Endocrine approaches to treatment of Alzheimer's disease and other neurological conditions: Part I: Some recollections of my association with Dr. Abba Kastin: A tale of successful collaboration.

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Festschrift for Dr. Abba Kastin

Endocrine approaches to treatment of Alzheimer’s disease and other neurological conditions Part I: Some recollections of my association with Dr. Abba Kastin: A tale of successful collaboration Andrew V. Schallya,b,c,d,e,∗ a

Endocrine, Polypeptide and Cancer Institute, Veterans Affairs Medical Center, Miami, FL, United States South Florida VA Foundation for Research and Education, Miami, FL, United States c Department of Pathology, University of Miami, Miller School of Medicine, Miami, FL, United States d Division of Hematology/Oncology, Department of Medicine, University of Miami, Miller School of Medicine, Miami, FL, United States e Division of Endocrinology, Department of Medicine, University of Miami, Miller School of Medicine, Miami, FL, United States b

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Article history: Received 3 March 2015 Accepted 12 March 2015 Available online xxx Keywords: AJ Kastin CNS effects of GHRH analogs Alzheimer’s disease TBI PTSD GWI

It is a privilege to contribute an article to the Festschrift honoring Dr. Abba J. Kastin for his 35 years of service as the Editor-in-Chief of the journal Peptides and for his enormous contributions to the field. However, in my case it is an exceedingly difficult task to adequately express a fitting recognition of my fruitful collaboration and close friendship with Abba that has existed for more than 50 years. My formal association with Abba started in 1964 when he joined my laboratory at the New Orleans VA hospital and Department of Medicine of Tulane University in New Orleans. I believe it would not have been possible to find a medical investigator with a comparable education, training, intelligence and pleasant personality such as Dr. Abba Kastin. His other assets included an excellent knowledge of endocrinology, experimental and clinical research approaches, and his ability for excellent planning and systematic organization.

∗ Correspondence to: Endocrine, Polypeptide and Cancer Institute, Veterans Affairs Medical Center, Miami, FL, United States. Tel.: +1 305 575 3477; fax: +1 305 575 3126. E-mail address: [email protected]

Dr. Kastin joined my group at the suggestion of Dr. Griff Ross of NIH, an outstanding endocrine clinician and one of the leaders of the Endocrine Society, to work on the search for the substances controlling the release of MSH. This was the only pituitary hormone the release of which was not being investigated elsewhere, and since my laboratory at that time already had a reputation for work on peptide hormones we headed in that direction. Subsequently with our exciting research program and hard work we succeeded in making many notable findings in endocrinology, and neuroendocrinology and we reported them in the various top level journals in the field. The total number of joint publications (articles, abstracts, reviews and book chapters) with Dr. Kastin for that period amounted to about 322 of which some 76 were for MSH and its release. This was Dr. Kastin’s original project, and then he continued on work which led to the subsequent discovery of MSH release-inhibiting factor, the tripeptide MIF-1, in my laboratory. It may be important to mention that our collaborative clinical publications on hypothalamic hormones involved TRH [2,13,14,30]. Our initial clinical publications on TRH were with my

http://dx.doi.org/10.1016/j.peptides.2015.03.009 0196-9781/Published by Elsevier Inc.

Please cite this article in press as: Schally AV. Endocrine approaches to treatment of Alzheimer’s disease and other neurological conditions. Part I: Some recollections of my association with Dr. Abba Kastin: A tale of successful collaboration. Peptides (2015), http://dx.doi.org/10.1016/j.peptides.2015.03.009

