the ventricles, choroids plexus, olfactory bulb (Anholt et al 1984; Benavides et al 1983; Cymerman et al 1986; Schoemaker et al 1983). The expression of PBR in vivo is reported to be increased in microglia activated by brain injury (Banati 2002; Banati 2003), and this increase has been used as an indicator of neuronal injury and neurodegenerative disease (Benavides et al 1987; Cagnin et al 2002). Several compounds are able to cross the blood brain barrier and specifically bind to PBR in vivo. The most widely used selective ligand is 1-(2-Chlorophenyl-N-methylpropyl)-3-isoquinoline carboxamide (PK11195), which has been labeled with [11C] for positron emission tomography (PET) studies. R-(-)[11C]PK11195 has been used in several neurological disordesr, including Alzheimer’s disease (Cagnin et al 2001a; Groom et al 1995), multiple sclerosis (Banati et al 2000; Debruyne et al 2003), stroke (Pappata et al 2000), Rasmussen’s encephalitis (Banati et al 1999), herpes encephalitis (Cagnin et al 2001b), amyotrophic lateral sclerosis (Turner et al 2004) and multiple system atrophy (Gerhard et al 2003). However, the brain uptake has a low ratio of specific to nonspecific binding (less than 20%), which is not high enough for stable quantitative analysis (Pappata et al 1991; Pappata et al 2000; Sauvageau et al 2002). In the last several years, a new class of high affinity PBR ligands has been radio-synthesized based on aryloxyanilides (Okuyama et al 1999), and some promising PET radioligands have already been developed from this class (Maeda et al 2004; Zhang et al 2003). [11C]DAA1106 (Fig. 1) is one of the published candidates and has recently been tried in human subjects, with no observable phramcological effects (personal communication, T. Suhara, MD, PhD; National Institute of Radiological Sciences, Chiba, Japan). We have also sought to develop PET ligands based on aryloxyanilides with high brain uptake and high ratio of specific to nonspecific binding to nondisplaceable radioactivity. The purpose of this IND is to study one promising candidate. [11C]PBR28 has high affinity (IC50 = 0.6 nM measured with [3H]PK11195), receptor selectivity, moderately fast washout from monkey brain, and a good ratio of specific to nonspecific binding (see Section V).


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The objective of this study is to characterize the pharmacokinetics of the brain uptake of [11C]PBR28 in healthy subjects by performing compartmental analysis with an arterial input function. We wish to know if time-independent values of brain distribution volume (i.e., a measure of receptor density) can be obtained in human subjects, as we have found for rhesus monkey (Section V.B).

• If [11C]PBR28 is amenable to quantitation in human subjects, then it may be a useful marker of neuroinflammation in disorders such as Alzheimer’s disease, stroke, and multiple sclerosis. We understand that the current exploratory IND application is for a limited number of healthy subjects. We will not proceed to any other studies without review and approval of such a plan by the FDA.

• On the other hand, if the brain washout of [11C]PBR28 is too slow to achieve stable values of distribution volume within 120 min scanning, we will discontinue studies with this radioligand. We would then seek to develop a longer-lived 18F-labeled analog of PBR28.

II. Study Protocol: PET Imaging Using [11C]PBR28.

The complete protocol is located in Appendix B. A brief summary is included below. The peripheral benzodiazepine receptor (PBR) is distinct from central benzodiazepine receptors associated with GABAA receptors. Although PBR was initially identified in peripheral organs such as kidneys, endocrine glands and lungs, later studies identified PBR in the central nervous system. In normal conditions, PBR is expressed in low levels in some neurons and glial cells. PBR can be a clinically useful marker to detect neuroinflammation because activated microglial cells in inflammatory areas express much greater levels of PBR than in microglial cells in resting conditions. PBR has been imaged with positron emission tomography (PET) using [11C]1-(2-chlorophenyl-N-methylpropyl)-3-isoquinoline carboxamide (PK11195). However, this classical ligand provides only low levels of specific signals and is not sensitive to detect changes occurred in vivo. Recently we developed a new ligand, N-acetyl-N-(2-methoxybenzyl)-2-phenoxy-5-pyridinamine [11C]PBR28, which showed much greater specific signals than [11C]PK11195 in non-human primates. In the present protocol, we plan to perform a kinetic brain imaging study in healthy human subjects to measure PBR in brain regions with [11C]PBR28. Successful development of a PET ligand to image PBR will have a strong impact on clinical management of brain disorders with inflammation such as multiple sclerosis and ischemia and neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease where inflammation is involved in the disease progression. III. Chemistry, Manufacture and Control.

