A Magic Bullet in Pain Relief?

The advancement of medicine has numerous accomplishments; however, one of the slower improvements involves addressing and managing pain. Significant instances of pain, both in acute and chronic form, afflict hundreds of millions of people worldwide, but most modern treatments struggle to demonstrate meaningful improvement versus past treatments. In fact it is estimated that at least half of surgical patients do not receive effective pain control after their treatments.1,2 Also addiction to pain medication has become a mounting problem in recent years making long-term pain management strategies more difficult.

One potential strategy for managing pain that has gained popularity in recent years is focusing on the activation of analgesic targets like sodium channels Nav1.7, Nav1.8 and Nav1.9. These sodium channels belong to a larger family of voltage gated sodium channels (Nav1.1-1.9) that each has specific locations and functional roles in the body. Among the aforementioned three sodium channels, Nav1.7 is viewed as the most important and its function was first identified from conditional knockout studies in mice expressing Nav1.8 after assumptions were raised from a small family appeared to have significant pain insensitivities via a loss-of-function recessive mutation in Nav1.7.3,4 The resultant study identified Nav1.7 playing a significant role relative to inflammatory pain and the conditional deletion of Nav1.7, not surprisingly, heavily reduced that level of pain to an almost non-registered symptomatic level.3,5,6

Nav1.7 and its 1.8 and 1.9 cohorts are present near the synapses of neurons that are commonly thought to be responsible for sending and receiving pain signals. Overall Nav1.7 appears to transmit action potentials via neurotransmitter release through a threshold managed by Nav1.9, which receives input from Nav1.8.7-10 However, it does not appear that Nav1.7 activation is exclusively reliant on Nav1.8 or 1.9.7

While one means to address pain in the past was the utilization of global sodium channel blockers, developing a drug that has strong specificity for Nav1.7 is thought to be a principal strategy for more effective pain management by localizing treatment to increase selectivity and reduce negative side effects, especially those involving the heart since Nav1.7 is not located near the heart. While not all forms of pain involve Nav1.7, which should surprise no one, a significant number of pain processes appear to incorporate Nav1.7, which has produced the aforementioned enthusiasm for producing a target therapy.4,7

Of course since the major discovery associated with Nav1.7 occurred in 2006,4 various drug development programs have been underway to produce an appropriate and effective treatment. Unfortunately despite the creation of numerous specific stable antagonists, the general results have been disappointing ranging from non-replicated results to unexpected negative side effects.11 One piece of information from these studies highlights an apparent contradiction where the more selective the antagonist for Nav1.7 the less effective the pain reduction versus less selective molecules like lidocaine being more effective.6

The major reason behind this result is thought to be a relationship between Nav1.7 and enhanced natural opioid signaling born from studies involving Nav1.7 null mutant CIP.4 Basically in null mutants an unknown biological relationship develops producing the dramatic change in opioid concentrations in a natural/steady-state condition that is responsible for blocking pain. This belief is supported by the ability of Naloxone, an inverse agonist for the u-opioid receptor (MOR) and antagonist for k and d-opioid receptors, to frequently reverse the pain insensitivity born from Nav1.7 null.7,12 However, oddly enough while knocking out SCN9A, a gene responsible for encoding Nav1.7, produces this enhanced opioid concentration state; simply reducing the activation efficiency of Nav1.7 after development does not seem to produce anywhere near the enhancement of opiods. Basically there is no proportional response.

One explanation for this result is look at how the null creature compensates for the loss of Nav1.7 during development. No Nav1.7 expression commonly results in transcriptional up-regulation of Penk, which is a precursor of met-enkephalin, but Penk was not up-regulated in Nav1.8 or 1.9 nulls.7,13 This result suggests that the neurotransmitter release associated with Nav1.7 is the critical step. Complete channel block of Dorsal Root Ganglion (DRG) neurons via high concentrations tetrodotoxin, this is relevant because a number of the neurons at this location have Nav1.7 channels, also creates a state of enhanced opioid expression.7 However, without a complete channel block there does not appear to be significant increase in opioid or enkephalin expression.7 Overall the increase in opioid concentration within null mice, and probably humans, target nociceptive input consistent with the expression of opioid receptors on small nociceptive afferents.7,14

This result seems to suggest that there is no middle ground in blocking Nav1.7; either the treatment has to produce a 100% channel block or there is no significant increase in pain insensitivity/pain relief.15,16 This issue is a problem for while some agents attempt to improve selectivity by binding to areas outside the pore-forming region on channels through less effective conservation producing inhibitory action independent of the channel’s functional state,6 it is highly unlikely that even these strategies will develop a molecule to create a 100% selective block without significant negative side effects. This challenge has lead researchers to focus on biologics, like venom toxins, over small molecules due to increased rates of selectivity even incorporating techniques like saturation mutagenesis;17-19 however, at this moment success appears improbable.

This result regarding full channel block produces two questions: first the interaction of Nav1.7 suggests that sodium can function as a secondary messenger with respects to the expression of enkephalin through the alteration of Penk mRNA expression levels. Such a belief is supported by the behavior of the ionophore monensin, which results in decreased expression of Penk whereas blocking the channel up-regulates Penk mRNA.13

If this is the case, then the importance of Nav1.7 over that of Nav1.8 and 1.9 may be directly attributable to the level of sodium that passes through Nav1.7, which has a greater effect on overall intracellular sodium concentrations versus other sodium channels. For example HEK293 cell lines with permanent expression of Nav1.7 establish a resting intracellular sodium concentration around double the level of control cells.7

Second, Nav1.7 could produce a form of some level of natural opioid inhibition or at least a form of negative feedback. This mindset seems to be supported by gain-of-function mutations in Nav1.7 typically producing conditions of erythromelalgia (PE), which is characterized by episodes of symmetrical burning pain of the feet, lower legs, and even hands and is tied to increased Nav1.7 channel activity.6 However, if this is the case it raises an interesting question to why null Nav1.7 seem to produce no inherent negatives born from the additional concentrations of opioids, i.e. no addiction or sensitivity. Perhaps in null cases other pathways form to provide a level of opioid feedback inhibition or “saturation” management.

Based on the above information it does not appear that producing a molecule to interfere with Nav1.7 activity can be effectively used to treat pain because full blockage is seemingly required to produce conditions associated with pain insensitivity and general pain treatment. Also blocking Nav1.7 over long and consistent periods of time may damage other important sensory processes. The reason Nav1.7 demonstrates success in knockouts, both cultured and natural, may be because the knockout mutation forces the body to focus on other pathways to manage the other systems that Nav1.7 would normally interact with if it existed. However, that does not exclude using information pertaining to Nav1.7 activity to identify a better pain management treatment.

A better strategy may be to pursue strategies to expand or mimic concentrations of met-enkephalin, which is directly influenced by Nav1.7 activity. Met-enkephalin is a strong agonist for the d-opioid receptor, has some influence on the u-opioid receptor and almost no effect on the k-opioid receptor.7 However, despite its meaningful opioid influence, met-enkephalin has low residence times in the body due to rapid levels of metabolization.20 Thus, simply injecting met-enkephalin into a person would serve little purpose in addressing pain because it would have to be done at large doses and too frequently. However, a synthetic enkephalin, [D-Ala2]-Met-enkephalinamide (DALA) has shown some positive attributes at managing pain by changing its rate of metabolism.

In the end despite the clear understanding that pain relief can be achieved by blocking a channel like Nav1.7, no compounds have been developed to effectively and easily take advantage of that reality. Due to the requirement of full channel block it is highly unlikely that a treatment involving small molecules will ever be successful, leaving the door open only for modified biologics. However, even with a successful “in lab” molecule the location of Nav1.7 in higher concentrations behind the blood brain barrier may make meaningful treatment difficult without some level of increased blood brain barrier penetration. Overall the allure of channel block pain therapy involving a specific location like Nav1.7 may need to be supplemented by further focus on the more downstream products associated with channel activation or inactivation like Met-enkephalin to complement pain relief strategies.


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Citations –

1. Chapman, R, et Al. “Postoperative pain trajectories in cardiac surgery patients.” Pain Research and Treatment. 2012. Article ID 608359. doi:10.1155/2012/608359

2. Wheeler, M, et Al. “Adverse events associated with postoperative opioid analgesia: a systematic review.” Journal of Pain. 2002. 3(3):159–180.

3. Nassar, M, et Al. “Nociceptor-specific gene deletion reveals a major role for Nav1.7 (PN1) in acute and inflammatory pain.” PNAS. 2004. 101(34):12706-11.

4. Cox, J, et Al. “An SCN9A channelopathy causes congenital inability to experience pain.” Nature. 2006. 444(7121):894-8.

5. Abrahamsen, B, et Al. “The cell and molecular basis of mechanical, cold, and inflammatory pain.” Science. 2008. 321(5889):702-5.

6. Emery, E, Paula Luiz, A, and Wood, J. “Nav1.7 and other voltage-gated sodium channels as drug targets for pain relief.” Expert Opinion on Therapeutic Targets. DOI: 10.1517/14728222.2016.1162295

7. Minett, M, et Al. “Endogenous opioids contribute to insensitivity to pain in humans and mice lacking sodium channel Nav1.7.” Nature Communications. 6:8967. DOI: 10.1038/ncomms9967

8. Eijkelkamp, N, et Al. “Neurological perspectives on voltage-gated sodium channels.” Brain. 2012. 135:2585–2612.

9. Akopian, A, et Al. “The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways.” Nat. Neurosci. 1999. 2:541–548.

10. Baker, M, et Al. “GTP-induced tetrodotoxin-resistant Naþ current regulates excitability in mouse and rat small diameter sensory neurones.” J. Physiol. 2003. 548:373–382.

11. Lee, J, et Al. “A monoclonal antibody that targets a Nav1.7 channel voltage sensor for pain and itch relief.” Cell. 2014. 157(6):1393-404.

12. Dehen, H, et Al. “Congenital insensitivity to pain and the "morphine-like” analgesic system.” Pain. 1978. 5(4):351-8.

13. Popov, S, et Al. “Increases in intracellular sodium activate transcription and gene expression via the salt-inducible kinase 1 network in an atrial myocyte cell line.” Am. J. Physiol. Heart Circ. Physiol. 2012. 303:H57–H65.

14. Usoskin, D, et Al. “Unbiased classification of sensory neuron types by large-scale single-cell RNA sequencing.” Nat. Neurosci. 2015. 18:145–153.

15. Minett, M, Eijkelkamp, N, and Wood, J, “Significant determinants of mouse pain behaviour.” PLoS One. 2014. 9(8):e104458.

16. Minett, M, et Al. “Pain without nociceptors? Nav1.7-independent pain mechanisms.” Cell Rep. 2014. 6(2):301-12.

17. Shcherbatkok, A, et Al. “Engineering highly potent and selective microproteins against Nav1.7 sodium channel for treatment of pain.” J. Biol. Chem. 10.1074/jbc.M116.725978

18. Harvey, A. “Toxins and drug discovery.” Toxicon. 2014. 92:193-200

19. Yang, S, et Al. “Discovery of a selective Nav1.7 inhibitor from centipede venom with
analgesic efficacy exceeding morphine in rodent pain models.” PNAS. 2013. 110:17534-17539

20. Minett, M, et Al. “Distinct Nav1.7-dependent pain sensations require different sets of sensory and sympathetic neurons.” Nature Communications. 2012. 3(4):791-799.

