Project: Novel PET Imaging Probe Delivery for Early Alzheimer’s Detection
Alzheimer’s disease is a neurodegenerative disorder that is thought to account for 60 - 70% of the approximately 47.5 million cases of dementia worldwide [1]. In addition to being dementia’s most common cause, it has also been identified as a major killer [2]. A disease of the predominantly elderly, it is characterized by the progressive decline in memory recollection and formation as well as cognitive ability. Symptoms of Alzheimer’s can widely vary according to numerous factors. Individuals who have achieved a higher level of education, for example, are far less at risk for developing symptoms of Alzheimer’s [3]. This fact has been attributed to the concept of the “cognitive reserve”, wherein the mental faculties of patients with abundant cognitive reserves are less vulnerable to the pathological effects of Alzheimer’s as a result of neurostructural differences acquired through years of study [4]. Patients who have maintained consistent social and mental stimulation have also been found to exhibit cognitive reserve and therefore fewer symptoms [5].
Though the effects of Alzheimer’s disease may vary on an individual basis, there is a collection of symptoms that are almost universally reported among sufferers, together with a characteristic progression of stages in which symptoms gradually worsen. In the “mild” stage of Alzheimer’s, sufferers may find it difficult to complete tasks that were once easy for them [6]. They may ask for questions to be repeated, have difficulty locating possessions, and experience behavioral changes [6]. In the “moderate” stage of Alzheimer’s, lucidity and memory continue to decline. Confusion begins to pervade and it may become difficult to recognize even close family members, learn new things, and complete basic multi-step tasks [6]. Finally, in the latest stage, the “severe” stage of Alzheimer’s, communication becomes a dubious prospect [6]. The sufferer may experience a loss of control of bodily functions (including bladder and bowel control and the ability to swallow at will), weight loss, seizures, and the increased need to sleep [6]. Ultimately Alzheimer’s results in death [2].
Alzheimer’s disease has been observed to be closely associated with the misfolding of amyloid precursor protein, or APP, a transmembrane protein predominantly located in the synapses [9]. APP is normally processed by alpha and gamma secretases, proteolytically cleaving the N-terminal domain, which is soluble and plays a role in neurotrophy [10]. In the brains of Alzheimer’s patients, however, another protein called beta secretase will occasionally process APP, and in doing so produce beta amyloid. The damage wrought by the faulty processing of APP is two-fold: 1. The beta amyloid protein fragments tend to aggregate into amyloid fibrils, resulting in the neurotoxic effects witnessed in Alzheimer’s Disease [11]. 2. The presence of beta amyloid in such large concentrations as in Alzheimer’s may trigger changes in Tau, a protein associated with microtubules, inducing it to form neurofibrillary tangles. Such tangles are actually linked to severe Alzheimer’s to a greater extent than beta amyloid plaque [12]. Beta amyloid plaque can hamper the transit of signals across the synaptic gap, impeding communication between neurons [13]. Neurofibrillary tangles, on the other hand, also degrade neural connectivity and can result in non-apoptotic cell death [14].
Although the signs of Alzheimer’s disease are discernible in the brain up to 20 years before the onset of symptoms, it is typically diagnosed at the “mild” stage, at which point irreversible neural atrophy has already led to the occurrence of obvious symptoms [7][6]. Unfortunately for patients, this places a severe restriction on the possibility for early therapeutic options and preventative measures against later symptoms (perhaps ones directed at bolstering cognitive reserves). As of now, diagnosis is typically made by primary care physicians, to whom family medical history is an important lens through which to view symptoms, as well as anecdotal evidence from those close to the patient [8]. Cognitive tests may be used to determine the extent of the dementia, while imaging of the brain via imaging modalities such as MRI or CT is useful in surveying the neurostructural effects [8].
The limiting factor in the early detection of Alzheimer’s disease is clearly the fact that one must wait until the “ship” has already hit the “iceberg” in order to react. What if, however, there were a way to screen for Alzheimer’s long before symptoms began to show up? What it there were a way to “spot the iceberg” of Alzheimer’s in the horizon and begin taking steps to minimize its impact? Through the combined use of several different technologies, some well established, some nascent, it may be possible to do just that.
PET, or positron emission tomography, is a widely used imaging modality that works off the principle of a positive beta-emitting radionuclide-tagged host molecule being introduced into the patient and being allowed to distribute itself within the body according to the host molecule’s usual metabolic fate. This technique is widely used in the detection of cancers, wherein either [18F]fluorodeoxyglucose (in order to target the highly glucose demanding cancer cells) or [18F]fluorothymidine (to target the high nucleic acid demand of cancer cells) are used as molecular imaging probes. The pattern is clear; a particular chemical reaction within the body may be “hijacked” for medical imaging purposes.
Beta amyloid plaque would be the obvious target for Alzheimer’s detection. Indeed, there have been a number of attempts to come up with effective molecular imaging probes for beta amyloid plaque. For example, 11C-labeled Pittsburg Compound B, or (PiB) is one such probe whose potential has been thoroughly explored [15]. PiB binding, however, has been found to be inadequately correlated with the structural changes associated with Alzheimer’s [15]. Florbetaben is another molecular probe that has been proposed, and it is actually reasonably successful at traversing the blood-brain barrier, with a good specificity for beta amyloid plaque once it has [16]. Unfortunately, neither PiB nor florbetaben have any sort of a “safety net” to prevent them from accumulating in off-target organs.