The CMC Section is a separate portion of this application. IV. Pharmacology and Toxicology.

A. General Description. PBR28 and DAA1106 are selective aryloxyanilide ligands for PBR (Figure 1).

Although called the “peripheral” benzodiazepine receptor for historical reasons, PBR is located both in the central nervous system (CNS) and many peripheral organs, including endocrine tissues, kidney, heart, liver and blood cells. In the CNS, PBR exists mostly but not exclusively in glial cells. In most of these organs, PBR is located in the outer membrane of mitochondria. In some organs, PBR shows other subcellular localizations. For example, PBR has been localized in mitochondrial inner membrane of guinea pig lung (Mukherjee and Das 1989). In rat liver, PBR has been localized in two sites: a mitochondrial and an unidentified non-mitochondrial location (O'Beirne et al 1990). In


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heart, PBR is located in the plasma membrane, where it is reported to be coupled to calcium channels (Mestre et al 1985).

PBR has been proposed to be involved in cellular proliferation, calcium channel activity, immune responses, transport of porphyrin and anion and regulation of steroid biosynthesis (Zisterer and Williams 1997). Because of the low mass dose administered in this protocol (10 μg per PET scan), an acute toxicity experiment in mice by a single intravenous administration was obtained from Tetsuya Suhara, MD, PhD (National Radiological Sciences, Japan) and literature search was performed on other possible pharmacological/toxicological effects studied in cultured cells, mice, rats, rabbits, guinea pigs and humans.

We have no formal toxicology data on PBR28. Instead, the following Section assesses the pharmacology and toxicology relative to two other PBR ligands: a) DAA1106, which is a close chemical analog of PBR28 (Fig. 1). b) PK11195, which is the prototypical PBR ligand and which has been used as a 11C-labled probe in several human studies (see Section I) – and also studied in pharmacological doses in human subjects (see Section IV.F).

All three ligands (PK11195, DAA1106, and PBR28) are selective for PBR. Thus, the expected pharmacology of PBR28 is based upon the known effects of PBR agents. In addition, we have limited animal toxicology data on the closely related analog DAA1106.

Comparison among these three ligands is made relative to their KIi values. PBR28 was measured in our lab using R-(-)-[3H]PK11195 as the radioligand and membranes prepared from rat brain, following a published procedure (Chaki et al 1999). The Ki values of the three compounds are fairly similar, with somewhat greater affinity for the two aryloxyanalides than PK11195:

PBR28: 0.2 nM (rat brain) (Briard et al 2005) DAA1106: 0.04 (rat brain) & 0.2 nM (monkey brain) (Chaki et al 1999) PK11195: 0.7 nM (rat brain) & 0.8 nM (monkey brain) (Chaki et al 1999)

[Unless otherwise noted, PK11195 refers to the racemic mixture.]

As described below (Section IV.F), PK11195 has been administered with no adverse effects to human subjects at doses of 10 mg IV and 100, 200, and 400 mg PO (with ~33% absolute bioavailability). By comparison, the currently proposed dose of up to 10 µg PBR28 is expected to be safe and lack pharmacological effects.

PBR28 DAA1106 Ki = 0.2 nM Ki = 0.04 – 0.2 nM


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R-[11C]-PK11195 Ki = 0.8 nM Figure 1. Structures of three PBR-selective ligands.