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collaborator at Tulane University in New Orleans, Dr. Cy Bowers, as the leading author, and myself. Dr. Carlos Gual and Abba Kastin were co-authors [2,13,14,30]. These studies on TRH have been cited in our various book chapters [30]. More than 100 joint publications with Abba were concerned LHRH and its agonistic and antagonistic analogs. Many of these papers were of clinical studies on LHRH which we carried out mostly in Mexico, at the enthusiastic invitation of and in collaboration with Dr. Carlos Gual, Dr. Arturo Zarate, Dr. Gonzales Barcena and others [26,43,47–52,92]. Other publications consisted of major articles, both basic and clinical, on LHRH and on which Abba was a co-author [71]. Other joint publications, mostly clinical, were devoted to somatostatin and its analogs, on which we worked, mostly in England, with Drs. Reg Hall, Mike Besser, M. Thorner, Steve Bloom, and Antonio Gomez-Pan [1,9,10,18,60,89]. In later publications on somatostatin analogs we collaborated with Dr. Gonzalez-Barcena [27,28]. This favorable situation and so many publications on hypothalamic hormones were of course due to our fruitful work on TRH [2,13,14,30]. There was also the issue of our clear leadership in the chemical, physiological and clinical advances on LH-RH [26,43,47–51,72,90,93]. Dr. Kastin’s work on MIF and MSH established his leading position in the fields of both MSH and MIF, as he was the first to show that a hypothalamic peptide could act “upward” on the brain, as we had previously shown for hypothalamic peptides acting “downward” on the pituitary [40–42,45,46]. As a result of realizing that peptides could act “upward” on the brain, Dr. Kastin became very interested in the behavioral and CNS effects of hypothalamic hormones and related compounds such as endorphins and enkephalins and so he started elaborate studies on the influence of our synthetic TRH, LHRH, somatostatin, and their analogs, on the brain [46]. As mentioned above his work with MSH was the first to show that any peripherally generated peptide could have direct effects on the brain. Abba carried out both basic

and clinical work on behavioral effects of MSH, with Curt Sandman and Lyle Miller [37,38]. Then he accomplished elaborate CNS studies on MIF, LHRH, Met-enkephalin (also called opioid growth factor) and somatostatin, with Nick Plotnikoff and Rudolph Ehrensing [20,44,62,63]. He also investigated various opiate peptides with Gayle A. Olson and Richard D. Olson [36,39,45], with whom he introduced the concept of endogenous brain anti-opioids. With Jim Zadina he described the naturally occurring, highly selective, high affinity Mu-opiate receptor agonist endomorphin; their original paper in Nature [92] has now been cited almost a thousand times. We also examined, with Bill Banks, penetration of somatostatin analogs through the blood–brain barrier [6]. These studies resulted in numerous publications and established Abba as a top expert in this field also. In particular, it was important conceptually to show that a peptide injected peripherally could have an effect on the brain, and then to show that this occurred by its passage directly across the blood–brain barrier. These, now well-established concepts were initially as controversial as that of my own work on control of pituitary function by hypothalamic peptides. I also collaborated with Prof. Gyula Telegdy and Prof. L. Vecsey from the University of Szeged, Hungary on CNS effects of a hypothalamic MSH analog and other peptides synthesized in our laboratory [84,85]. This collaboration on CNS effects of peptides with Abba Kastin, and later Gyula Telegdy, turned out to be not only important and influential but also exceedingly useful for my future work when I decided to investigate, in my current laboratory, the effects of our antagonistic analogs of growth hormone-releasing hormone (GH-RH) in experimental models of Alzheimer’s disease. This decision was made on the basis of our discovery of anti-inflammatory and antioxidative effects of GHRH antagonists and the potential for applying these in the CNS. I will always remain deeply grateful to Dr. Kastin and Prof. Telegdy for introducing and exposing me to these investigations of CNS effects of peptides.

