31. August 2008 · Comments Off on Structure-function analysis of neuroprotectants · Categories: Cryonics, Neuroscience, Science · Tags: , , , ,

In “The chemistry of neuroprotection”, the author argues convincingly that there could be great benefit from a systematic and rigorously scientific study of the physical chemistry of putative neuroprotectants vis-à-vis their pharmacological effect. However, the first example used of the earliest thinking in this direction (which comes, not surprisingly via V. A. Negovskii, the father of resuscitation (1) medicine) is instructive as to some of the potential barriers standing in the way of this approach.

“It is not surprising that all the agents which are effective in shock carry a negative charge. This applies both to heparin, which possesses a very strong negative charge, and to hypertonic glucose solution. The same may be said about a substance now in wide use – dextran – which has small, negatively charged molecules, and also about the glucocorticoids 21, 17, and 11, which also have a negative charge.” – Professor Laborit in: Acute problems in resuscitation and hypothermia; proceedings of a symposium on the application of deep hypothermia in terminal states, September 15-19, 1964. Edited by V. A. Negovskii.

In the intervening decades since Laborit wrote the words quoted above, supraphysiologic (high) steroids have not only failed to demonstrate benefit in cerebral resuscitation and shock, they have been found to be actively harmful in every well designed RCT undertaken to test their utility (a). This also extends to their lack of utility in trauma, spinal cord injury and sepsis. Similarly, the utility of heparin in treating the encephalopathy of the post-resuscitation syndrome, or improving survival after cardiac arrest has recently been called into question. Glucose, hypertonic or otherwise, was long ago demonstrated to markedly increase neurological injury if given immediately after reperfusion following cardiac arrest, and elevated blood levels of glucose, both pre- and post cardiac arrest have a strong negative correlation with both survival and neurological outcome.

Determining the seriously harmful effects of steroid administration in critical illness took decades. Despite the compelling evidence for their injurious effects, administration of large, supraphysiologic doses of steroids is still a practice both used and defended by some clinicians (albeit not ones who rely on evidence based criteria) and the use of glucose in shock, trauma and cardiac arrest took a nearly comparable period of time to discredit. These two examples are noteworthy because they comprised mainstays of therapy for most kinds of neuroinjury for decades, and they had compelling theoretical appeal, as well as many positive small clinical and animal research studies. Indeed, the debate continues to this day with controversy centred mostly on the use of low or “physiological replacement” doses of steroids in critical illness. As the eminent pulmonologist and intensivist Neil Macintyre observed in 2005, “Patients die, but steroids never do.”  This raises the twin problems of bad research (i.e., junk science) and statistically under powered or otherwise flawed studies. Combined, it has been estimated that these two types of defective studies comprise the bulk of published peer-reviewed scientific work.

High dose corticosteroid therapy for neuroinjury offers another complication in determining the therapeutic efficacy of any drug that merits consideration as a neuroprotectant (new or old). While there is no doubt that high-dose corticosteroids are ineffective and deleterious in the clinical setting, there is also little doubt that these agents are neuroprotective in the laboratory setting under certain conditions and for discrete subpopulations of neurons. The reasons for the failure of translational research in the case of corticosteroids are complex, but are mostly attributable to crucial differences between the laboratory and the real world of clinical medicine. In the case of corticosteroids these differences are most significantly:

a.    Delay from time of insult to time of treatment; in the laboratory the timing of interventions is uniform and is typically much shorter than is the case in the clinic where delays in both presentation and treatment are both long and highly variable.
b.    Heterogeneity of injury in humans compared to animals; animal models of neuroinjury are highly standardized (location, extent, mechanics) whereas human patients present with diverse injuries inflicted in many complex and often poorly understood ways.
c.    Species differences; not only are there large genetic differences between humans and rodents in general, there are dramatic differences in the native ability of rodents to both resist and overcome infection in comparison to humans.
d.    Demographics and comorbidities: laboratory animals are comparatively very uniform genetically, are typically young and healthy and of the same age, do not have comorbid conditions such as hypertension, diabetes, atherosclerosis, obesity or the diminished physiological capacity and repair and regenerative capacity increasingly present in humans over the age of 25.
e.    Rodents aren’t people and do not interact with investigators in ways that facilitate straightforward determination of an adverse affect such loss of short term memory, or other cognitive deficits. It is now understood that the corticosteroids are toxic to the neurons of the hippocampus in both rodents and men. However, injury from this adverse effect is not only more evident in men than in mice (or rats for that matter), it is only men who are capable of complaining about it.

It is notable that all of these effects, with the possible exception of increased resistance to steroid-induced immunosuppression-mediated infection, obtain in the case of other translational models of drug development. The conclusion that corticosteroids are very likely neuroprotective in humans (in terms of the direct pharmacological effect on selected subpopulations of neurons in injured central nervous tissues under ideal conditions) is highly likely. However, the confounding realities of the clinic and the genetic differences between men and rodents (the animals almost exclusively used in this type of research) mask this effect. This poses yet another serious challenge to investigators seeking to establish common moieties in prospective neuroprotective molecules.