Treating Cancer Through Metastasis Neutralization and Possible Activation

While cancer in any form is potentially dangerous to a patent, it is widely acknowledged that only a small percentage of primary tumors are threatening to the life of a patient in the interim. A vast majority of cancer patient deaths occur due to cancer metastasis. Metastasis is a complex pathway of molecular interactions that produce an end result that involves the departure of a group of tumor cells from the primary tumor into the bloodstream and eventual invasion into other tissues resulting in the formation of additional tumors. Not surprisingly the process is governed by a number of complex pathways that are not fully understood. Despite this lack of knowledge regarding metastasis, is it clear that one of the best strategies for addressing cancer would be to create a therapeutic regimen that would prevent metastasis from occurring in the first place allowing physicians ample time to eradicate the primary tumor with no legitimate threat of reoccurrence.

Of the agents thought to be involved in cancer metastasis chemokine receptor CXCR4 is a promising agent of study and potential therapeutic target. Chemokines are a group of low molecular weight cytokines that induce chemotaxis, most of the time as chemoattractants, largely in leukocytes, endothelial and epithelial cells.1 Chemokines are commonly classified into CC, XC, CXC or CX3C designations based on the positioning of their respective conserved cysteine residues.1 One of the key normal functions of chemotaxis is to facilitate the movement of pro-inflammatory cells to the site of inflammation, including immune cells, normally after some form of injury. CXCR4 is an attractive target because of both strong anecdotal and experimental evidence regarding its overall expression and role in tumor malignancy and metastasis.1-5

CXCR4 functions as a G protein-coupled receptor (GPCR) that principally binds stromal cell-derived factor 1 (SDF-1), which is also known as CXCL12. With regards to cancer, CXCR4 plays a significant role in directional migration though activation of actin polymerization6,7 as well as invasion and adhesion, which influence the overall level of aggression for a tumor. There is also some evidence suggesting that CXCR4 plays a role in angiogenesis as well.4,5

CXCR4 can undergo four major changes to influence its functionality: homo or heterodimerization (with CCR2, CCR5, CXCR7 or CD4), phosphorylation, glycosylation, or sulfation.8-14 Unfortunately limited information is known about functional changes associated with dimerization in cancer for almost all studies involving CXCR4 dimerization relate to HIV, but it is thought that such changes enhance CXCL12 binding. It is also believed that dimerization typically occurs internally before CXCR4 is expressed on the cell surface, typically as oligomers. This oligomeric structure persists in the plasma membrane.14

Phosphorylation occurs principally at serine residue number 339 (Ser339) after exposure to either CXCL12, epidermal growth factor (EGF), or phorbol ester and it is believed that phosphorylation may also occur to a much smaller extent at Ser324, Ser325, and Ser330.12 Phosphorylation is important for increasing the probability of receptor internalization and secondary messenger activation. On a side note mono-ubiquitination occurs at Lys327, Lys331 or Lys333.13 Glycosylation of human born CXCR4 only appears to occur at Asn11 and seems to serve no unique function other than stabilizing CXCL12 binding (lack of glycosylation reduces binding efficiency).1

Sulfation, which takes place primarily at Tyr21 and does not appear to occur on two other potential sites (Tyr7 and Tyr12),15 may the most interesting modification regarding CXCL12 binding probability and functionality.16 When CXCL12 binds to CXCR4 there is a specific site interaction between sulfated Tyr21 on CXCR4 and Arg47 on CXCL12.15 One of the reasons that sulfation appears important is that one study demonstrated that while highly metastatic NPC cells and non-metastatic NPC cells expressed similar levels of CXCR4, both via mRNA and protein, only high levels of sulfinated CXCR4 resulted in high metastatic potential.17

While there are a number of tyrosine sulfation pathways, with regards to CXCR4 one of the more prominent interactions involves the action of the latent membrane protein 1 (LMP1). While LMP1 concentration changes are not universal to CXCR4 activity increases, LMP1 interacts with EGF receptors, which is thought to be one of the early steps in inducing Tyrosylprotein sulfotransferase-1 (TPST-1) dependent tyrosine sulfation of CXCR4.15 However, there is the lingering question of whether sulfation is the chicken or the egg, is it a chief agent in how CXCR4 activation influences the potency of cancer or is it a result of that activation?

CXCR4 activation also significantly increases the overall expression of matrix metalloproteinases (MMP), especially MMP-2, MMP-3, MMP-7 and MMP-9.18,19 One of the major triggers for this action could involve the activated CXCR4 guiding Bone marrow-derived cells (BMDCs) to their pre-metastatic niche, which then triggers BMDCs to initiate the release various metastatic positive elements including various MMPs most notably MMP-7 and MMP-9.20,21 CXCR4 is also involved in the homing of cells into the endosteal HSC,22 which facilitates the expression of SUMO-specific protease 1 that regulates MMP-9 as well.23

As stated above one of the key steps in metastasis is the proteolytic degradation of the extracellular matrix (ECM) in which various MMPs are critical agents. Initially MMPs were thought to be degenerative proteases that were limited to cleaving matrix components, but that role has expanded to include the release of growth factors and other bioactive peptides localized at cleaved extracellular matrices.24-26 While there are up to 26 known MMPs (MMP-1, MMP-2, MMP-3, …) only a few have demonstrated significant roles in both cancer growth and cancer metastasis. Of these select few MMPs that play a prominent role in cancer, MMP-3 and MMP-7 appear to be the most important.

MMP-7, (a.k.a. matrilysin) is the smallest MMP and is commonly expressed in epithelial tumor cells instead of interstitial cells27 and has numerous substrates in the ECM namely collagen fibers, laminin, gelatin, proteoglycan and elastin, etc.28,29 MMP-7 is commonly over-expressed in various types of cancer including, but not limited to non-small cell lung, pancreatic, oral squamous cell carcinoma, colorectal, prostate, stomach and papillary thyroid carcinoma.28,30-34 In addition to its ECM degradation role, MMP-7 can also breakdown cell surface proteins, which aids cancer cell proliferation through the regulation of apoptosis and angiogenesis as well as help evade immune system detection.34-36

Furthermore, as mentioned above, MMP-7 is thought to increase expression of MMP-2 and MMP-9 to aid in ECM degradation and other pro-cancer actions.37,38 Finally not surprisingly MMP-7 levels increase in response to decreased blood glucose levels for this increase is tied to a low quality or deteriorating principal environment for the tumor, which should trigger elements responsible for assisting metastasis like MMP-7. However, while MMP-7 appears to be the most active MMP in most cancers, its activation may only be a downstream event caused by MMP-3.

An early action taken by a member of the MMP family typically involves MMP-3 cleaving decorin, releasing growth factor-beta and cleaving transforming growth-factor-alpha (TGF-a), which activates the MAP-kinase pathway.39 This activation can later activate MMP-7, which as previously mentioned leads to the activation of MMP-2 and MMP-9.40 In addition to activating other MMPs like -2, -7 and -9, MMP-3 can also promote genomic instability and epithelial–mesenchymal transition (EMT) through the activation of Rac1b, which stimulates both the production and release of intercellular mitochondrial superoxide.41,42 MMP-3 appears to be the dominant expression route for Rac1b.41

Due to the important role MMPs play in inducing both metastasis and possible anti-apoptosis protection, a number of researchers have thought of MMP inhibition as a promising treatment option. However, clinical trials investigating the viability of MMP inhibitors, commonly known as tissue inhibitors of metalloproteinases or TIMPs, have not proven successful.43 One of the major theories behind this failure is that during tumor development MMPs have different roles depending on tumor progression and the other molecules present in the tumor microenvironment. Some information has demonstrated anti-tumor effects for certain MMPs, most notably MMP-3, MMP-8, MMP-9, MMP-12, and a newer MMP, MMP-26, which may be a natural protectorate MMP.43-47

Clearly this dual behavior makes targeting MMPs directly difficult for therapeutic reasons, as demonstrated clinically, thus it could be more important to focus on important MMP triggers like CXCR4, which appear to activate MMPs during a time when their interaction with the tumor microenvironment will produce a net negative for the patient.

CXCR4 also can activate the P110-beta isoform of PI3K resulting in the eventual synthesis of phosphatidylinositol (3,4,5)-triphosphate, which leads to the phosphorylation of protein kinase B/Akt and mTOR pathways most notably activation of p70S6K and 4E binding protein 1.7,48,49 Not surprisingly mTOR inhibitor, rapamycin, reduces the extent of p70S6K and 4E binding protein 1 activation in a CXCL12/CXCR4 environment.7,19,50 Furthermore CXCR4 also activates elements in the Src family of protein tyrosine kinases, which aid the activation of focal adhesion elements like Crk, paxillin, and tyrosine kinase/Pyk2.51

For a long time CXCR4 was thought to be a unique target for CXCL12 until CXCR7 was identified, potentially complicating the role of CXCR4. Similar to CXCR4, CXCR7 is expressed at a much higher rate in malignant cancer cells versus normal cells and binds CXCL12 with high affinity.10,52 However, despite the significant similarities between CXCR4 and CXCR7, CXCR7 does not appear to play a meaningful role in cancer development or metastasis.53,54 The role of CXCR7 appears to involve the migration of primordial germ cells or interneurons.55 Its increased expression simply may be the result of dramatically increased levels of CXCL12 in the localized environment. Interestingly enough CXCR7 may prove to be a possible therapeutic element as an indirect natural CXCL12 competitive inhibitor of sorts for every CXCL12 molecule that binds to CXCR7 is no longer available to bind to CXCR4.

Earlier it was mentioned that CXCR4 might have a relationship in tumor growth and/or angiogenesis. If true, then this result is complicated because cancer growth at established tumor sites is more rapid in the absence of CXCR4 rather than its presence.53 Therefore, it may be that while CXCR4 assists growth immediately after invasion, its continued presence becomes detrimental for the tumor because it helps induce metastasis, thereby diverting resources like recruited and differentiated endothelial cells or progenitors from the original tumor towards elements that will be involved in the metastasis or even the attraction of metastatic elements already in the bloodstream.

CXCR4 also appears to interact with another potential important factor in cancer metastasis, macrophage migration inhibitory factor (MIF). MIF is a pro-inflammatory cytokine that plays an important role in inflammation and immune response and is expressed at a higher than normal rate during numerous cancer stages like cell proliferation, angiogenesis and anti-tumor immune interaction.56-58 High MIF concentrations have also been associated with poor outcomes in lymphoma, melanoma and colon cancer.59,60 CXCR4 interacts with MIF through the formation of a MIF receptor complex with CD74, which further enhances MIF-stimulated AKT activation.61

There is some thought that when MIF lacks its traditional activation pathway it requires caspase-1 activity for proper secretion.62 Also Golgi-associated protein p115 may be essential for the transport of MIF from the perinuclear ring to the plasma membrane and then out of the cell.63 In addition to aiding metastasis, MIF is also thought to apply some level of apoptosis resistance to cancer cells, favoring those with androgen-dependency over those with androgen-independence, but that resistance may be tied to CXCR4 interaction.64

With CXCR4 having its “fingerprints” over a number of pro-cancer processes, fortunately an additional element that makes it an attractive therapeutic target is its natural role in the body. In non-cancerous tissue CXCR4 is expressed on hematopoietic cells like CD34+ HSC, B-lymphocytes, neutrophils, monocytes, macrophages, and microglia, etc.65 CXCR4 or CXCL12 knockouts in mice result in impaired hematopoiesis through reduced hematopoietic stem cell (HSC) trafficking, which results in heart and brain defects as well as vascularization commonly producing embryonic death;66 in adults CXCR4 is important in HSC homing for the bone marrow microenvironment and lymphocyte trafficking.65 However, most of the time CXCR4 expression in normal cells is low, unless the body has been recently injured. Therefore, treatments that limit CXCL12/CXCR4 pathway activation should result in limited negative side effects for healthy non-injured individuals.