Please cite this article in press as: Schally AV. Endocrine approaches to treatment of Alzheimer’s disease and other neurological conditions. Part I: Some recollections of my association with Dr. Abba Kastin: A tale of successful collaboration. Peptides (2015), http://dx.doi.org/10.1016/j.peptides.2015.03.009

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ARTICLE IN PRESS Festschrift for Dr. Abba Kastin / Peptides xxx (2015) xxx–xxx

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Part II: Some features of Alzheimer’s disease Andrew V. Schallya,c,d,∗ Luis M. Salgueiroa a


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Article history: Available online xxx Keywords: AJ Kastin CNS effects of GHRH analogs Alzheimer’s disease TBI PTSD GWI

Alzheimer’s disease (AD) is the most common disorder of the central nervous system (CNS) and the most frequent cause of devastating dementia in the elderly. Alzheimer’s disease is typified by loss of memory and by cognitive decline [15,31,64,68,94]. It is estimated that more than 5 million Americans were suffering from Alzheimer’s disease in 2014 [68]. Nearly two-thirds of Alzheimer’s patients are women. Alzheimer’s disease dramatically lowers the quality of life of patients, puts a major burden on the family, costs countries around the world billions of dollars annually and eventually has a sad outcome. In the USA alone, the current costs amount to more than 300 billion dollars per year and surpass those for cancer. Because people are living longer, the number of individuals affected by Alzheimer’s disease is continuing to increase, thereby augmenting an already major world-wide health problem [68]. Among recent victims of Alzheimer’s disease were politicians such as

∗ Corresponding author at: Endocrine, Polypeptide and Cancer Institute, Veterans Affairs Medical Center, Miami, FL, United States. Tel.: +1 305 575 3477; fax: +1 305 575 3126. E-mail address: [email protected] (A.V. Schally).

Ronald Reagan and Margaret Thatcher, William Proxmire and Cyrus Vance, artists such as Willem de Kooning, composers such as Aaron Copeland, actors such as Jimmy Steward, Charlton Heston, Charles Bronson and Peter Falk, actress Rita Hayworth, painters such as Norman Rockwell, singers such as Perry Como, Glenn Campbell and Etta James, fighters such as Sugar Ray Robinson, columnists such as Pauline Friedman Phillips (Dear Abby). We are losing beloved and productive members of our society. It is imperative, therefore, to find new and more effective means of treating this devastating disease. Considerable efforts should also be allocated to prevention as well as improvement of early detection before the later stages of the disease develop and the condition becomes irreversible. Unfortunately, the research funding for these efforts continues to be inadequate. Alzheimer’s disease is a neurodegenerative one typified by behavioral changes, progressive loss of cognitive function and a debilitated physical condition. Ultimately AD patients are unable to survive without assistance [15,29,31,54,64,68,74,78,94]. The present conceptions of AD are based on neuropathologic autopsy findings [15,29,31,54,64,68,74,78,94]. Alzheimer’s disease leads to loss of nerve cells and tissue in the brain and causes cortical shrinkage. The brains of Alzheimer’s patients exhibit extracellular neuritic plaques and intracellular neurofibrillary tangles as its two major pathologic hallmarks. These neuropathologic findings have generated numerous hypotheses of pathogenesis and led to much research attempting to elucidate the molecular mechanisms of the disease [29,54,64,74,77,78,94]. The extra-cellular neuritic plaques in the brains of AD patients are composed of ␤-amyloid (␤A) peptides. These neuritic plaques result from the aggregation of ␤-amyloid (␤A) peptide, which itself is generated

Please cite this article in press as: Schally AV, Salgueiro LM. Part II: Some features of Alzheimer’s disease. Peptides (2015), http://dx.doi.org/10.1016/j.peptides.2015.03.009