B. In Vitro Receptor Binding. Radioligand-binding assays were performed by using the resources of the NIMH-

PDSP (Pharmacology Drug Screening Program). Detailed on-line protocols are available at the NIMH-PDSP web site (http://pdsp.cwru.edu). PBR28 was tested at 10 µM and found to have < 50% displacement of the target radioligand for the following receptors: 5HT1a, 5HT1b, 5HT1d, 5HT1e, 5HT2a, 5HT2b, 5HT2c, 5HT3, 5HT5a, 5JT6, 5HT7, alpa1a, alpha1b, alpha2a, alpha2b, alpha2c, beta1, beta2, brya3, D1, D2, D3, D4, H1, H2, H3, H4, M2, M5DAT, NET and SERT.

As expected, PBR28 showed Ki > 10 µM for several “central” benzodiazepine receptor subtypes (i.e., the GABA-A receptor): alpha1beta1gamma2, alpha2beta2gamma2, alpga5beta2gamma2, and alpha6beta2gamma2.

Only one receptor showed >50% displacement at 10 µM: the kappa opiate receptor. A full displacement study showed Ki = 2.2 µM.

C. Acute 7-Day Intravenous Toxicity Study of DAA1106 in Mice Dr. Tetsuya Suhara (Director of Neuroimaging at the National Institute of

Radiological Sciences in Chiba, Japan) provided the following information on DAA1106. Translation of the summary originally written in Japanese: Solutions of 20 mL/kg

(10 mg/kg DAA) and the maximum injectable volume of 40 mL/kg (20 mg/kg DAA) were administered to mice through a tail vein at a rate of 1 mL/min. Three mice were used for each dose and DAA solution was administered once. To control mice, solutions of 20 and 40 mL/kg without DAA were administered. Mice were observed for 2 h after the administration, and once a day in the morning for seven days. Body weight was measured on the day of the administration and once a day in the morning for seven days after the administration. Even the maximal dose of 20 mg/kg (corresponds to DAA1106 and PBR28 1.4 g/70 kg) DAA1106 caused only temporal decrease of locomotor activity and respiration,


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which were caused by the vehicle but not by DAA1106. No animal died. DAA1106 did not change body weight, and autopsy did not show significant changes. Therefore, it was concluded that the toxicity of DAA1106 was weak. These results indicate that the mass dose of 740 MBq [11C]DAA1106 with specific activity of more than 3.7 GBq/μmol to a 60 kg human subject is at least 1.5 × 104 smaller than the mass dose that did not show pharmacological effects. Therefore, no pharmacological effects are expected in human studies.

D. In Vitro Experiments 1. Cellular proliferation.

A selective antagonist at PBR, PK11195 inhibited cell proliferation of C6 glioma, neuro-2A neuroblastoma, SP2 hybridoma and NCTC epithelial cells with ED50 of > 27 μM (Gorman et al 1989).

2. Mitochondrial oxidative phosphorylation in rat liver, kidney and adrenals.

PK11195 did not change mitochondrial respiration rate up to a concentration of 10 μM (Zisterer et al 1992).

3. Immune response: Increase in resistance of the malaria parasite. PK11195 dose dependently inhibited proliferation of Plasmodium falciparum

(Dzierszinski et al 2002). Almost complete inhibition was achieved at a concentration of 35 μg/mL and 20% inhibition was achieved at 10 μg/mL in the cultured medium.

4. Immune Response: Inhibitory action of PK11195 on phagocyte function. At 5 × 10-6 M, PK11195 did not induce changes in respiratory burst, glutamate

production or IL-1b production in mononuclear phagocytes (Klegeris et al 2000).

E. Animal Studies 1. Nerve-evoked contractions of ex vivo rabbit bladder.

At a concentration of 10 μM, PK11195 showed less than 10% changes in the contractions (Smyth et al 1994).