Another potential benefit from CXCR4 inhibition could involve reducing the probability of chemotherapy agent resistance, including Docetaxel (DTX) resistance. Some research suggests that the CXCL12-CXCR4 pathway interacts with p21-activated kinase 4 (PAK4)-induced LIM domain kinase 1 (LIMK1) via phosphorylation to reduce the ability of DTX to destabilize microtubules, which typically results in cell cycle arrest during the G2/M phase.67 Basically CXCR4 activation provides additional protection against cell death for tumors when exposed to DTX. This result suggests that LIMK1 could have a role similar to microtubule-associated protein (MAP) depending on whether or not it is phosphorylated. Therefore, this chemotherapy resistant pathway has two principal inhibition targets in CXCR4 or PAK4 to negatively influence prospective chemo resistance.

Existing potential therapies involving CXCR4 have focused largely on inhibiting the binding capacity of CXCR4 most notably either through the use of AMD3100, a specific CXCR4 antagonist, or synthetic peptide TM4.14,68,69 AD3100 (a.k.a. (Plerixafor) is a small molecule with two cyclam rings connected by a phenylene linker that have nitrogens on each ring that have charge-charge interactions with carboxylate groups on CXCR4, which inhibits CXCL12 binding.70-72

Plerixafor is most commonly used as a pre-treatment element for chemotherapy where, as mentioned, CXCR4 disruption reduces the probability of hematopoietic stem cells homing to bone marrow, thus it increases their circulation in the blood stream allowing for their collection for transplantation after chemotherapy regimens.73,74 Plerixafor has also proven promising as an anti-cancer treatment via its ability to reduce cancer cell chemotherapy resistance by either neutralizing the CXCL12-CXCR4 pathway or reducing the physical attachment of various micro-environment critical cells, similar to what is expected for a CXCR4 inhibitor.

An interesting side effect in plerixafor treatment is that surface expression of CXCR4 increases both in vitro and in vivo.65 One possible explanation for this outcome could be that principal signals that induce CXCR4 expression continue while plerixafor prevents CXCL12 from binding CXCR4; CXCL12 binding leads to internalization and activation of secondary pathways. When there is no CXCL12 binding there is no CXCR4 internalization, but the pathways governing CXCR4 expression towards the cell surface continue, thus explaining the overall increase in CXCR4 expression. If this tendency is accurate then long-term treatment with plerixafor alone may not be beneficial because the increased surface expression will substitute for the CXCR4 “removed” by plerixafor interaction. Basically plerixafor works well alone in the short-term, but may not work well alone in the long-term, which may be the same fate of all substrate based CXCR4 inhibitors.

Apart from preventing metastasis, treating the primary tumor is also an important task, especially when surgical options are unavailable. One promising potential therapeutic agent that can influence both primary tumors and metastasis is salinomycin (SAL), which has demonstrated effective ability to kill cancer via a perceived mixture of apoptotic and autophagic cell death in breast, prostrate, brain, blood, liver, pancreatic and lung cancers with no immediate lethal toxicity.75-77 Initially it was reported that SAL was toxic to certain neuronal cells (dorsal root ganglion in mice) at 1 uM, but this toxicity was neutralized when paired with factors that inhibited mitochondrial Na+/K+ exchangers with no resultant change in cancer cell cytotoxicity.78,79

One of the chief advantages of SAL in treating cancer is that it uses a different methodology apart from more common chemotherapy drugs like Doxorubicin, Cisplatin, Gemcitabine, Temozolamide, Tratsuzumab, Imatinib, etc.75,80-82 SAL has a preference for targeting cancer stem cells (CSCs), which reduces the probability of cancer reoccurrence after its primary removal.75 CSCs are important to address in treatment because they are commonly thought of as another element responsible for driving the core of cancer metastasis after responding to various signal triggers as well as driving cancer recurrence after the primary tumor is eliminated due to their ability to more frequently resist anti-cancer therapies. Thus, addressing CSCs, either directly or indirectly, is a critical part to addressing both cancer itself as well as its metastasis.

Another interesting behavioral aspect of SAL is amplified effectiveness under hypoxia or starvation conditions. This result makes sense on two different levels: first, it is thought that a means in which SAL triggers cell death is through damage to the mitochondria, in part to being a potassium ionophore, promoting hyperpolarization in the mitochondria, which decreases ATP availability and triggers caspase-3, 8 and 9, a consequence worsened by starvation conditions.76,83 Second, its effectiveness against CSCs is enhanced by the reaction of the tumor to hypoxia. In hypoxia the primary tumor will begin to focus an effort to metastasize due to the negative environment that currently exists; one step in this process requires the recruitment and creation of CSCs which reduce resource availability for the primary tumor, yet those CSCs are more effectively eliminated by SAL versus the cells that comprise the primary tumor.

The ability of SAL to “cooperative” with other anti-cancer drugs directly is questionable for most of the benefit from co-therapy between SAL and given drug x appears indirect.84 This result is somewhat interesting because there is some evidence to suggest that SAL can also function as an efflux pump inhibitor, which is commonly operated by a p-glycoprotein;85-87 efflux pumps increase the ability of cancer cells to remove chemotherapy agents before the inducement of cell death, so inhibiting them would increase tumor susceptibility to these chemotherapy agents. However, Metformin (METF), which is though to lower circulating insulin levels and stimulate AMPK-mediated suppression of mTOR, along with having some anti-cancer properties in thyroid, prostate, gastric, breast and glioblastoma,88-90 seems to have some form of direct enhancing cooperative relationship with SAL.84

This combination activity results in the “unspecific” inhibition of EGFR and HER2/HER3 leading to reduced concentrations of AKT and ERK1/2 via an unknown mechanism.84 However, it seems appropriate to suggest that based on SAL activity when acting alone that this “inhibition” is born from a reduction in available receptors due to cancer cell or associated cell death.

Another mechanism for inducing cancer death that includes SAL interaction is autophagy. In its most basic form autophagy involves the “self-digestion” of intracellular elements via the vacuolar lysosomal degradation pathway to recycle cytoplasmic constituents.91 Autophagy is typically used to prevent the accumulation of damaged proteins and organelles, largely born from cell damage due to outside agents; for cancer it would be anti-cancer drugs. This process also reduces the production potential of reactive oxygen species (ROS) that negatively impact cell survival.

There is reason to believe that SAL can interfere with autophagy in cancer cells by inhibiting lysosomal activity driven by cathepsins.85 Interestingly enough this activity occurs without impacting the lysosomal compartment.85 This aspect of SAL interaction is not surprising due to structural similarities with both nigericin and monensin, which have similar behavior as anti-porters themselves, but the lack of change in pH of the lysosomes from SAL treatment belies a different pathway.

Also the behavior of SAL runs contrast to ATG7 expression where ATG7 acts as a protectorate of sorts ensuring the proper functionality of autophagy. For breast cancer cells and more than likely other forms of cancer, aldehyde dehydrogenase 1 positive cells (ALDH+) promote autophagy.85,92 Thus, SAL directly works against ALDH as well as competes to “thwart” ATG7 autophagy protection. Therefore, ATG7 in some instances is able to neutralize the dual ability of SAL to kill cancer cells via induction of apoptosis while inhibiting autophagy inhibition. Therefore, inhibiting the activity of ATG7 may provide a useful co-therapy with SAL to significantly neutralize the ability of cancer cells to build resistance to SAL-related apoptotic activity.

Another popular method of action for SAL against cancerous agents is thought to be its interaction with Wnt signaling and its relation to b-catenin. The interaction between Wnt and b-catenin begins when Wnt binds to frizzled (Fzd) receptor and then that complex binds to lipoprotein receptor-related protein 5 or 6 (LRP5/6) co-receptors leading to a ternary complex that typically exists at the cell surface.93 The presence of this complex can trigger phosphorylation of either LRP leading to the recruitment of axin, which then undergoes endocytosis.93 The end result of this entire process is the breakdown or inactivation of the Adenomatous polyposis coli (APC)-Axin complex, which is responsible for b-catenin elimination.

This processes is important because b-catenin accumulation leads to its nuclear translocation and can even increase expression of Wnt genes via those tumors located at the invasive front, which have more interaction with growth factors and cytokines including hepatocyte growth factor.94 This interaction may even create a positive feedback loop of sorts.94,95 This nuclear translocation of b-catenin is thought to play some role in tumor cells experiencing cell-cycle arrest and EMTs via the loss of E-cadherin expression creating some form of cancer stem cell, with increased migration/metastasis potential.96,97

SAL interferes with the Wnt pathway by degrading the LRP6 protein and possibly LRP5 protein, which obviously reduces the probability that they form a complex with Wnt and are later phosphorylated activating the complex.85 LRP protein importance is further supported by a level of suppression in breast cancer tumor growth after treatment with a LRP antagonist, Mesoderm development (Mesd).98,99

However, SAL is not a cure-all when it comes to this supposed pro-cancer pathway, for Wnt and its associated complex is not the only significant destruction inhibition interaction experienced by b-catenin. Expression of platelet-derived growth factors (PDGF) can induce the tyrosine phosphorylation of p68 via c-Abl kinase.100 After phosphorylation p68 can bind b-catenin and inhibit GSK3b mediated phosphorylation reducing the probability that it is eliminated, thereby increasing the probability of b-catenin nuclear location.100 It is also thought that EGF and TGF-b can induce p68 phosphorylation via receptor tyrosine kinases.100,101

Another possible strategy to deal with LRP protein complex interaction involves the inhibition of vacuolar H+-adenosine triphosphatase using an agent like archazolid.102 LRP6 phosphorylation and internalization appears to require V-ATPase. The general role of V-ATPase is transport of both intracellular and extracellular organelles near the plasma membrane. Not surprisingly it also pumps protons leading to the acidification of vesicles, which promotes endocytosis.103

Another element in cancer development that has garnered attention for metastasis is the role of carcinoma-associated fibroblasts (CAFs). Tumor invasion is heavily influenced by the tumor microenvironment, especially the types of non-tumor cells. Various types of fibroblast recruitment lead to the production of soluble factors and extra-cellular matrix (ECM) remodeling usually through actin changes and cell migration born from MMPs, Rho targeted via ubiquitination and SUMO pathways104 as well as global DNA hypomethylation and recruitment of mesenchymal stromal cells; these changes increase the viability of future invasion.105-108

The cancer stroma is typically populated by various concentrations of fibroblastic cell groups that make up CAFs and are commonly divided into myofibroblast (MFs) and non-myofibroblast populations (non-MFs).105 MF populations have received much more attention than non-MF populations more than likely due to the diversity of the non-MF populations. It is thought that CAFs differ significantly from normal fibroblasts and myoblasts, but there is little information regarding the extent of these differences.105,109

CAFs are heavily involved in various pro-cancer pathways like tumor necrosis factor alpha (TNFa), IL-1 and IL-6,105,110,111 which lead to promoting invasion, immune suppression and angiogenesis through the secretion of SDF-1, TGF-b, hepatocyte growth factor (HGF), PDGFs, or vascular endothelial growth factors (VEGFs) principally driven by FSP-1- or PDGF receptor alpha-positive stromal fibroblasts.112-116

Not surprisingly if CAFs are thought to play a role in all of these pro-cancer processes, targeting them would prove useful for developing effective therapies. A number of proposals have been made regarding PDGF receptor inhibitors, SUMO inhibitors, Met receptor inhibitors or HGF inhibitors; however, on its face it is difficult to envision how to effectively target the “right” CAFs due to the widely diverse population of cells within the stroma. Interestingly enough some possibly contradictory research could prove some insight.