by proteolytic cleavage of physiologically normal amyloid precursor protein (APP), a transmembrane protein. The “amyloid cascade hypothesis” proposes that aggregation of ␤A peptides cleaved from APP, and the subsequent dissemination of these ␤A peptide deposits through the brain, represent the initial events in the progression of the pathophysiology of Alzheimer’s disease [31,74,77,78,94]. A protein complex including the protease, ␥secretase, is required for this cleavage of APP to occur and to produce the pathogenic ␤A peptide. In AD an imbalance exists in the production and clearance of ␤-amyloid [58]. Mutations in the three genes encoding APP, presenilin-1, and presenilin-2 (needed for ␥-secretase activity) lead to autosomal-dominant Alzheimer’s disease, the least common type [78]. This type of AD mutations account only for about 10% of AD patients but provide evidence that the production by ␤A peptide, by proteolytic processing of APP, results in the disease being manifested [15,29,31,54,64,74,78,94]. The ␤A peptide aggregates contribute to the physical disruption of the synaptic connections and have been inculpated in the etiology of dementia [54,78]. Thus, the genetics of familial AD strongly support the amyloid cascade hypothesis [64,78]. It has also been reported that ␤-amyloid deposits in transgenic mice are reminiscent of prions [77]. Thus, some evidence exists that ␤-amyloid aggregates involve prion-like propagation [77]. Other recent findings suggest that Tau, a microtubule associated protein (MAP), may be a critical mediator of ␤A toxicity [53]. The brains of Alzheimer’s disease patients also contain tangles of hyperphosphorylated Tau protein [29,74,77]. Tau protein is a soluble protein that modulates the stability of microtubules and the cellular cytoskeleton. Phosphorylation modulates the direct integration of Tau into microtubules (MTs), allowing their stabilization and assembly. This protection facilitates autophagy and clearance of ␤A1-42. The loss of Tau function by mutation, deletion or hyper-phosphorylation may lead to decreased binding of Tau to microtubules and subsequent impairment of autophagic flux which may alter the distribution of intracellular and extracellular ␤A1-42. Reduction of endogenous Tau levels prevents ␤-amyloid induced deficit in brains axonal transports and attenuates ␤A-induced neurodegeneration in experimental models of Alzheimer’s disease [73,76,88]. Defects in Tau protein and microtubule malfunction have also been postulated to play a role for AD development. In transgenic mouse models of AD, ␤A assembly has been shown to precede Tau protein pathology [29,54].

One theory suggests that the accumulation of intracellular fibrous tangles of the hyper-phosphorylated form of Tau (insoluble p-Tau) within the neuron play a major role in the onset and progression of AD and leads to neuronal dysfunction and death [29], correlates with cognitive decline, increasing ␤A-toxicity and neurodegeneration and postulates that dysfunctional p-Tau leads to neuronal death resulting in dementia [22,25,29]. There could also be a link between inflammation and Alzheimer’s disease. Thus, another, more recent hypothesis is that neuronal damage activates brain immune cell microglia and astrocytes, stimulating them to produce interleukin-1 (IL-1) and S-100 inflammatory cytokines [29]. In turn, the resultant excess of inflammatory cytokines then leads to an increase in the production of ␤-amyloid plaques. This suggests that neuro-inflammation may be another possible culprit in AD and therefore another potential therapeutic target. It is known that abnormal levels of circulating inflammatory cytokines are found in patients with AD and therefore possibly play a role in its pathogenesis [29]. These mechanisms have not been broadly elucidated, however. Various studies have demonstrated that some hypothalamic neurohormone analogs can affect CNS functions [20,36–42,44–46,62,63,84,85,91]. Thus, these studies suggest that neuroendocrine mechanisms could play a contributive role in the development of AD. At menopause, for example, the endocrine system undergoes profound changes with dysregulation of pituitary-gonadal axis; major decreases in estrogen secretion occur [3,11]. The accompanying high levels of the gonadotropins, LH and FSH in serum or in neurons, may contribute to or be associated with the increase in formation of ␤-amyloid plaques in the brain [8,33,79]. These gonadotropins also show some rise after andropause. LHRH agonists such as leuprolide have beneficial effects on the Alzheimer’s disease related symptoms of neurodegeneration in animal models [83]. Bowen RL et al., proposed an association between elevated serum gonadotropin concentrations and Alzheimer’s disease [12]. This may connect thru the fact that high levels of gonadotropins may also contribute to inflammation. However, several Phase I, II clinical trials were conducted on the effectiveness of the LHRH agonist, leuprolide, in retarding mental deterioration in patients with Alzheimer’s disease and no improvement was found [21]. In our considered opinion these trials should have been conducted with a modern LH-RH antagonist such as Degarelix, which induces an immediate blockade of LH and FSH release and has minimal side effects and my function preferentially.