2. Anxiolytic effects: Sniffing and ambulation in open field (rat).

PK11195 (20 mg/kg/day) did not show effects on sniffing and ambulation in open field (Rago et al 1992).

3. Anxiolytic Effects: Exploration time spent in the light area (mouse).

A single oral dose of DAA1106 was given 30 min prior to the test. There was no effect with 0.03 mg/kg and a significant increase in the time spent in the light area with 0.1 mg/kg (Okuyama et al 1999).

4. Intracranial self-stimulation in rat.

There was no effect with 60 mg/kg PK11195 (Pellow et al 1986). The route of the administration was not described in the paper.

F. Human Studies.

1. Pharmacokinetics and cardiovascular effects of PK11195 at reast and after exercise.

PK11195 was administered intravenously (10 mg) and orally in three single dosages (100, 200 and 400 mg) to each of 10 healthy volunteers (Ferry et al 1989).


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• The elimination T1/2 was 3.7 ± 3.0 h, with high interindividual variability. • The pharmacokinetic parameters Cmax and AUC were linear over the range of 100

to 400 mg PO. • Oral doses showed ~33% absolute bioavailability. • None of these doses of PK11195 (IV or PO) caused a significant change in heart

rate or blood pressure at rest or during exercise 1, 3, 6, and 24 hours after drug administration.

The peripheral vascular effects of PK11195 (200 mg PO) were examined in six

healthy human subjects (Thuillez et al 1989). PK11195 significantly increased carotid and brachial artery flow (20 and 39%, respectively) and diameter (4 and 7%, respectively). PK11195 decreased forearm vascular resistance, without changing heart rate or blood pressure. There was no change in plasma rennin activity or plasma aldosterone concentrations. The authors reported: “No serious untoward effects were encountered during this study.” However, there was one case each of facial flushing, headache, and asthenia.

NOTE: Appendix F contains a spread sheet that lists of the studies cited above in Section IV. V. Animal Experimentation.

A. Metabolism and Clearance of PBR28 We have investigated the in vitro and in vivo the stability of [11C]PBR028. In

vitro, [11C]PBR028 was stable in both rat and rhesus monkey whole blood confirmed by radio-HPLC analysis. In vivo, we studied the rat brain radiochemical composition using radio-HPLC at two different mass dose concentrations of the [11C]PBR028 . After administering 1.4 mCi (mass dose of 0.86 µg/kg) and 2.3 mCi (mass dose of 4.83 µg/kg) to two healthy rats, they were killed 30 min later. Whole blood was collected from both animals and the brains were then resected for analysis. Plasma samples were isolated from whole blood, excised brains were homogenized and all were deproteinated and analyzed by radio-HPLC for their composition. The plasmas radiochromatographic profile was that of two radioactive peaks; one associated with a polar radiometabolite and another associated with the parent [11C]PBR028 ligand.

The rat brain, at a high specific activity of [11C]PBR028 (mass dose of 0.86 µg/kg), showed radiochemical composition that was 94.4% of [11C]PBR028. Increasing the mass dose associated with [11C]PBR028; up to 4.8 µg/kg, rat brain radioactivity remained at 89.8% composition of the parent compound. In monkeys, after bolus injection of [11C]PBR028, the radioligand was quickly metabolized and represented 86.2±6.8%, 20.2±4.0% and 8.8%±4.7% of total plasma activity at 5, 30 and 60 min, respectively. Plasma activity of [11C]PBR28 peaked approximately 1 min and decreased rapidly to 65.2±5.3%, 18.1%±4.4%, 6.9±1.5% and 3.1±0.9% at 2, 10, 30 and 60 min, respectively (Fig.2).