As mentioned above CAFs in the stroma are typically defined as either MF or non-MF, but both of these groups can be activated and/or transformed as well. One particular group to question is activated non-transformed MFs, which express alpha-smooth muscle actin (a-SMA). There are some that believe that these cells have “anti-cancer” activity instead of “pro-cancer” activity. Support for this mindset comes from studies of early and late stages of pancreatic cancer outcomes, clinical correlation between high a-SMA levels and improved survival on a general level, and studies of resected tumors.117-120 Also there is some question to whether or not a-SMA positive MF cells increase hyaluronic acid concentration.121 The anti-cancer attributes of MFs appear to stem from aiding both innate and adaptive immune response via increased fibrosis.117

Whether or not these MFs are pro-cancer or anti-cancer elements is important to deduce because anti-cancer therapies tend to kill indiscriminately around the principal tumor and its microenvironment, including a-SMA positive MFs. If these particular MFs are anti-cancer then these drugs are inherently less effective because while they are killing cancerous elements they are also killing anti-cancer elements.

The final possible important element to CAFs and their role in cancer is their ability to produce exosomes. For example in breast cancer CAFs secrete Cd81+ exosomes that can induce the planar cell polarity (PCP) signaling pathway targeting Wnt and influencing the polarity of carcinoma cells.122 Internalization of these exosomes also promotes Wnt11-PCP induction via autocrine through Frizzled receptor signaling leading to increased probability of pulmonary metastases.112 Targeting these exosomes may be a valid therapeutic strategy for reducing cancer potency.

As mentioned above directly targeting CAFs via therapies may be difficult, but one potential candidate could be TNF receptor associated factor 6 (TRAF6). At least for squamous cell carcinoma (SCC), TRAF6 plays a role in enabling nuclear factor kappa beta (NF-kB) signaling to activate a number of downstream pathways for CAFs, like Akt, Src-family kinases, IKK, IL-1beta and p38 and can regulate the formation of Cdc42-dependent F-actin microspikes.105,107,123 While the role of Cdc-42 is exactly unclear, TNFa plays a large role in promoting invasion in SCC and TRAF6 plays a significant role in producing sufficient TNFa concentration. The reduction of either one of these pathways significantly reduces cancer invasion, possibly due to the K63 ubiquitin ligase activation associated with TRAF6.124

The final issue when addressing cancer metastasis is developing a strategy to promote delivery of the anti-cancer agents to increase the probability of positive action, especially through ensuring proximity action. A reason behind this strategy is that some agents, like the rather useful SAL, demonstrate low quality aqueous solubility, which restricts their ability to be injected through a more standard IV strategy.125 Not surprisingly nanoparticles have become the most attractive vessel for transporting anti-cancer agents to cancer sites.

Overall nanoparticles are advantageous due to their low to non-immunogenic activity reducing complications and increasing their lifespan in the bloodstream, their natural and generally safe biodegradability and biocompatibility and their general design flexibility for producing the right type of particle for the given job. For example some nanoparticle structures use polypeptides with elastin and hydrophilic properties in effort to produce immune-tolerant elements. These elements are commonly referenced with the acronym, iTEP.126 However, nanoparticles need a form of “navigation” system to reach the appropriate target. The two most common targeting strategies are the use of antibodies or the use of aptamers.

Antibody targeting in some respects is the “old reliable” while aptamer targeting is somewhat new. Aptamers are comprised either of oligonucleic acid (DNA or RNA) or a peptide that are able to bind a specific target molecule. The major advantages of aptamers are their molecular specificity, their lack of immunogenicity, and their low molecular weights. These two latter advantages along with ease of production have further increased the popularity of aptamers versus antibodies regarding therapeutic targeting strategies including those dealing with cancer. A number of aptamers have already been developed for use in cancer treatment.127,128

Regardless of navigation methodology, the drug delivery vessel must have the right navigation point. One molecule that has drawn interest for potential cancer targeting ability is hyaluronic acid (HA). HA typically binds to CD44, a receptor commonly over-expressed on numerous types of tumors.129 Furthermore HA is frequently broken down by tumor cells by hyaluronidases (Hyals), which is widely thought to experience concentration increases in various cancers including prostate, bladder, colorectal, brain and breast due to the presence of increased low weight HA fragments found in these tumors versus normal cells.130-133

The general process of HA catabolism involves binding to CD44 resulting in its breakdown into smaller elements by Hyal-2 while still on the cell surface forming what is known as a caveolae. This caveolae eventually becomes an endosome that fuses with lysosomes resulting in the further degradation of HA fragments into tetrasaccharides by Hyal-1.134,135 Based on this process one could theorize that a self-assembled nanoparticle comprised of HA would serve as an effective means of drug delivery to the tumor site both tracking and through its degradation, a belief that has been supported with early empirical results.129

In addition to HA, it is widely thought that cluster of differentiation 133 (CD133) is a positive stem cell marker for both normal and cancerous tissue and is thought to be a critical agent in identifying CSCs. For example it is common for CD133+ cancer cells to form mammospheres that can initiate tumor growth in non-tumor cells. Due to the importance of CD133 expressing cells, an RNA aptamer (A15) has already been developed that binds to CD133 for use as a “tracking” marker of sorts and some groups have already explored the idea of using A15 as a drug delivery targeting agent.136

However, it must be noted that while CD133 expressing cells appear to be the most important in the CSC pool, tumors do produce CSCs that express other surface receptors while not expressing CD133. For example in osteosarcoma, CD133, CD117 and Stro-1 are all considered to be legitimate CSC markers.48 Additionally there is some evidence to support the idea that CSCs can convert to non-CSCs and back again.137 Therefore, while targeting CD133 is clearly an appropriate strategy for treating CSCs, it may not be the only targeting strategy necessary to eliminate CSCs.

The type of nanoparticle is only part of the issue involving drug delivery. Another important element is whether or not the principal drug should have other elements encapsulated with it to increase efficacy. For example SAL delivery involves a charged hydrophobic drug trapped inside a hydrophobic core of micelle-like nanoparticle; these interacting charges can increase destabilization potential, leading to ineffective drug application, which has been seen in past studies.125,126 Therefore, this charge interaction needs to be neutralized.

An early candidate for cooperation with SAL stability was N,N-dimethyloctadecylamine (DMOA) due to its similar hydrophobic nature, yet positive charge which is obviously counter to the negative charge of SAL. Unfortunately DMOA proved too toxic for this role.126 Fortunately it has a less toxic analogue in N,N-dimethylhexylamine. However, this reduced toxicity comes at the price of reduced hydrophobic strength due to a shorter hydrocarbon chain.126 Thus, researchers have added alpha-tocopherol as a second hydrophobic agent to enhance internal hydrophobicity to increase stability, which appears to work well.126

In the end while metastasis is still a process with a number of question marks associated with its occurrence and action there does appear to be certain elements that have important roles in its successful occurrence and function regardless of these question marks. First, it is quite clear that any therapy will have to involve some form of drug cocktail to cover multiple metastasis pathways including treatment of the principal tumor via either drugs or surgery. With this strategy in mind one interesting combination would involve the use of SAL in HA nanoparticles, some form of CXCR4 inhibitor, something like Plerixafor should be sufficient when not applied by itself, and a standard chemotherapy drug like Docetaxel.

Another possible addition to this cocktail could be an anti-angiogenesis drug. In recent treatment history anti-angiogenesis drugs have had a negative history of being useful anti-cancer agents despite the sound theoretical reasoning that reducing growth resources should reduce cancer growth potential. The failure of anti-angiogenesis drugs more than likely occurred due to the induced hypoxia environment increasing rates of metastasis. However, this increased metastasis may be a benefit when the anti-angiogenesis drug is used in combination with a CXCR4 inhibitor and/or SAL, which could speed cancer death by eliminating the metastatic elements versus attempting to eliminate the principal tumor. Of course this combination and the possible positive outcome is only theoretical, without appropriate empirical evidence the addition of an anti-angiogenesis agent may not provide a benefit, similar to how it functions currently.

While promising gains have been made in recent years in immunotherapy-based techniques to combat cancer, it is important to acknowledge that overall there is no magic bullet, but the above potential cocktail should be able to overlap the important negative cancerous element of both primary tumor elimination through multiple destruction pathways and metastasis neutralization via elimination of CSCs as well as the major pathways the drive the preparation and activation of metastasis itself. This method alone or in combination with a proven effective immunotherapy technique could provide a legitimate anti-cancer therapy for various stages of cancer development.



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The Current State of Alzheimer’s Disease Treatment

Additional Alzheimer’s disease Blog Post here



Regrettably there remains no effective treatment for Alzheimer’s disease (AD). Current therapies target cholinergic (acetylcholine esterase inhibitors) and glutaminergic (NMDA receptor antagonists) neuronal activity in an attempt to improve symptoms largely associated with cognitive decline.1-3 Unfortunately these treatments are limited in their effectiveness because they do not address the cause of the disease, but rather the symptoms. There are other non-pharmacological treatments that address the detriments of cognitive decline like social measures through various support groups and more personal individualized care. However, while these interventions do what they can to help manage AD, without the development of a viable disease-modifying therapy the natural expansion of AD cases, due to an increasing elderly population, will significantly increase global healthcare costs especially in high healthcare cost countries like the United States. In addition to increasing healthcare costs across the board these cases will also significantly reduce the quality of life for millions.

Based on the success of the amyloid beta (Abeta) cascade theory regarding the development of AD one of the principal recent strategies for creating a future treatment has been utilizing an Abeta antibody that will either prevent plaque formation or break plaques apart hopefully producing positive cognitive remediation for those suffering from AD. Unfortunately while this theory appears reasonable, positive empirical evidence supporting this strategy has proven lacking. In fact numerous Phase II and Phase III studies have failed to demonstrate significant positive cognitive outcomes for these types of drugs versus placebo controls.1,4,5 The two most notable recent failures have been Bapineuzumab and Solanezumab; both were able to reduce fibrillar amyloid concentrations, but demonstrated no significant benefit to cognitive processes.5,6

These results should not be surprising because these drugs represent an older way of thinking about Alzheimer’s disease where plaques are the principal deleterious agent and their elimination is essential for recovery. Unfortunately there is ample evidence that soluble Abeta oligomers, and not their fibrillary associates, are the actually deleterious agents responsible for a significantly level of the symptomology of AD. If this different pathway is correct then the elimination of Abeta plaques should serve little benefit, as seen in the multiple Phase II and III trial failures, and could even been considered negative depending on how those plaques are broken apart (possibly increasing the available concentration of Abeta oligomers).

Now it is believed that Solanezumab can bind to soluble Abeta, which could explain why it performed better than Bapineuzumab, which binds to aggregate/fibrillar Abeta.6 However, the binding activity of Solanezumab was still insufficient to produce a meaningful benefit. Solanezumab supporters believe that if applied early, before symptoms, it may be able to produce a meaningful benefit; however, this belief may be misplaced. If this treatment has to begin that early to produce valid benefit then it will not help many individuals overall, assuming that it ever works for at the moment its “potential” is still theoretical.

There is an additional concern that simply changing the strategy from a fibrillary antibody to a soluble one may cause as many problems as it solves. Despite its fame as the chief element responsible for initiating AD, Abeta has innate roles in the brain that could cause problems if natural concentrations are significantly reduced as would occur in a preventative vaccination/treatment strategy. The two major natural roles for Abeta in the brain appear to be that of an indirect neuronal inhibitory agent and an anti-microbial agent.7-10 This anti-microbial activity may be why producing success from a direct antibody therapy is difficult for numerous previous attempts at active immunization against Abeta has resulted in numerous cases of aseptic meningoencephalitis.11

Regarding the issue of Abeta as an anti-microbial agent, there is a wealth of circumstantial evidence that seems to support such a conclusion and small amounts of direct evidence that demonstrate anti-microbial behavior against certain specific targets.7-9 For example one typical piece of evidence is that AD temporal lobe homogenates contain about 25% more activity against C. albicans on average over non-AD samples.7 However, while Abeta is thought to react against C. albicans it is also suggested that microglia are more active in AD patients than in non-AD patients, thus this increased activity maybe derived from the microglia instead of the Abeta. On a side note this anti-microbial behavior has lead some to conclude that AD can be induced by pathogenic response. Overall there could be two different methodologies behind how bacteria and other pathogenic agents could induce AD.