Please cite this article in press as: Schally AV, Salgueiro LM. Part II: Some features of Alzheimer’s disease. Peptides (2015), http://dx.doi.org/10.1016/j.peptides.2015.03.009



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Part III: Experimental studies on antagonists of LH-RH and GH-RH in animal models of Alzheimer’s disease: Projections for treatment of other neurological conditions Andrew V. Schallya,c,d,∗ Luis M. Salgueiroa a


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Article history: Keywords: AJ Kastin CNS effects of GHRH analogs Alzheimer’s disease TBI PTSD GWI

In view of our interest in the possible use of peptide analogs for the treatment of Alzheimer’s disease, and we have collaborated with Prof. Gyula Telegdy in Szeged, Hungary in a preclinical evaluation of the LHRH antagonist, Cetrorelix in mice and rats [80,83]. The LHRH antagonist, Cetrorelix (Fig. 1) was previously shown to inhibit gonadotropin and sex-steroid secretion in rats and human beings [21]. We evaluated the effects of Cetrorelix on the ␤-amyloid2535-induced impairments of the consolidation of passive avoidance, memory, anxiety, and depression in mice and rats. With Prof. Telegdy we showed that Cetrorelix, in CSF mice, blocked the impairment of the consolidation of passive avoidance (CPA) learning when given intracerebro-ventricularly (icv) into the lateral cerebral ventricle 30 min following ␤-amyloid25-35 administration [83]. Cetrorelix elicited anxiolytic action in the plus-maze test and in the forced swimming and tail suspension tests it showed anti-depressive-like activities [82]. These results suggest the need of further tests of Cetrorelix in models of Alzheimer’s disease [82]. Similar findings to those in mice were obtained in Wistar rats. Thus,

∗ Corresponding author at: Endocrine, Polypeptide and Cancer Institute, Veterans Affairs Medical Center, Miami, FL, United States. Tel.: +1 305 575 3477; fax: +1 305 575 3126. E-mail address: [email protected] (A.V. Schally).

in rats, Cetrorelix also blocked the effects of ␤-amyloid25-36 and was able to correct the impairment of the memory consolidation caused by ␤-amyloid. This suggests that the effects of Cetrorelix are not species specific [80]. These results illustrate that Cetrorelix, among various neuroendocrine peptides acting through their receptors present in brain cortex, affects brain function and has the ability to correct the impairment of the memory consolidation caused by ␤-amyloid [80,83]. We have also investigated the role of growth hormone-releasing hormone (GH-RH) antagonists, and the changes related to aging, in the levels of GHRH as well as growth hormone (GH) and insulin-like growth factor 1 (IGF-1) which may also play a role in the development of AD [19,33]. Receptors for GH-RH present in brain cortex, may mediate these changes [33,55,79]. The administration of GHRH or its agonists has been reported to produce a favorable effect on cognitive function in adults with mild cognitive impairment [4,86]. Since centrally released GH-RH controls the GH-IGF-1 axis, we decided to carry out studies with Prof. Telegdy’s group, in mice and rats, with an early GHRH antagonist, MZ-4-71, (Fig. 1) which was shown to inhibit GH release. In CSF mice, this GHRH antagonist facilitated the consolidation of passive avoidance learning. ␤-amyloid25-35 impaired this effect [82]. GHRH antagonists blocked this impairment when given icv simultaneously or within 30 min following ␤-amyloid25-35 administration. In the forced swimming tests, MZ-47-1 demonstrated antidepressive-like action

