Exploratory IND Application: Evaluation of [11C]PBR28
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. / Section1 / 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
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).
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 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
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, 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/mmol 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).