The first method involves observations that several bacteria contain amyloidogenic proteins. For example the periplasmic outer membrane lipoprotein of E. coli demonstrates a similar amino acid sequence to Abeta peptides and have similar visual structures similar to amyloid.12,13 In addition there appears to be pathological similarity between herpes simplex encephalitis (HSE) and AD.13-15 With this information one could come to the conclusion that either certain bacteria have the ability to mimic Abeta and facilitate extracellular receptor interactions similar to that of extracellular Abeta or the bacteria produce their own Abeta. Either scenario would enhance the probability of an individual developing AD in the presence of a specific infection.

The second method involves the overexpressed synthesis and release of Abeta produced in response to an infection. If Abeta does in fact have an anti-microbial effect then it would make logical sense from a biological standpoint that the body would release Abeta in response to an infectious agent, especially in the brain where immune response is limited. Cumulative infections or a single long duration infection could result in an increased probability to develop AD due to the excess production of Abeta to fight off the infection.

Not surprisingly there are some significant concerns about claims that infections play a significant role in the development and progression of AD. Simply from a general understanding it appears that too often researchers want to tie a particular pathogen to the cause of a given disease, “this bacteria/virus causes this particular type of cancer” is one of the most popular. The problem is that most of the time there is no direct evidence to support such a conclusion beyond the fact that the pathogen is present in individuals who have the condition. Another piece of reasoning that individuals who are pro-infection like to report is along the lines of “well, virus/bacteria x has a similar symptomology and/or pathology in the brain” as was previously mentioned above. The problem with that claim is that there are hundreds of diseases that are very similar to each other in many symptomatic respects, yet have small very meaningful differences in how they originate.

Whether or not infection plays a meaningful role in AD is still under debate, but is more unlikely than likely because early developing cases rarely demonstrate any large bacteria/viral concentrations and a significant number of late-developing cases also have a lack of any abnormal bacteria/viral concentrations relative to the age of the patient. The lack of large bacterial concentrations in the early stages of AD progression, and especially for late-developing cases, leads to one of two conclusions regarding any increase in bacteria concentration in AD patients. First, this increase occurs as a later development due to what could almost be viewed as a weakened immune system because microglia are busy trying to clear the excess Abeta. Second, the increase has little to do with AD and can be considered a coincidental occurrence. Neither of these possibilities involves bacteria as a causal agent.

Overall there is sufficient direct and indirect evidence to support the idea that Abeta has anti-microbial properties. An example of indirect evidence is the increased susceptibility to infection possessed by beta-secretase or gamma-secretase knockout mice.16 Also Abeta, in vitro, is clinically active against at least eight common microorganisms, similar activity to pleiotropic LL-37, a common ‘‘antimicrobial peptides.7

However, despite this biological role, there is currently no published evidence that demonstrates an increase in Abeta synthesis and secretion that later leads to the development of AD. Therefore, it is difficult to conclude that infections genuinely facilitate AD through additional Abeta synthesis. This highlights an interesting aspect of Abeta in that eliminating it or dramatically reducing it increases infection susceptibility, yet infections seem to be unable to produce sufficient concentration changes in AD development.

Regarding the role of Abeta as an “inhibitory” agent in neuronal processes one of the most telling pieces of evidence is the fact that benzodiazepines, an inhibitory agent, reduce secretion of Abeta peptides from hippocampal slice neurons.10,17 Also overexpression of amyloid peptide precursor (APP) significantly reduces excitatory activity.10 Other evidence suggests an “inhibitory” similar role through excitatory depression, but under normal physiological conditions this excitatory depression possesses a level of minute control resulting in enough of an impact to quell hyper-excitation, but not enough to cause short-term or long-term damage.

There is some research that suggests a connection between epilepsy and AD. A number of studies claim that increasing Abeta42 concentration in mice increases the probability for progressive epilepsy.18,19 One interesting component is whether the role of Abeta with regards to its influence on neuronal excitability changes as it changes states from monomeric to oligomeric to proto-fibrillar/fibrillar. While Abeta as a monomer/oligomer construct appears inhibitory, the ability of fibrillar Abeta to interfere with membrane fluidity allows it to influence neurons in an excitatory manner.

Another piece of information that supports a role for Abeta in neuronal firing is that calcium imaging shows hyperactive neurons clustering around amyloid plaques in the cortex of APP/PS1 mice.20 There are two common explanations for this result: 1. the neurons begin to hyperexcite, which prompts the release of Abeta peptides (40, 42, 43, etc.) in an attempt to inhibit further excitation. However, during the process of inhibition these peptides begin to coalesce into proto-fibrillars and plaques which then somehow rejuvenate hyper-excitation (most likely by changing membrane fluidity and resting potential) creating a small, but progressively positive feedback loop; 2. the neurons begin to release Abeta peptides in larger than normal concentrations, perhaps due to infection or genetic mutation, these Abeta peptides then form proto-fibrillars and plaques to facilitate a new found hyper-excitation.

One method to determine which of the above explanations is more logical is to distinguish between the influence of the proto-fibrillar Abeta and monomer/oligomer Abeta. Basically ask the question: do proto-fibrillars really induce additional excitatory behavior or are they simply blocking the ability of the smaller Abeta species (monomers, dimers and oligomers) to reduce excitatory activity?

For example it makes sense that an Abeta oligomer binding to an extracellular receptor induces a neuronal depressed response with the receptor eventually discarding the bound Abeta, but that unbound Abeta could still be available to bind another receptor if necessary until it is cleared. If proto-fibrillars act as a form of blockade it would be expected to limit the binding ability of oligomer Abeta reducing the ability to lessen excitatory behavior. However, the reduction of this damping ability does not explain why non-epileptic individuals would develop epilepsy, there must be an additional excitatory agent. Thus it stands to reason that the first answer is more favorable in that proto-fibrillar Abeta induces excitatory activity.

However, there are questions regarding the timing of epilepsy development because hippocampal neurons become hyperactive early in transgenic mice whereas hyperactivity in the cortex is temporally linked to plaque formation.20 One possibility to explain this apparent contradiction is that soluble Abeta has a higher probability of inhibiting inhibitory elements in the hippocampal space versus the cortex due to neuronal architecture. Regardless of this hyperexcitation issue, typically epilepsy in AD patients increases in probability with disease progression, which corresponds to a greater development and concentration of proto-fibrillar Abeta, especially relative to oligomeric Abeta. Therefore, it stands to reason that under normal conditions and early to moderate AD Abeta concentrations are chiefly inhibitory agents (regardless of what type of neurons they are inhibiting), but due to the differing activity Abeta slowly becomes more excitatory as the AD advances.

This method of inhibitory action extends not only to competitive binding between glutamate and Abeta on NMDA and AMPA receptors, but also induce endocytosis of AMPA receptors reducing excitatory binding probability.10,21-23 The endocytosis of AMPA receptors may explain why neurons that are hyperexcited with APP over-expression mutations are not immediately reversible, but over time become reversible with cessation of immediate neuronal firing.10 The lack of Abeta also induces GABAergic neuron sprouting, which may be driven to compensate for the hyperexcitation.24

In the end any future attempts to develop an antibody-based therapy for Abeta will have to determine how the presence of the antibody will influence the two natural Abeta processes. While there has been some initial and isolated success from studies that have demonstrated some protective benefits for auto-antibodies of a AB oligomer subset,25-26 it is difficult to “hand wave” away the negative results associated with previous antibody tests that resulted in cases of aseptic meningoencephalitis.

Recall that the production of Abeta begins with APP, which is a transmembrane protein that has three principal isoforms, 695, 751 and 770, each containing the 4 kDa Abeta peptide and is synthesized in the rough endoplasmic reticulum and glycosylated in the Golgi apparatus.10 Three types of secretase enzymes interact with APP. Endopeptidase alpha-secretase cleaves within the Abeta region, eliminating any opportunity to form an Abeta peptide. If APP is not cleaved by alpha-secretase then APP can be incorporated into endocytic compartments for cleavage by beta-secretase and/or gamma-secretase.

Beta-secretase cleaves APP at the N terminus of the Abeta peptide sequence and gamma-secretase cleaves at the C terminus. When beta-secretase cleaves APP it generates a secreted ectodomain beta-APP and a 10-kD COOH terminal fragment (beta-CTF).27,28 This beta-CTF fragment is the substrate for gamma-secretase, which cleaves the transmembrane domain of APP producing an Abeta fragment.28 Gamma-secretase can cleave at multiples sites creating multiple length Abeta peptides (typically 40, 42 and 43).27 However, if gamma-secretase cleaves APP before beta-secretase, the end product cannot be converted to Abeta. Therefore increasing alpha-secretase concentration/activity will decrease Abeta concentration, increasing beta-secretase concentration/activity will increase Abeta concentration, and increasing gamma-secretase may or may not (depending on other factors and just simple luck) increase in Abeta concentration.

While the functionality of the various secretases is straightforward, the development of a viable inhibitor is challenging for it faces three separate problems. First, secretases, especially beta, have multiple substrates and large substrate binding domains, so competitive inhibitors are typically short-lived and non-competitive inhibitors have difficulty achieving full inhibition. Second, inhibitor candidates must be able to cross the blood-brain barrier. Third, secretases are also involved in other important biological processes, thus inhibition must be conducted carefully otherwise numerous unfavorable side effects will accrue making long-term treatment difficult. For example beta-secretase is thought to be necessary for proper function of muscle spindles due to its interaction with Neuregulin-1, thus long-term beta-secretase inhibition may already be a non-starter.29

Another problem with secretase interaction is the relationship between gamma-secretase and intramembrane cleavage of Notch receptors, most notably Notch1.30 The Notch pathway in general is important for neuronal function, cell communication and cell homeostasis in developed brains. Therefore, due to this relationship while gamma-secretase inhibitors typically reduce Abeta concentrations in plasma and cerebral spinal fluid (CSF) they also produce side effects like haematological and gastrointestinal toxicity, skin rashes and changes in skin color.30 The failure of a Phase III trial of Semagacestat (a highly touted gamma-secretase inhibitor), after a successful Phase II trial, was due to the above mentioned side effects including the development of non-melanoma skin cancer as well as a dose-related worsening of cognitive measures.31

The above problems make it difficult to believe that a secretase inhibitor will be a long-term answer for AD treatment. There was a growing trend towards redirecting attention away from an inhibitor agent and towards a modulator agent. A modulator can shift the APP cleavage site maintaining the relationship with the Notch receptor, thus creating a possibility to reduce Abeta concentration while reducing side effects. Unfortunately one of the first modulators, Tarenflurbil, failed to produce any positive results regarding AD treatment;32 thus modulators may not be a strong choice for future AD therapies.

Also research has been invested in a naturally occurring monosaccharide, NIC5-15, that can function as a gamma-secretase inhibitor that somehow avoids interfering with the Notch relationship as well as increasing tissue sensitivity to insulin reducing insulin concentration.33,34 However, there is the lingering concern about how this inhibitor will influence the natural roles of Abeta in the body and its lack of any meaningful studies beyond a very simple Phase II trial.