Please cite this article in press as: Schally AV, Salgueiro LM. Part III: Experimental studies on antagonists of LH-RH and GH-RH in animal models of Alzheimer’s disease: Projections for treatment of other neurological conditions. Peptides (2015), http://dx.doi.org/10.1016/j.peptides.2015.03.009



Fig. 1. Structures of LH-RH antagonists cetrorelix and GHRH antagonists used in experimental studies on Alzheimer’s disease.

and in the plus-maze, it elicited mild anxiolytic action [81,82]. These results suggested the merit of further investigation or a clinical trial of GHRH antagonist, MZ-4-71, in patients with the anxiety, depression and cognitive impairment observed in Alzheimer’s disease. In another study in collaboration with Bill Banks and John Morley at the VA Hospital in St. Louis [5], we evaluated our GHRH antagonist, designated MZ-5-156, on age-related changes in SAMP8 mice, a strain that develops cognitive deficits with aging. The SAMP8 mouse strain is used to model Alzheimer’s disease and has a natural mutation that results in increases in amyloid precursor protein (APP) and ␤-amyloid peptide during aging. The DNA repair enzyme, telomerase, maintains eukaryotic chromosome stability by ensuring that telomeres regenerate DNA each time the cell divides, protect chromosome ends from damage, help to preserve genomic integrity and prevent senescence. Mice treated subcutaneously with 10 ␮g/day of GHRH antagonist MZ-5-156, for 4 months showed increased telomerase activity in heart, aorta, liver and stomach, improvement in some measures of oxidative stress in the brain, and improved pole balance [5]. MZ-5-156, used for 2–4 months of treatment, also improved cognition, increased mean life expectancy by 8 weeks, and decreased tumor incidence. Thus, we can conclude that GHRH antagonists affect brain function and demonstrate positive effects on some of the characteristic conditions associated with aging [5,81,82]. The blood–brain barrier is formed by brain endothelial cells joined by tight junctions and separates the CNS from the general circulation. It was previously not known, whether peripherally administered GHRH antagonists can cross the blood–brain barrier. In view of the apparent responses to GHRH analogs even when they were administered subcutaneously, we thought that we should confirm experimentally that antagonists of growth hormone-releasing hormone can cross the blood–brain barrier. For that, we administered a GHRH antagonist radio-iodinated JV-1-42 and showed that, after i.v. injection (I-JV-1-42) enters the brain intact. This demonstrates that i.v.-administered I-JV-1-42 readily crosses the blood–brain barrier and accumulates in the brain [32]. We have reported previously that GHRH antagonists inhibit the expression of enzymes involved in the generation of reactive oxygen species (ROS) and thus appear to have antioxidative activities [7]. We then extended our observations on the associated anti-inflammatory effects of GHRH antagonists [61,69]. In several studies we evaluated the effects of more recently