We intend to test a solution that will address all of the most important concerns when considering a beta amyloid imaging probe. The proposal is to “hijack” bapineuzumab, a monoclonal antibody that targets beta amyloid protein fragments, in order to highlight beta amyloid plaque in PET images. Although monoclonal antibodies address the molecular affinity problems of other probes, they have little capacity to traverse the blood-brain barrier alone [17]. With the aid of the 3HM, a nanoparticle carrier that comes in the form of a hybrid micelle, bapineuzumab can traverse the blood-brain barrier with a high degree of fidelity to the target-organ (in this case the brain) [18]. Thus, the problems of molecular binding affinity, transport across the blood-brain barrier, and target-organ specificity are all addressed by our solution.
To arrive at the final design of this novel solution, we carefully considered several technical challenges. The selection of a proper molecular imaging probe was our first concern. After careful consideration, we eventually settled on monoclonal antibodies. Although monoclonal antibodies have been used to treat Alzheimer’s therapeutically, they are not common imaging probes due to the issue of crossing the blood-brain barrier. Our approach, however, is not hindered by such an inconvenience. Out of the numerous therapeutically popular monoclonal antibodies that target the Aβ peptide, Bapineuzumab was chosen to serve as our molecular imaging probe due to its advancement in the clinical trial phase [28]. It is a humanized monoclonal antibody, which means it is obtained from a non-human species, and its protein sequence is modified to make it compatible with the human body. It follows the common route of detecting and preventing the formation of amyloid fibril like most other therapeutic antibodies [30]. The fragment crystallization domain of this antibody, which is responsible for detecting and attaching to the Aβ peptide, recognizes the linear amino terminus of the peptide. Most therapeutic monoclonal antibodies are proven to be active on this terminus of the Aβ peptide. It is capable of binding to most of the forms of Aβ including “prefibrillar aggregates and plaques” [31]. This makes it especially well suited to early detection of Alzheimer’s, as beta amyloid only becomes neurotoxic in its fibrillarform [11]. In terms of removing the amyloid plaque, our monoclonal antibody follows the two most popular proposed mechanisms. First of all, upon sufficient entry, the antibodies will bind to the amyloid plaque. It will initiate phagocytosis of the plaques by the microglial cells and increasing macrophages [30]. This has the side effect of removing the antibody from the brain. Secondly, it is associated with changing the concentration of free Aβ peptide in the blood, which initiates an Aβ peptide concentration gradient across the blood-brain barrier [29][30]. In terms of biodegradability, studies have shown that the half-life of Bapineuzumab ranges from 21 to 26 days [28], although it is dose dependent. Furthermore, multiple studies in phase 1 clinical trial have been performed to assess the biocompatibility, pharmacological efficiency and tolerability of our antibody [20].
After settling on our choice of molecular imaging probe, there was the challenge of passing the blood-brain barrier - no small task for a monoclonal antibody. The blood-brain barrier is the wall that separates the neuronal tissue in the central nervous system and the extracellular tissue in the brain from the regular blood circulation. Structurally it comprises astrocytes, pericytes, microglial cells and endothelial cells that are closely attached to each other via tight junctions [19][25]. As a result, the blood-brain barrier is selectively permeable to certain types of molecules (and hence the design challenge). Different mechanisms that are responsible for mediating the transport of various molecules include: a paracellular pathway controlled by tight junction, passive transport of lipophilic molecules, transcytosis due to nonspecific adhesion between charged molecules and the receptors of endothelial cells, and transcytosis due to specific interaction between molecules and the receptors of endothelial cells [23][25]. It was crucial to understand and take into account the mechanisms of these transport pathways while considering various therapeutic nanocarriers.
Firstly, the nature of the surface of the nanoparticle was an important consideration to make. Modification of the surface of the nanoparticles and enabling them to pass the blood-brain barrier is an essential step in the therapeutic nanoparticle fabrication method. The structure and the surface properties of the nanoparticles determine the route of transport they take. In terms of structure, the 3HM is a self-assembled hybrid micelle [24]. It is a tertiary structure of a protein that is formed by two steps of coiling. The hybrid structure of a single particle consists of three different components. The head group, which is the bulky part, consists of a self-associating 3-helix bundle. The second component, which is a polyethylene glycol (PEG) chain, is attached to the middle of the bundle [24]. The third component is a smaller PEG chain, which is attached to C terminus of the peptide. This is the hydrophobic component of the amphiphilic micelle, which creates a sheer layer on the surface of the actual nanoparticle. On the N terminus of the peptide is attached a double alkyl chain which is attached in order to modify the permeability of the blood-brain barrier [24].