One of the more interesting and potentially important elements in the progression of AD is the location of beta-secretase and gamma-secretase relative to each other and APP. A point of interest is what role lipid rafts play in dictating how APP is processed. Lipid rafts are lateral assemblies of cholesterol and sphingolipids which form ordered platforms that move through the matrix of a cellular membrane that can compartmentalize various membrane processes. Due to this compartmentalization they can produce microdomains that provide an efficient environment for molecule assembly and membrane protein trafficking as well as influencing membrane fluidity.

The involvement of lipid rafts in Abeta processing is supported by multiple stages of evidence. First there is reason to suspect that beta-secretase needs to be associated with lipid rafts to even be active let alone interact with its APP substrate.28 Whether or not gamma-secretase is also inactive when outside of a lipid raft is unclear, but it appears that significant activity takes place on lipid rafts.35-37 Bolstering support for the involvement of lipid rafts is the identification of various AD related proteins in lipid rafts from both human and mice brain. Currently Abeta40, Abeta42, presenilin 1, beta-secretase, APP, beta-CTF and alpha-CTF have all been isolated from lipid rafts.28,38,39

In addition beta and gamma-secretase induced cleavage seems to depend on endocytosis of APP. The requirement of endocytosis suggests that beta-secretase interaction does not occur at the cell surface. This requirement may be because surface APP and beta-secretase are either floating freely in the cellular matrix or on separate lipid rafts.28 Therefore, it seems to make more sense that the interaction between beta-secretase and APP occurs after endocytosis during the amalgamation of various lipid rafts within endosomes. Interestingly the appearance of larger endosomes is a typical precursor to AD progression, which could support this idea, i.e. the endosomes grow larger to accommodate the coalescence of the lipid rafts due to greater cholesterol and/or Abeta levels.

There is also evidence that significantly increased concentrations of Abeta begin to appear in lipid rafts before even symptoms begin, but these studies did not compare concentrations of Abeta in the lipid rafts to concentrations of Abeta in the intracellular or extracellular matrix, thus this result cannot be used as conclusive evidence that Abeta synthesis originates or is dependent on lipid rafts.40 Another important distinction is that some estimate slightly over 20% of brain Abeta on lipid rafts,40 with lipid rafts only constituting 0.4 to 0.8% of a given plasma membrane.41 While the estimate relative to lipid raft compartment space is for only one particular cell type, there is little reason to believe that the amount of lipid rafts varies significantly between different cell types.

For the moment assume the following regarding Abeta synthesis and lipid rafts:

- The overall size of a lipid raft is largely dictated by the total amount of elements available to form it;
– beta-secretase is only active on a lipid raft and produces beta-CTF;
- gamma-secretase is in close proximity to lipid rafts; whether or not it only active on a lipid raft is unknown;
- alpha-secretase is not localized on lipid rafts;

Based on the above information it makes sense that removing cholesterol from plasma membranes would significantly increase membrane fluidity (by shrinking the total number and size of lipid rafts). Increasing membrane fluidity would increase lateral movement of APP and alpha-secretase in the plasma membrane possibly increasing alpha-secretase activity. Also reducing the availability of lipid rafts should also limit beta-secretase activity making alpha-secretase interaction more likely, which has been supported experimentally.42

There is an interesting side point here in that recall fibrillar Abeta in the extracellular matrix is thought to change membrane fluidity with a higher probability for an increase in fluidity over a decrease. If this change in fluidity actually occurs then it could act as a negative feedback mechanism. After enough Abeta is secreted into the extracellular matrix that leads to the formation of fibrillar elements, like plaques, an increase in membrane fluidity should occur that would negatively influence lipid rafts reducing the probability for further Abeta synthesis until the fibrils are cleared from the extracellular matrix. If this is the case then plaque busting drugs could worsen AD in multiple ways, not only by breaking down fibrils and plaques making more toxic oligomers, but also eliminating a negative feedback mechanism which could limit Abeta synthesis.

There appears to be two different avenues when APP can associate with a lipid raft: 1) during transit between emergence from the Golgi body and becoming a transmembrane protein, newly synthesized APP could interact with lipid rafts; 2) during the endocytosis process where the APP is re-internalized through clathrin-coated pits.43,44 Without direct evidence it appears more reasonable to assume endocytotic recycling as the dominant lipid raft interaction process simply because it is more frequent of the two.

Further support for the importance of lipid rafts involves the behavior of its building blocks. Cleary numerous studies have demonstrated that increasing cholesterol leads to an increase in AD development probability in addition to lipid rafts, but cholesterol is not the only element that makes up lipid rafts. What happens if sphingomyelin levels are altered? The initial assumption would be that increasing sphingomyelin levels would lead to a corresponding increase in lipid rafts and Abeta synthesis. However, this does not appear to be the case in at least one study. When down-regulating sphingomyelinase (SMase) and up-regulating sphingomyelin-synthase activity, both actions increase available sphingomyelin, intracellular and extracellular Abeta levels decrease.45 The same result was acquired when foregoing enzyme manipulation and directly increasing sphingomyelin levels.

There are two immediate possibilities that could explain this result. First, the ratio between the total levels of cholesterol to sphingomyelin may influence the structure of the lipid raft where higher sphingomyelin levels create a raft formation that reduces beta and/or gamma-secretase activity. Second, maybe cholesterol is not directly responsible for changing Abeta concentrations, but instead an associated molecule that frequently increases and decreases in consort with cholesterol levels is actually influencing Abeta concentrations. While this second possibility is possible it must also address how different variations of ApoE dramatically change the probability of developing AD, which makes it unlikely.

Increasing sphingomyelin also appears to increase concentrations of C99, the byproduct of beta-secretase processing.39,45 Therefore, it can be reasoned that these changes in both C99 and Abeta concentrations are the result of a reduction in gamma-secretase activity more than likely due to a reduced ability to interact with C99 due to lipid raft proximity issues rather than direct reduced gamma-secretase synthesis or increased inhibition.

Unfortunately there could be another positive feedback effect relative to Abeta42 as Abeta42 directly increases SMase activity and reduces sphingomyelin-synthase activity.45 Such a result is interesting because does that mean SMase is also located on lipid rafts? There is no reason to immediately assume that this inhibition/activation will lead to a significant increase in Abeta concentration because if the change between the destruction/creation dynamic is altered too much in favor of destruction it will lead to the breakdown of lipid rafts halting Abeta synthesis. There could be a problem in that any loss of sphingomyelin in the rafts may be accommodated by an increase in cholesterol deposition. If in fact the lipid raft ratio between cholesterol and sphingomeylin does matter with respects to gamma-secretase activity and overall Abeta production then such an outcome could indeed worsen the progression of AD.

Another important element involving lipid rafts is that Abeta appears to inhibit sphingosine kinase-1, an enzyme that is chiefly responsible for balancing ceramide and sphingosine 1-phosphate (S1P).46 Ceramide significantly influences many stress signals, which can result in ceasing cellular growth or even cell death where S1P neutralizes the effects of ceramide.46 Thus Abeta, separate from other AD-related mechanisms, can induce cellular death by increasing ceramide concentrations and decreasing S1P concentrations.

There may also be a relationship between SMase and ceramide, which could also act as a positive feedback mechanism relative to the toxicity of Abeta.46 Finally IGF-1 is able to stimulate sphingosine kinase-1 activity neutralizing ceramide, thus this method may be how IGF-1 provides its neuroprotective effect relative to AD. Overall a strategy that focuses on influencing lipid raft configuration or their associated elements may be an interesting means to help neutralize AD because it avoids direct inhibition of the secretases and could allow for more fine control of Abeta concentration.

As mentioned above although there are still numerous concerns with implementing an Abeta antibody treatment strategy, another potential antibody strategy that has gained some favor in recent years is treatment with intravenous immune (or immuno) globulin (IVIG). In 2002 it was determined that the IVIG element Octagam had the tendency to possess antibodies for Abeta, which fostered the idea that IVIG could be used to treat AD.47 Early evidence from a small Phase II IVIG efficacy study showed improved cognition in mild to moderate AD patients and reduced Abeta in CSF.48,49

Unfortunately this initial promise was marred by the failure of IVIG to demonstrate any improvement in cognitive scores in a larger Phase III study and an additional Phase II study.50,51 The failure to reproduce the positive results from the Phase II study in the Phase III study limits the hope that IVIG could be a useful treatment in the future. Some argue that one bright spot is that IVIG did improve cognitive ability in ApoE4 carriers, otherwise commonly regarded as those who are genetically inclined to develop AD versus more spontaneous development.52 Note that from a safety standpoint both studies did support a positive safety profile for IVIG in AD patients.

Unfortunately even if the Phase III results were positive, one of the major obstacles to producing an effective IVIG based treatment is that lack of uniformity in the concentrations for different samples. According to FDA regulations IVIG samples must be prepared from the plasma contents of at least 1,000 individuals with all IgG subgroups (1-4) present, purified by removing all other blood elements and must be utilized or properly stored within 21 days of its creation. This process produces a non-uniform IVIG product that may have differing concentrations and types of antibodies versus another sample produced in a different laboratory. One sample may contain antibodies to Abeta and tau whereas a second sample may only contain antibodies for tau.53

Another drawback is that it takes approximately 9 months to produce an IVIG sample, thus mass production on a typical pharmaceutical scale is not possible creating a dearth in supply potential. Some have suggested alleviating this problem through new manufacturing processes or use of recombinant strategies, but neither of these suggestions have been incorporated into a large-scale production line, thus limiting their predictive power.54 Also unless more people donate blood in general increasing production with a natural product base will be very difficult.

This supply crunch creates various problems both ethically and economically. Not surprisingly IVIG treatment is expensive, costing about $75 per patent gram or $7,500 - $15,000 for the average person; with a fixed price for reimbursement from Medicare it stands to reason that a number of low-income individuals could be priced out of long-term IVIG therapy55 (new infusions would be expected between every two to five weeks), which is standard protocol and would be applicable for AD. IVIG is also used to treat acute infections and some other conditions most notably immune deficiencies and autoimmune diseases. With the limited supply availability transferring IVIG samples to AD patients would hurt these other patients, that is of course if IVIG was an effective treatment for AD, which has not been sufficiently or appropriately demonstrated.

Finally IVIG treatment is not without its own side effects most notably increased probability for thromboemboli due to increased serum viscosity reducing blood flow, especially in individuals with existing vascular difficulties and/or abnormalities.56,57 Another concern is the increased probability for a decrease in white blood cell, red blood cell and platelet concentrations including increased platelet aggregation, which could also exacerbate thromboemboli potential.58-60 A decrease in hematocrit levels is thought to occur through high-molecular weight IgG complexes binding red blood cells increasing sequestration.61 Despite these drawbacks one positive produced from the IVIG clinical studies is the idea that a multi-antibody therapy should be superior to a monoclonal antibody therapy. However, one of the new problems with such a strategy is identifying the antibodies, outside of Abeta, that should be included in such a therapy.