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Fig. 2 Time-dependent change of radioactivity and non-metabolite ratio of [11C]PBR28 in plasma

B. PET Imaging of Nonhuman Primates

Two rhesus monkeys (Macacca mulatta, body weight) were used (Table 1). Anestehsia was initiated with i.m. injection of ketamine (10 mg/kg) and then maintained under anesthesia with 1.6% isoflurane and 98.4% O2. The electrocardiograph (ECG), body temperature, heart and respiration rates were measured throughout the experiment. Body temperature was maintained at 37.0-37.5°C. All of PET scans were performed on a GE Advance scanner (General Electric Medical Systems, Waukesha, WI), with reconstructed resolution of 6 mm full-width half-maximum in all directions in 3D mode by applying scatter correction. The high resolution research tomograph (HRRT, Siemens/CPS, Knoxville, TN, USA) scanner (Schmand et al 1998; Wienhard 2002). All scans of HRRT were acquired in 64-bit list mode format. Data were reconstructed into a 256x256x207 image matrix with pre-determined frame schedule using a list mode OSEM algorithm (Carson et al 2003), resulting in an image resolution of 2.5 mm FWHM. No scatter correction was applied. After a transmission scan, the radiopharmaceutical (dose: 4.17±1.36 mCi, specific activity: 1530±580 mCi/μmol) was intravenously injected. Coronal slices covering the whole brain were obtained. PET scans were acquired for 120-180 min (33-45 frames with longer scan duration at later time points). To measure plasma concentration of [11C]PBR28 and the metabolites, a second line, intra-arterial, in the contralateral limb was used to obtain 14 blood samples. Eight samples (0.5mL each) were drawn at 15 s intervals until 2 min, followed with 1-mL samples at 3, 5, 10, 30, 60, 90, 120min in heparin-treated syringes. Each blood sample was separated into plasma and blood cell fraction by centrifugation. The tomographic images were analyzed with PMOD 2.65 (pixelwise modeling computer software; PMOD Group, Zurich, Switzerland) (Burger et al 1998). All frames of the original reconstructed PET data were summed, and this summed image was coregistered to a T1- weighted magnetic resonance (MR) image acquired separately on a GE 1.5 T Signa MR scanner (SPGR, TR/TE/flip angle = 13.1 ms/5.8 ms/45°, 0.4 x 0.4 x 1.5 mm and coronal acquisition on a 256 x 256 x 60 matrix) (GE Medical Systems,Waukesha, WI) using SPM2 (Wellcome Department of Cognitive Neurology, London, U.K.), and regions of interest were defined on the frontal, temporal , parietal and occipital cortices, cerebellum, putamen, thalamus, 3rd ventricle and 4th ventricle of the MRI. To normalize brain uptake relative to the injection dose and the body weight, standardized uptake values (SUVs) were determined as (% Injected activity/ g brain) × g body weight.

0 20 40 60 80 1000

20

40

60

80

100

120 TotalParent

Time (min)

%SU

V

0 20 40 60 80 1000

20

40

60

80

100Non-Metabolite ratio (%)

Time (min)

Non

-met

abol

ite r

atio

(%)


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Estimation of distribution volume with arterial input function:

One-tissue (1C) and unconstrained two-tissue compartment (2C) models were applied. Rate constants (K1, k2, k2', k3, and k4) were defined as described previously (Laruelle et al 1994). In the 1C, VT=K1 / k2'f1 where VT is the distribution volume for the single tissue compartment. In the 2C, VT is described separately by the distribution volumes in nondisplaceable (VN) and specific binding compartments (VS).

VN = K1/k2'f1 VS = K1k3/k2k4f1 = Bmax'/Kd VT = K1(1+ k3/k4) k2f1 where Bmax' is unoccupied binding site density. Under tracer conditions, Bmax' = Bmax. In these two models, although the definition of K1 is the same, that of k2 and k2'is different. That is, k2 is transfer rate to the vascular compartment from the nondisplaceable compartment in the 2C, and k2'refers to the transfer from the total tissue compartment (Laruelle et al 1994). Vi' is defined as Vi' = f1Vi These definitions indicate that Vi values are expressed relative to the free fraction of radioligand in plasma and that VT' values are expressed relative to the total (free plus protein-bound) concentration of radioligand in plasma. Non-linear least-squares analysis was performed on the VOI-generated time-activity data using PMOD 2.65. Parameters were estimated using the Marquardt algorithm (Bevington and Robinson 2003) with constraints restricting parameters to positive values. Statistical analysis