Secondly, we needed to consider the size of the nanoparticle, which plays a crucial role in the pharmacokinetics and the biodistribution of the molecule. It determines the mechanism of the transcytosis that is used for sending the nanoparticle across from the blood-brain barrier. The size of the 3HM is approximately 20nm, which is smaller than any other nanoparticle that are currently used for the therapeutic delivery to the brain [25]. Upon dissolving the amphiphilic compounds in an aqueous solution, they self-assemble to form micelles which have a size that varies between 20nm and 18nm. The particle showed fairly uniform size distribution, which is confirmed by dynamic light scattering and small angle X-ray scattering method [24].
Finally, it was crucially important that the nanoparticle be biocompatible in every sense of the word. Cytotoxicity and interaction with immune response system are two important aspects of biocompatibility. Generally after therapeutic nanoparticles enter into human body, mononuclear phagocyte system recognizes them. It is a part of the human immune response system, which is responsible for the accumulation of nanoparticles in the spleen, liver and bone marrow [27]. PEGylation of the 3HM helps overcome this problem. The addition of these long PEG chains protects the nanoparticle from proteolytic enzyme attack. This increases the circulation time and the stability of the particle. These chains are also non-antigenic. This prevents the mononuclear phagocyte system from recognizing the nanoparticles, initiating any immune response and accumulation of nanoparticles in non-targeted areas, which make them nontoxic in general [26]. Biodegradability is another critical component of biocompatibility. The 3HM nanoparticles have been tested for biocompatibility on mouse models of Glioblastoma cancer, with no adverse effects being reported. In terms of biodegradability, the 3HM has a half-life of 25h, which is reasonably high. After crossing the blood-brain barrier however, the nanoparticles reach the central nervous system where they initiate release of the drug (in this case Bapineuzumab) and eventually degrade [26].
Once we knew that it was possible to transport Bapineuzumab across the blood-brain barrier, the only thing left to do was to choose which radionuclide to attach to the monoclonal antibodies. 18F, an artificially made positive beta emitter, is one of the most common radionuclides used for neuroimaging purposes. The half-life of this radioactive element is 1.83 hours, which is short compared to other radionuclides [32]. We will use a common bioconjugation technique to attach the monoclonal antibody to the radionuclide, and this process involves chelating agents [33]. They attach with the monoclonal antibody and form a complex, which is capable of attaching to the radionuclide. One possible approach is the aluminum fluoride 18F chelation method in which chelating agent is 1,4,7- isothiocyanatobenzyl triazacyclononane-1,4-diacetic acid(NODA-Bz-SCN). It is a bifunctional chelating agent [33]. In previous experiments, this method has successfully conjugated peptide with 18F.
Thus the feasibility of our design has been effectively demonstrated. The 3HM nanoparticles together with 18F-labeled Bapineuzumab form a basis for a novel approach that has been custom tailored to the needs of neuroimaging in early Alzheimer’s detection. Putting it all together, the 18F-labeled Bapineuzumab will be coated with the hybrid 3 helix micelle nanoparticles using an appropriate emulsion and solvent evaporation technique - a common technique for nano-encapsulation of drugs or monoclonal antibodies before targeted delivery [34]. The purpose of this encapsulation is to ensure a safe transport of the antibody within blood circulation, as well as to decrease the risk of disruption of the blood-brain barrier by the antibody. Furthermore, it ensures proper delivery of the monoclonal antibody to the targeted site, with a decreased chance of interacting with any non-targeted component. After intravenous injection of our nanoparticles, they are expected to travel mostly towards brain, since they have less specificity non-targeted regions [35]. In addition, due to PEGlaytion, fewer nanoparticles are expected be accumulated in the non-targeted region, which decreases cytotoxicity. Renal and hepatic clearance will aide in eliminating whatever off-target nanoparticles there are [36]. The nanoparticles upon reaching the brain are expected to cross the blood-brain barrier without any disruption and subsequently reach the cerebral cortex, which is the site of beta amyloid plaque. Due to the biodegradation properties, the nanoparticles are expected to release the 18F-labeled Bapineuzumab with little difficulty. As the antibodies will be accumulated around the amyloid plaques, the 18F will emit positrons which will very quickly annihilate with neighboring electrons to produce gamma rays, which will be detectable via PET imaging. Physicians can then interpret the outcome of such PET scans.
With our hypothesis established, this leads us to the experimental methods phase. Extensive in vivo and in vitro testing would need to be performed in order to ensure safety and efficacy. The ultimate goal is to lay out a full testing framework that can be used in preparation for eventual clinical trials. The truly novel aspect of our technique is the use of nanoparticles in conjunction with 18F-labeled Bapineuzumab for imaging purposes. The main advantage of nanoparticles is that they reduce the chances of off-target accumulation. Therefore, in addition to our primary concerns, safety and efficacy, we hope also to test whether or not we have been successful in our primary innovation of greater target fidelity. The basic structure of our trials will be as follows: First, in vitro testing will be used as a proof of concept and to confirm the results of previous studies which indicated that monoclonal antibody accumulation correlates well with beta amyloid plaque deposits. The next stage will involve a variety of in vivo testing methods to investigate the safety of our technique, as well as determine the correlation between images of high activity in PET scans and beta amyloid plaque in the brains of animal test subjects. Mice will be used most extensively in early in vivo testing, whereas primates will become relevant as human trials become a more likely prospect.