If IVIG is not a direct treatment option, an indirect treatment option could be gleamed from how IVIG affects the inflammatory response where IVIG inhibits complement activation, modulates chemokine expression and regulatory T cell subsets, and negatively influences inflammatory cytokines.52,62,63 One of the more notable results is a decrease in the ratios of IL-5 and IL-12 relative to IL-10 (i.e. either IL-5/IL-12 concentrations decreased or IL-10 concentrations increased), which is thought to decrease the rate of atopy.64,65

Another important element influencing the inflammation response of IVIG is the role of IgG Fc fragments, which involve a glycan component with a terminal sialic acid.66,67 IgG fragments with sialic acid bind to human receptor dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) or its murine orthologue (SIGN-R1).68 These binding targets are thought to stimulate immunosuppressive action reducing inflammation, which could reduce AD severity. However, this anti-inflammatory behavior requires a high dose, if IVIG is the source provider, because only a very small percentage of IVIG contains elements with sialic acid. This high dose may be also be prohibitory for treatment due to side effects.69

Another theory behind the effectiveness of IVIG is how it interacts with a more exotic version of Abeta. For example some evidence has demonstrated a significant decrease in soluble Abeta56 oligomer concentration after IVIG treatment. Similar to Abeta42, Abeta56 is another abnormal Abeta isoform that is thought to increase the probability of AD development and influences cognitive impairment on a concentration dependent level.68 One reason for this influence is that Abeta56 increases the expression rate of tau and its effect is negatively influenced by drebrin and fyn kinase availability available in IVIG treatments.65

A more controversial issue with IVIG treatment is whether or not any positive influence is drawn from its facilitated decrease in CD4/CD8 ratio. Various research has produced results were AD patients have increased,71-72 no significant changes73 or decreased74 CD4/CD8 ratios. Overall it is difficult to conclude, either through IVIG treatment or in general, whether influencing CD4/CD8 ratios is an intelligent therapy strategy for treating AD. In addition to CD4/CD8 ratios, IVIG also appears to decrease the concentration of YKL-40, but outside of being a marker for advanced AD there is little belief that manipulating its concentrations could prove useful as a therapeutic.75

With the failures of Abeta antibiotic therapies and the difficulties associated with IVIG confirmation and production some researchers have turned their attentions to attacking tau as a treatment methodology. One of the major reasons tau looks promising is that its pathology appears to correlate better with dementia severity than Abeta. Based on some research tau supporters argue that tau is actually responsible for Abeta toxicity.4 However, there is a significant problem with this enthusiasm namely that while misfolded and hyperphosphorylated tau does lead to generic dementia, it fails to develop into AD without the influence of Abeta.76,77 Also the argument that Abeta toxicity is dependent on tau only appears applicable to fibrial Abeta not soluble Abeta, which is of little consequence because fibrial Abeta has low direct toxicity overall.78 These issues have created conflict between Abeta and tau proponents regarding which element is worth neutralizing.

Regardless of its lack of AD initiation tau could be an important theoretical therapeutic target. A quick reminder that tau is a microtubule-associated protein (MAP), which is important for the proper stability and functioning of microtubules. The general understanding behind tau toxicity follows a similar pattern to that of AD. Hyperphosphorylation of tau negatively influences its affinity for microtubules increasing microtubule structural degradation and increases the probability that monomer tau form oligomers, paired helical filaments (PHF) and neuron fibrial tangles (NFTs). The breakdown of microtubules reduces in axonal transport leading to synaptic starvation and retrograde degeneration.

However, originally most believed that the neurotoxicity of tau was born from NFTs whereas more and more recent evidence supports the tau oligomers being responsible for a majority of damage.79-81 Some may suggest that this more severe oligomer toxicity does not correlate with increased NFT load and distribution markers for AD progression. This result is not contradictory because more tau oligomers equals more damage, but also increases the probability for more NFTs.

One of the interesting elements of tau is the idea of a positive feedback mechanism that makes it self-propagating, similar to a prion.82,83 If such a methodology is correct, then AD treatment would require one of two strategies: 1) treat AD before this self-propagating positive feedback mechanism is activated by Abeta; 2) the tau mechanism must be neutralized to a point that disallows the occurrence of this self-propagating mechanism. Otherwise tau is not addressed, the treatment may neutralize AD, but the continued expression of tau could lead to another form of dementia.

There is two major schools of thought with regards to neutralizing the effects of tau: 1) influence tau phosphorylation; 2) influence tau aggregation. The first option typically involves using elements that will inhibit phosphorylation of tau whereas the second option typically involves using elements that will either prevent tau aggregation or enhance aggregate disassembly.

Not surprisingly the first option has been explored on a greater level than the second owing to the idea that hyperphosphorylation stems from an abnormal ratio of activation between glycogen-synthase-kinase-3 (GSK3), which is responsible for phosphorylation, and phosphatase PP2A, which is responsible for removing phosphates from tau.84 There was some early promise seen for influencing tau through the inhibition of GSK3 by way of either lithium or valproate, two treatments that are commonly used in psychiatric disorders with relatively stable safety histories and protocols. In addition both are thought to enhance neuroprotective effects by upregulating anti-apoptotic factor BCL2.85

Unfortunately despite these positive effects, small studies involving lithium treatment in patients with mild Alzheimer’s disease demonstrated no change in CSF biomarkers or any cognitive benefit.86 Granted some explanations for this result could be the study’s short time frame (6 weeks) and the mild condition of Alzheimer’s could limit the overall effectiveness of a tau-based therapy because the detrimental nature of hyperphosphorylation has yet to fully occur. Studies with valproate have generated similar results with no positive effects on cognitive or functional status.85,87

Another natural compound that has been targeted as a potential tau therapy is nicotinamide, the biologically active form of niacin (Vitamin B3) and precursor of coenzyme NAD+. Studies in mice have demonstrated that orally administered nicotinamide limits cognitive deficits and reduces concentrations of phosphorylated tau.30,88 There is also some evidence that nicotinamide upregulates acetyl-alpha-tubulin, protein p25 and MAP2c, which are all thought to increase microtubule stabilization, thus increasing the probability of neuron survival.88

There is limited understanding regarding the biological effects of nicotinamide in AD methodology; however, nicotinamide appears to exert two effects relative to tau and microtubule stabilization. First, it upregulates p25 and dowregulates p35, which is thought to increase microtubule stabilization.88 Second, it inhibits SIRT2, which functions as an alpha-tubulin deacetylase.88 While the exact method is still unclear, increasing acetylated alpha-tubulin levels, along with alpha-synuclein activity, increases microtubule stabilization and reduces cognitive degradation, possibly through aggregation stimulation among microtubules.88

The principal method in which Abeta influences the progression of tau phosphorylation leading into hyperphosphorylation and possible altered tau conformations is increasing activation of GSK3, which is activated downstream of NMDA-receptor signaling.89 Other more minor signaling pathways that are also involved are CAMKK2-AMPK kinase and C-Jun N-terminal kinase.90,91 Obviously multiple pathway activation eliminates the ability to fully neutralize Abeta activation of tau with a single molecule, a result that explains in part why a treatment like Mematine is not as effective as it should be theoretically.

In general the progression of tau to a hyperphosphorylated deleterious agent occurs in consistent manner where concentrations of tau dramatically increase in the transentorhinal cortex eventually producing sufficient quantities of NFTs, then concentrations increase in the hippocampal CA1 (II-IV) later advancing into the temporal (V) and isocortical areas (VI).92-94 While this progression can occur through normal aging it is dramatically accelerated in the presence of Abeta despite a lack of direct proximity/compartment relationship, i.e. brain regions low AB concentrations with no plaques can still see accelerated tau phosphorylation due to only a neuronal connection with a concentration heavy region.95,96

The exact method in which Abeta induces greater tau phosphorylation through the above enzymes is unclear, but the three major options are: 1) direct interaction from specific binding of monomeric and oligomeric Abeta to a variety of neuronal receptors; 2) indirect action involving induced inflammation via glial and microglial cells; 3) cross-seeding between Abeta and tau dramatically increasing misfolding and hyperphosphorylation probabilities for future tau proteins.84

Overall the chief problem with attempting to utilize a treatment for tau as a principal therapy is that it is a downstream actor. While tau may produce a meaningful amount of neuronal damage in advanced versions of AD, it is not the only damage producing agent and any treatment would be a chronic one for it would not influence Abeta concentration, the chief upstream effector of tau toxicity. This reality should not eliminate the idea for a tau based aspect to an AD treatment, but should end the idea that only neutralizing tau would be enough.

The importance of ApoE is clear in the development and progression of AD. Therefore, there has been significant study regarding its transcription including the important elements of activation and heterodimerization of the nuclear receptor retinoid X receptor (RXR) along with peroxisomes activated receptors (PPAR) or liver X receptors (LXR).97 In addition to facilitating ApoE transcription these elements also activate lipidators which are thought necessary for the proper functionality of ApoE.98,99 Thus, it seems reasonable that increasing RXR agonists should increase ApoE transcription and possibly even ApoE efficiency. This was the thought process that lead to the utilization of Bexarotene, an older cancer drug, as a possible new therapy.

Early in its testing a result produced by Bexarotene appeared very promising as it upregulated ApoE and other lipidators like ABCA1 in transgenic mouse models of Abeta amyloidosis.100 Furthermore after this upregulation there was rapid reduction in Abeta plaques and increased cognitive abilities.100 However, this success was short-lived for a number of other groups have failed to replicate this plaque reduction and improved cognition result despite also replicating the increased upregulation of ApoE and ABCA1.97,101,102

This lack of replication is troubling because various testing has demonstrated that Bexatrotene is able to active its RXR and lipidator targets effectively, but despite this action it appears that this increased upregulation is not able to consistently and/or effectively remove Abeta. Part of the problem is that there is no single formulation for Bexatrotene, thus the one used in the original research demonstrating a positive Abeta removal result may be significantly different from the formulations used by later work attempting replication.97 Unfortunately it appears that the researchers in the original work have yet to release their Bexatrotene formulation eliminating the ability to address this potential discrepancy.

Another possibility to explain the differences may be the interaction between Bexarotene and the blood brain barrier. Studies identified enhanced Abeta peptide clearance at the blood brain barrier moving peptides from the brain into the blood through an ApoE and LRP1-mediated process.103,104 What this result means is still unclear because of the overwhelming failure to replicate the original results. An additional concern created by this discrepancy is that Bexarotene has some negative side effects like weight loss, hyslipidemia, hypersensitivity, hypothyroidism and leukopenia.105 Therefore, as it stands Bexatrotene or other agents influencing RXR do not appear to be viable treatment agents.

As mentioned numerous times the interaction with cholesterol and AD is important, especially with regards to ApoE, thus some believed that statins could provide an effective means to manage or even treat AD. Despite evidence in animal models supporting the neuroprotection and improved pathology for statins, these benefits have not consistently or effectively transferred to human trials.106 Thus, the question of whether or not statins are an effective therapy option for AD patients is a controversial issue.

If one ignores the results from animal models and humans the initial premise seems plausible in that statins reduce available cellular cholesterol concentrations, which based on the relationship between cholesterol and ApoE, or even cholesterol and lipid rafts, should reduce Ab levels. Lower Ab levels should reduce, if not outright cease, the progression of AD, if provided at an early enough stage. While the initial premise seems to flow logically there are questions to whether or not a high enough concentration of statins enters the brain. In addition cholesterol accumulation and behavior function differently between the brain and the rest of the body due to the blood brain barrier.

In the brain cholesterol is produced almost exclusively from de novo synthesis instead of relying on a combination of de novo synthesis and lipoprotein uptake through LDL, HDL, etc. Without this particular lipoprotein cholesterol relationship the efflux of cholesterol from the brain utilizes 24-S-hydroxycholesterol.106 Not surprisingly patients with early-onset AD have elevated concentrations of 24-S-hydroxycholesterol, which suggests a higher intracellular cholesterol level.106

Suppose that statins are unable to pass through the blood brain barrier at high enough concentrations to significantly influence cholesterol levels in the brain, how can one explain the results that statins do provide some effect, especially in non-AD individuals? If one is to believe that there is an effect, then one possible explanation is that by reducing the cholesterol level in the body, the natural synthesis of Ab outside of the brain is reduced. Thus in individuals with ApoE4, which can bind Ab and transport it across the blood brain barrier, less Ab will be available for transport potentially reducing the amount of Ab inside the brain.107

However, the same probably cannot be said for those with ApoE2 or E3 as there does not appear to be significant Ab transport into the brain from these versions of ApoE. Therefore, if this assessment is accurate then statins could provide a small therapeutic effect for individuals with AD and the ApoE4 isoform, but would be relatively useless for individuals with AD and the ApoE2 or E3 isoform. This theory could also explain why animal models typically demonstrate positive results because a number of models stimulate AD development with ApoE4 mutations.