Clinical trials of putative neuroprotective substances have been overwhelmingly negative. This has been the outcome despite often stellar results achieved in animal models; often in diverse species in studies conducted by multiple investigators in different institutions and sometimes in different countries; none of whom have any obvious relationship, let alone one that might raise the specter of conflict of interest. In the last 6 years alone, over 1000 experimental papers and over 400 clinical articles have appeared on this subject. What this suggests is that the same deficiencies seen in studies reported upon in rest of the peer-reviewed biomedical literature also apply to studies of pharmacological intervention in neuroprotection. An inevitable conclusion is that until the signal to noise ratio improves, attempts to draw general conclusions about  the shared, essential properties of neuroprotective molecules will be difficult at best, and unreliable or misleading at worst.

Perhaps a good place to start this kind of analysis is in an area where the molecular structure of the agent(s) is extraordinarily simple and the animal and clinical data are both robust and show good to fair agreement. Hypertonic sodium chloride solutions have demonstrated efficacy in providing both systemic (splanchnic) and cerebral protection in a broad class insults including hemorrhagic/hypovolemic shock, closed head injury and less robustly in stroke and global cerebral ischemia. Interestingly, other cation salts of chloride given at comparably high tonicity do not have this effect. Furthermore, animal as well as small human clinical studies have demonstrated isochloremic hypertonic solutions to be as effective as hypertonic sodium chloride at restoring microcirculatory flow and reversing metabolic acidosis in haemorrhagic shock without the potentially troublesome side-effect of raising the mean arterial pressure to levels where re-bleeding may occur in trauma or subarachnoid haemorrhage.  A relative lack of effectiveness of the chloride salt of magnesium compared to the sulfate salt of this ion has also been noted. Understanding the mechanics of these paradoxes would seem to be a worthwhile and comparatively straightforward place to begin such structure-activity relationship analyses.

17β-Estradiol

Cerebroprotective drugs not infrequently possess a multiplicity of pharmacological effects that are known to be neuroprotective but that may be accomplished by very different and even indirect means in terms of their structure-function relationship. Some cerebroprotective molecules, such as the female hormone 17β-estradiol and the mixed estrogen antagonist-agonist tamoxifen share common physiochemical properties such as free radical scavenging, N-methyl-d-aspartate (NMDA) receptor inhibition, and modulation of volume regulated anion channels (VRAC); which play a role in ischemia-induced release of excitatory amino acids. There is considerable evidence that some of 17β-estradiol’s neuroprotective effect is via signal transduction as well as its neurotrophic effects, even at doses below those necessary for its direct effects on reactive oxygen species production and its NMDA receptor inhibiting effects. While the structure of the molecules shares some important features, they are also structurally very different and the signal transduction and neurohormonal effects are almost certainly very different. Thus, these molecules also present a fascinating opportunity to probe structure-function relationships in neuropharmacology.

Tamoxifen

Finally, an admission, or perhaps a confession is order in ending this discussion. This author has been responsible for the application of at least one putative neuroprotective drug to cryopatients which ultimately proved ineffective in human clinical trials when administered during and after cardiopulmonary resuscitation (CPR). This drug, nimodipine, performed well in animal trials, but failed to show benefit in human trials, possibly as a result of its hypotension-inducing effect. Adequate mean arterial pressure (MAP) following resuscitation from cardiac arrest is essential to survival and a post arrest bout of hypertension has been demonstrated to provide substantial cerebral rescue in animal models of global cerebral ischemia. Reduction of MAP in cryopatients is a serious concern because achieving adequate perfusion pressure is problematic under the best of conditions. It is also worth noting that cryopatients have been given a variety of other ineffective neuroprotective drugs over the past 30 years, including the opiate agonist naloxone, the corticosteroid methylprednisolone and the iron chelating drug desferroxamine.

While these drugs, with the possible exception of nimodipine, are not likely to have been injurious (except perhaps to the pocketbook), their use raises important questions about when and how promising animal research should be translated to the setting of clinical cryonics. Unique among all other populations of human and animal patients, cryopatients have the opportunity to be treated with neuroprotective drugs that show great promise, absent the long delays of regulatory vetting, and independent of the economic pressure experienced by pharmaceutical companies to not only market drugs that are effective, but to market ones that are also profitable. The question thus becomes what criteria do we use in applying these drugs absent the extensive pre- and post marketing evaluation that obtains with approved ethical drugs? In essence the question we must ask and answer is “can we do better, much better in fact, than our colleagues in conventional critical care medicine?

Michael G.  Darwin, Independent Critical Care Consultant

Click here for this entry with references in PDF format.

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(1) Resuscitation medicine is properly termed reanimatology, and is so-called in the non-English speaking world

(a) The one condition in which there is unequivocal benefit to supraphysiologic administration of steroids is meningococcal meningitis with substantial evidence also supporting a similar degree of efficacy in Typhoid  and Pneumocystis carinii pneumonia.