Goodness-of-fit by nonlinear least squares analysis was evaluated using the model selection criterion (MSC), which is a modification of the Akaike information criterion (AIC) (Akaike 1974). MSC gives greater values for better fitting. Goodness-of-fit by 1C and 2C was compared with F statistics (Hawkins et al 1986). The standard errors (SEs) of non-linear least squares estimation for rate constants were given by the diagonal of the covariance matrix (Carson 1986) and expressed as a percentage of the rate constants (coefficient of variation, %COV). In addition, %COV of VT' was calculated from the covariance matrix using the generalized form of error propagation equation (Bevington and Robinson 2003), where correlations among parameters were taken into account. A value of P<0.05 was considered significant.


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Table1. List of nonhuman primate [11C]PBR28 PET imaging

Study # Blocking Agents

Blocking Agent Dose

(mg/kg) Monkey Wt. (kg)

Specific Activity

(mCi/µmol)

Injected Dose (mCi)

Mass dose (µg)

PBR_M10 - P41 16.1 1122 4.23 1.31 PBR_M11 Cold PBR28 3 P41 16.1 1260 5.47 1.51 PBR_M18 - - P41 15.3 1285 1.95 0.53 PBR_M21 - - R16 9.6 2549 5.06 0.69 PBR_M22 - - P41 14.2 1435 4.15 1.01

Brain Uptake of [11C]PBR28

[11C]PBR28 showed high brain uptake of approximately 300-500 SUV% (SUV% = % injected activity/cm3 brain × body weight (g)). The distribution of brain uptake for the [11C]PBR28 were consistent with the distribution of PBRs. That is, regions with high receptor densities (e.g., 4th Ventricle) had highest levels of activities at late time points of the scan (Fig.1). The brain uptake of [11C]PBR28 was blocked with a receptor saturating dose of non-radiolabeled PBR28 (3 mg/kg i.v., injection at the same time of [11C] PBR28 administration; Fig. 3).

Fig. 3 Time activity curves of [11C]PBR28 baseline and blocking studies. Nonlinear least squares compartmental analyses

Convergence was achieved in all regions (n=6) of all studies (n=3) with both one-tissue (1C) and two-tissue (2C) compartment models. 2C did not provide significantly better fitting than 1C. The difference between 1C and 2C fitting was not significant by F-test in all regions of all animals. Average MSC (Model Selection Criteria) values were 3.88±0.23 and 3.74±0.24 for the 1C and 2C fits, respectively. VT' estimated by 1C was consistent among 3 experiments performed using the same animal with COV values less than 10%.

After 100 min, VT' of [11C]PBR28 by 1C and 2C became independent of scan length in cerebellum and 4th Ventricle (Fig. 4). The changes of VT' between 100 and 120 min by 1C and 2C were less than 1.5%. Therefore, the binding of [11C]PBR28 was accurately measured with 1C using arterial input function.

Baseline Study

0 25 50 75 100 125 150 1750

100

200

300

400

500

Time (min)

%SU

V

Blocking Study

0 25 50 75 100 1250

100

200

300

400

500

600

700FrontalTemporalParietalOccipitalCerebellarPutamenThalamus4th_Ventricular

Time (min)

%SU

V


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In summary, these data show that stable levels of distribution volume are achieved in monkey brain with 100 min scanning length. The major goal of the proposed study is to determine whether human brain and plasma show similar pharmacokinetics and thereby permit accurate quantitation of PBR densities in brain.