In the brain ApoE also uses its lipid transport function to aid in the repair of neuronal cells. This attribute was hypothesized when experimenters identified a rapid and dramatic increase (200 fold) in APOE concentration after neuron injury followed by a return to normal levels after sufficient time for repair had pasted.108 The reason behind this dramatic increase is that under normal conditions almost all of the ApoE in the brain is produced by astrocytes, but under states of stress neurons start rapid synthesis of ApoE and more ApoE can be produced by active microglia and even neurons.109

The ability to aid in neuronal repair from most helpful to least helpful among the various ApoE isoforms is E2 > E3 > E4.106 Interestingly it appears that ApoE4 has a negative effect on neuronal repair where ApoD has to fill the repair facilitation role.110 ApoE4 is also thought to play a role in the general mental decline that occurs with normal aging. The principle element of misrepair seems to stem from an inability to support neurite outgrowth, which leads to loss of synapto-dendritic communication in certain parts of the brain.109

With the general failures of existing therapy strategies, some unconventional therapies have been explored like Latrepirdine. Latrepirdine was first introduced in Russia as a non-selective anti-histamine.111 Its mechanisms of action involve the weak inhibition of acetylcholinesterase and butyrylcholinesterase along with inhibition of NMDA receptors and voltage-gated calcium channels.112 Interestingly it also has a secondary mitochondrial protective effect preserving structure and function, especially under stressful conditions. This protection is thought to occur through inhibition of the mitochondrial permeability transition pore, which can be activated by Abeta.113 Based on the reasonable success of Memantine along with the ability of other anti-histamine drugs to demonstrate some positive benefits in treating neurodegenerative disorders, some believed that Latrepirdine could be a boon in AD treatment.

An initial Phase II study demonstrated safe tolerance and a statistically significant improvement in cognitive function and psychiatric symptoms, including an anti-depressive effect, for patients with mild or moderate AD.114 Unfortunately like so many other treatments before it Latrepirdine failed to carry these improvements over in Phase III studies.115,116 Some claim that the failure was in the design of the Phase III protocols not in Latrepirdine. However, looking at the result the benefits seen in the Phase II study were amplified by the placebo group worsening versus having no significant loss of cognitive function in the Phase III study. Therefore, the significance of the Phase II success may have been derived from mischaracterization of how severe the AD cases were in the placebo group versus the actual efficacy of Latrepirdine. Despite this drawback there is still hope that Latrepirdine could be a useful therapeutic agent in the future.

Another somewhat less conventional therapy strategy was the utilization of thiazolidinediones. The two most notable thiazolidinediones available for treatment are rosiglitazone and pioglitazone, which were originally developed to treat type 2 diabetes. Both function by stimulating nuclear peroxisomes proliferator-actived receptor gamma (PPARg) which reduces the expression probability of beta-secretase and APP as well as increasing the probability of APP degradation through ubiquitination.117

In addition to the direct action against APP, the loose connection between insulin action and AD lead some to believe that both of these agents could be used to increase insulin sensitivity reducing insulin concentration. Interestingly enough there is some similarities in the degradation of insulin and Abeta, thus leading to the belief that reducing the concentration of insulin would eliminate an “indirect” inhibition effect on Abeta degradation enzymes.117 Unfortunately neither of these agents have demonstrated positive clinical trial results with rosiglitazone reporting no improvement in cognition or global function and has been further derailed by new cardiac risks from the FDA.118

While developing quality therapies for AD is the principal goal, the extent of damage that is produced during the progression of AD makes the timing of therapy application critical. Therefore, it is important to develop diagnostic methodologies that can detect AD development at early enough levels so a therapy can be utilized to ensure no significant change in quality of life rather than simply hoping for some quality of life. The most reliable non-genetic means to determine if an individual is at an increased risk for developing AD is to observe Abeta42 or tau (both total and phosphorylated concentrations) in CSF. CSF is utilized because despite having a lower protein content versus serum, CSF directly interacts with the extracellular space in the brain, thus it produces an accurate assessment regarding the biological contents of the brain.

As previously mentioned the generally accepted neuropathology of AD occurs decades before the expression of symptoms leading to three main phases of AD development: 1) pre-symptomatic (where most of the damage is conducted); 2) prodromal (mild symptoms mostly focused around episodic memory failures); 3) large-scale memory issues and similar symptomatic features common with dementia;119 Interestingly enough among these three phases CSF derived Abeta42 and tau concentrations only appear to significantly change during the pre-symptomatic stage, i.e. there are only minimal changes during the prodromal and dementia stages.120,121 In addition outside of very specific genetic conditions, Abeta42 concentrations change (increasing then decreasing) before significant changes are seen in tau concentrations.122

The decrease in Abeta42 concentration is thought to occur due to oligomeric concentrations being removed from circulation when they become incorporated into plaques. However, if this behavior is accurate then Abeta42 production must decrease at a greater level than plaque formation during the advancement of the condition; this result would speak to a negative feedback associated between plaque formation and Abeta synthesis similar to the one discussed earlier.

Unfortunately the characteristics of these changes make meaningful detection difficult. The general accuracy of current diagnostic methods is actually rather low with sensitivities ranging from 71% to 88% and specificities ranging from 44% to 71%.123 In general diagnostic biomarkers should produce a sensitivity and specificity of at least 85% to be medically useful.124 Another problem is that inter-assay and inter-laboratory variability produces additional inaccuracies ranging from 20% to 35%.125,126 Note that a sensitivity of 100% indicates a 100% identification of subjects with AD where a specificity of 100% indicates a 100% accuracy in distinguishing between AD patients and non-AD patients.

Overall these inaccuracies creates problems in clinical drug testing as 10% to 35% of individuals clinically diagnosed with AD have negative amyloid PET scans, which calls into question whether or not these individuals actually have AD.127 Some have assumed that if these diagnostic tests are accurate then there should be serious consideration to divide AD diagnosis into two sub-categories: “amyloid-first” and “neurodegeneration-first”.5 Another problem with using Abeta and tau as biomarkers is differentiating between simple old age and AD as old age appears to follow a similar pattern.128

A newer direct method to produce information regarding early progression of AD is amyloid imaging. Researchers at the University of Pittsburgh were the first to produce a reliable imaging strategy by modifying the structure of thioflavine T to include 11C has a positron emitter.5 This altered thioflavine T could cross the blood brain barrier and selectively bind to Abeta. Various other imaging methodologies have been commercially developed, but these strategies forego the use of 11C in favor of 18F because the short half-life of 11C demands PET imaging with immediate access to a cyclotron for accurate measurements versus PET imaging alone.5 The first commercial compound to receive FDA approval was Florbetapir, but the Center for Medicaid and Medicare Services have yet to approve its coverage.5 However, that may have changed recently due to the passage of the Affordable Care Act.

One of the chief concerns about using direct Abeta imaging for diagnosis or even detection purposes is that it tends to prefer fibrils instead of oligomers. However, fibrils tend to form after oligomers and there is ample evidence to suggest that oligomers play an important role in the development and progression of AD. Therefore, not only could imaging fail to properly capture the full extent of Abeta expression, but also will lag behind identifying the actual progression of AD.

Another concern for this lag is that neutralizing Abeta strategies will have to proceed before any other negative methodology, like tau progression, accelerates otherwise treatment becomes much more difficult. For example model mouse studies demonstrated that vaccination prior to plaque initiation prevented all amyloidosis versus vaccination after initiation only eliminating about 50%.129 Another important element in diagnosis that has emerged in recent years is that approximately 1/3 of patients with clinical AD do not produce Abeta plaques in the brain, which would make differentiating between AD and non-AD (normal aging) more difficult for this method.

Another strategy to rectify the time delay for an individual between AD development and displaying symptoms of AD is to identify biological and genetic biomarkers that demonstrate significantly increased probability of AD development or currently active AD development.
Unfortunately most attempts to identify genetic biomarkers involve genome-wise association studies, which can produce erroneous results or produce more broad results with little known probabilities. For example one of the more important identified genes is CLU, which encodes clusterin (a.k.a. apolipoprotein J (ApoJ)).130,131 Clusterin is important because it is involved in Abeta clearance, inhibition and neuronal apoptosis and while it is expressed in numerous tissues throughout the body, expression is higher than average in the brain.132-134

This higher than average brain expression, especially in patients with AD, has raised hopes that clusterin could be used as an early identification biomarker for AD.135,136 Unfortunately this hope has not faired well against empirical evidence where higher levels of brain clusterin have not consistently preceded AD development.137,138 In fact as previously mentioned in the blog post linked to above, biomarker analyses in general, including meta-analyses, are plagued by larger, typically bias induced, effect estimates.

However, research has shown an increase in clusterin concentration in association with depression.138 This could explain some of the contradicting results between clusterin concentration and AD for some individuals with AD get depressed, for obvious reasons, and some do not. One of the disappointments with the failure to confirm plasma based clusterin as a biomarker for AD is the plasma aspect for the ease and efficiency of plasma testing is superior to collecting CSF.

Research on disease modifying drugs for AD has covered a lot of ground in recent years, but unfortunately unlike the existing symptomatic treatments there was yet to be a significant success. Even more troubling is that available results from the multitude of Phase III studies on disease modifying drugs suggest that a quality drug is not forthcoming. One strategy to improving the probability of developing a critical treatment is to ensure proper coordination between Phase II and Phase III studies as it is sometimes difficult to reconcile a glowing success in a Phase II study with a significant failure in the corresponding Phase III study.

Another issue that was previously discussed on this blog is studying the importance of multi-drug therapies in clinical studies. While individuals like to think of AD as an Abeta disease that later involves tau, there are numerous pathways involved in the development and progression of AD that can inflict significant cellular damage and produce neurological degeneration. Attacking and neutralizing Abeta is clearly the optimal solution, but current research implies that unless this neutralization is achieved very early in the disease progression, long before the development of symptoms, then it may not be an effective target. Therefore, it stands to reason that AD will commonly involve attacking multiple neurodegenerative pathways. However, there are almost no clinical trials involving treating AD patients with multiple drugs at the same time, clinical trials continue to be conducted with only one drug versus a placebo.

Some may conclude that this multi-drug therapy could be better consolidated into a single drug that attacks multiple targets (multi-target directed ligand design), which would make treatment less complicated from the patient’s perspective. However, such designs are more complicated from a regulatory standpoint and a biological one as the combined effects of a single drug may prove less potent in triggering each pathway versus two separate drugs, one for each pathway.

Despite significant levels of effort and research the immediate future for developing an effective treatment for AD does not seem promising. Some claim that AD research is woefully under-funded given the potential havoc that AD could bring against the healthcare system in the near future. However, the cry for more funding does not appear to be a valid response to the setbacks currently experienced in the AD research community. It may be that the focus of research must change from attempting to find a single compound that will address AD to creating a multi-drug treatment regimen and this multi-drug treatment may require investigating more indirect compounds.

For example flotillin 1 knockout mice express less Abeta and less amyloid plaques, but in levels that are not sufficient for treatment.139 However, if flotillin 1 inhibitors were paired with another Abeta therapy a therapeutic level result could be produced. Also the important of lipid rafts are somewhat acknowledged in the research community, but their important component and interactive elements are typically not investigated for future therapeutic effect. There are high hopes that adherence to a Mediterranean diet will reduce the probability of developing AD, but there have been mixed results regarding whether or not the diet provides a significant protective effect.140-143 Overall while more funding would be nice, a change in perspective regarding how to treat AD may be the most important step to producing an effective treatment.

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