25. August 2008 · Comments Off on The chemistry of neuroprotection · Categories: Neuroscience · Tags: , , , , ,

In a review of the 1998 21st Century Medicine seminars, Cryonics Institute president Ben Best writes:

“The presentations impressed upon me how much witchcraft and how little science has gone into the study of cryoprotectant agents (CPAs). This might be understandable in light of the fact that most cryobiologists are, in fact, biologists. I suspect that a great deal could be accomplished by a thorough study of the physics of the chemistry of CPAs.”

Such an observation could equally apply to the study of neuroprotectants in cerebral ischemia. There has been a growing literature investigating the potential of numerous molecules for the treatment of stroke and cardiac arrest. Although some approaches have been more successful than others, systematic reviews of the chemical and physical characteristics of effective drugs are lacking and discussion of the topic is  often confined to isolated remarks.

A number of examples:

“It is not surprising that all the agents which are effective in shock carry a negative charge. This applies both to heparin, which possesses a very strong negative charge, and to hypertonic glucose solution. The same may be said about a substance now in wide use – dextran – which has small, negatively charged molecules, and also about the glucocorticoids 21, 17, and 11, which also have a negative charge.” – Professor Laborit in: Acute problems in resuscitation and hypothermia; proceedings of a symposium on the application of deep hypothermia in terminal states, September 15-19, 1964. Edited by V. A. Negovskii.

“We report that estrogen and estrogen derivatives within the hydroxyl group in the C3 position on the A ring of the steroid molecule can also act as powerful neuroprotectants in an estrogen-receptor-independent short term manner because of to their antioxidative capacity.” Christian Behl et al. Neuroprotection against Oxidative Stress by Estrogens: Structure-Activity Relationship. Molecular Pharmacology 51:535-541 (1997).

“Minocycline’s direct radical scavenging property is consistent with its chemical structure, which includes a multiply substituted phenol ring similar to alpha-tocopherol (Vitamin E)” – Kraus RL et al. Antioxidant properties of minocycline: neuroprotection in an oxidative stress assay and direct radical-scavenging activity. Journal of Neurochemistry. 2005 Aug;94(3):819-27.

“It is notable, however, that NAD+ and minocycline share a carboxamide and aromatic ring structure. A common structural feature of competitive PARP inhibitors is a carboxamide group attached to an aromatic ring or the carbamoyl group built in a polyaromatic heterocyclic skeleton. This structure is also present in each of the tetracycline derivatives with demonstrated PARP-1 inhibitory activity. Alano CC et al. Minocycline inhibits poly(ADP-ribose) polymerase-1 at nanomolar concentrations. Proceedings of the National Academy of Sciences of the United States of America. 2006 Jun 20;103(25):9685-90.

Systematic study of structure-activity relationship of neuroprotectants would not only contribute towards the development of a general theory of neuroprotection in cerebral ischemia, it would also contribute to the design of multi-functional neuroprotectants. Although it is now increasingly accepted that combination therapy offers more potential for successful treatment of stroke and cardiac arrest than mono-agents, parallel or sequential administration of multiple drugs present non-trivial challenges in research design and clinical application. Such problems may be better addressed by designing molecules with different mechanisms of action in the same structure, an approach that is currently recognized and investigated by forward-looking biomedical researchers.

Although the field of cerebral resuscitation has known some notable researchers  like Vladimir Aleksandrovich Negovskii and Peter Safar, who devoted their lives to a thorough study of the mechanisms of cerebral ischemia and its treatment, the field as a whole shows a never ending stream of trial and error publications to investigate yet another drug (before moving on to other areas in neuroscience and medicine). Although there is an increased interest in meta-analysis of all these experiments, meta-analysis that places its findings in a broader biochemical and pharmacological context is rare.

The emphasis on theory and research design can be taken too far. As Nassim Nicholas Taleb recently argued, the role of design in biotechnology is overestimated at the expense of chance observations and unexpected directions. But in the area of cerebral resuscitation the risk of too much theory and systematization is low at this point. As evidenced by the successful development of vitrification agents with low toxicity in cryobiology, a committed long-term and systematical effort to find solutions to human medical needs can pay off.

05. August 2008 · Comments Off on Recent developments in the treatment of Alzheimer's · Categories: Health, Neuroscience · Tags: , , ,

The full text of the Life Extension Foundation magazine article (August 2008) describing the use of Enbrel for the treatment of Alzheimer’s disease and announcing LEF’s new Enbrel trial, is now available. As previously discussed, Enbrel (entanercept) has been shown to provide immediate benefits in Alzheimer’s patients, improving memory performance and less frustration and agitation within minutes of treatment.

The more recent publication (pdf document) of additional data from the same patients in the previously reported six month Phase II trial adds further evidence to these results, specifically noting a rapid improvement in the verbal fluency of patients undergoing weekly perispinal Enbrel injections. Additionally, case studies of two more patients are given in the text of the report, and a stronger case for carrying out larger scale studies (including Phase III clinical trials) is made.

A blog post at Al Fin reports on other promising Alzheimer’s treatments such as the drug Rember, which “appears to target ‘Tau tangles’ in the portion of the brain most active in memory formation.”