Fig. 4 The change of VT’ by 1 tissue compartment and 2 tissue compartment model of [11C]PBR28

C. Pharmacological Effects in Nonhuman Primates

A total of 4 PET scans were performed in monkeys: all involved injection of tracer doses of [11C]PBR28. One of the studies was involved with blockade with non-radiolabeled PBR28 (3 mg/kg i.v.). In these PET scans, the average injected mass dose of [11C]PBR28 was 1.0 µg for an average body weight of 14.0 kg (corresponding to 0.07 µg/kg). In all cases (for injection of both radiolabeled and non-radiolabeled PBR28), the difference of measurement between pre- and post-injection were: < 15 mmHg for systemic blood pressure, < 10/min for pulse, < 5/min for respiratory rate and < 0.3 Cº for temperature.

D. Estimated Receptor Occupancy

The peak uptake of [11C]PBR28 in monkey cortical regions was ~300% SUV%. For the proposed injection of no more than 5 µg, this corresponds to a concentration of 0.56 nM. This is calculated as follows: Assumptions: body weight = 70 kg = 70 L, SUV = 300%, molecular weight = 348 Concentration in brain = (5 × 10-6 g)/(348 mol/g) × 1/70 L × 300%/100% = 0.62 nM. The density of PBR in cortical regions is 440-800 fmol/mg protein (Cymerman et al 1986). By assuming for brain tissue that 1 g = 1 cm3, the density of PBR is 44-80 nM. If all tracer bind to the receptor (which is not true because of nonspecific uptake), the occupancy would range from 0.8 % (=0.62 nM/80 nM) to 1.4% (=0.62 nM/44 nM).

E. Radiation Dosimetry Estimation from Nonhuman Primates. The complete report is located in Appendix E. A brief summary is included below.

50 70 90 110 130 15075

100

125

150Cerebellum4th Ventricle

1 Tissue Compartment Model

Time (min)

VT' (

mL/

cm3 )

50 70 90 110 130 15075

100

125

150Cerebellum4th Ventricle

2 Tissue Compartment Model

Time (min)

VT' (

mL/

cm3 )


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Two male rhesus monkeys (4.1 and 5 kg) underwent whole-body PET scans after intravenous injection via the posterior tibial vein of 8.9 and 11.2 mCi [11C]PBR28. 2D dynamic scans were acquired on the GE Advance tomography (GE Medical Systems, WI) in 4 segments of the body (head to upper thigh) in frames of increasing duration (75 s to 15 min) for a total scan time of 120 minutes. A urinary catheter was inserted and clamped so that the radioactivity overlaying the bladder represented the total urinary excretion during the scanning interval. The data were analyzed with OLINDA 1.0/EXM, as described in Appendix. The estimated human Effective Dose was 40.8 mrem/mCi. In the current protocol, the injected activity will be ≤ 20 mCi; therefore, the effective dose per administration will be ≤ 0.82 rem.

VI. Human Experience.

We are not aware of any human studies with PBR28. However, the related analog PK11195 has been given with NO adverse effects to human subjects at doses of 10 mg IV and 400 mg PO. Please see Section IV.F.

VII. Environmental Assessment.

A categorical exclusion is requested regarding the environmental assessment. Activity excreted by the subjects is disposed through the sewage system at such low amounts that there will be no detrimental effects to the environment.

All potentially contaminated laboratory materials are held at least 10 half lives for decay. In addition, these materials are surveyed to ensure background levels of activity prior to disposal through regular waste systems.

VIII. Case Report Form.

The CRF is in Appendix D.

IX. References. Akaike H (1974): A new look at the statistical model identification. IEEE Trans Automat

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X. APPENDICES. A. Investigator C.V.


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B. Study Protocol.


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C. IRB Protocol Approval. This protocol has not yet been approved by the IRB of NIMH. This study will not be

initiated until it is approved by both the FDA and the IRB.


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D. Case Report Form.


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E. Dosimetry Report from Nonhuman Primates


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F. List of Cited Pharmacology / Toxicology Studies.

CHEMISTRY, MANUFACTURING, AND CONTROLS

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