synthesized antagonistic analogs of GHRH, such as MIA-602 (Fig. 1) MIA-313, and MIA-459, on the expression of inflammatory cytokine genes in models of human breast cancers (TNBC) [61] and benign prostate hyperplasia (BPH) which was induced in Wistar rats with testosterone [69]. Treatment with GHRH antagonists of mice bearing breast cancer reduced tumor growth and suppressed several important inflammatory signaling pathways including IL-1, IL-4, IL-6 and TNFa [61]. In Wistar rats, reductions of prostate weights were observed after 6 wk of treatment with GHRH antagonists as were significant reductions in protein levels of IL-1␤ [69]. This confirmed the anti-inflammatory activity of GH-RH antagonists, such as MIA-602. The neurohormones, such as GHRH in addition to controlling the GH/IGF-1 axis could also have intrinsic activity on both cognitive processes and the development of Alzheimer’s disease. Since early antagonists of GHRH MZ-4-71 and MZ-5-156, showed a positive impact on learning memory we decided to test a new more potent antagonist of GHRH, MIA-690, synthesized by us (Fig. 1), in different models of Alzheimer’s disease. The antioxidative effects of the GHRH antagonists, discussed above [7,61,69], also contributed to this decision. These studies were expertly performed in our laboratory by Dr. Miklos Jaszberenyi [33] physician scientist, using transgenic mice (5XFAD strain), which develop neurodegenerative symptoms characteristic of Alzheimer’s disease [33]. These genetically engineered mice accumulate amyloid-␤1-42 plaques as a result of presenilin-1 (PSEN-1) and APP amyloid precursor mutations. The effects of the GHRH antagonist,MIA-690,were evaluated in vivo, by observing the behavior, special learning and memory of the genetically modified “Alzheimer’s” 5XFAD mice in a Morris water maze (MWM) [24,59,87]. For the Morris water maze experiments the mice were divided into 4 treatment groups which received subcutaneous daily injections of vehicle solution or MIA690 at the doses of 2, 5 and 10 ␮g/day. The indices of cognitive performance (latency, cumulative index etc.) were followed in the MWM tests. During the 6 month experimental period, a marked deterioration of spatial learning and the latency of the animals was observed during the training trials.MIA-690 at the dose of 10 ␮g/day abolished the progressive decrease in the amplitude of the latency curve seen in training trials [33]. Accumulation of ␤amyloid1-42 rafts and Tau filaments in necropsied brain samples of control and treated animals was verified with the help of ELISA. Our result showed that the antagonist MIA-690 inhibited the concentration of ␤-amyloid1-42 and Tau filaments in the brains of




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Fig. 2. Effect of the GHRH antagonist MIA-690 on the ␤-amyloid distribution of 5× FAD mouse hippocampus. The figure shows the immunohistochemistry (IHC) of both, (A) hippocampal section of a MIA-690 treated (10 ␮g/day) and (B) the untreated control B6SJL-Tg mouse. Mice brains sections were obtained at 10 months, immunostained with anti-␤-amyloid antibody and developed with peroxidase-DAB. Nucleus was co-stained with hematoxylin. The untreated control hippocapus showed a higher density of A␤ plaques deposit (brown colored areas). 200× magnification (Courtesy of Dr. T. Reiner, Research Health Scientist, Miami VAHCS).

treated mice. Fig. 2 shows the ␤-amyloid distribution in the control animal and an animal treated with MIA-690, as revealed by immunohistochemistry [33]. In genomic and proteomic studies (PCR Array) the GHRH antagonist inhibited the transcription of various putative Alzheimer’s disease markers. The PCR array studies revealed statistically significant changes in the expression of 22 Alzheimer’s disease related genes in the brain samples of the 5XFAD mice following treatment with 10 ␮g MIA-690 for six months [33]. These transcriptional studies showed an inhibition of the expression of amyloid precursor proteins, the amyloid-␤ precursor protein-binding proteins (APP-BP)s, the amyloid-␤ generating BACE2 and several components including presenilin 1, and 2of the ␥-secretase complex. MIA-690 was also evaluated in vitro using HCN2 human cortical cell cultures treated with ␤-amyloid1-42. The formation of reactive oxygen species, markers of inflammation and neuro-hormonal signaling were measured by fluorescent detection, PCR, and ELISA [33]. In cell cultures, the GHRH analog, MIA-690, showed antioxidative and neuro-protective properties and also inhibited the GHRH/GH/IGF-1 axis [33]. The attenuation of oxidative stress by GHRH antagonists may play a role in the protection against amyloid-␤ proteo-toxicity. These studies [33] demonstrate that the GHRH antagonist, MIA690, has beneficial effects in various features of Alzheimer’s disease [33]. In studies in vivo, MIA-690 significantly delayed the Alzheimer’s disease-related deterioration of the cognitive performance in Morris water maze [33]. Thus our investigations suggest that GHRH antagonists can penetrate the blood–brain barrier and affect the cascade of Alzheimer’s disease by decreasing the aggregation and proteo-toxicity of ␤amyloid and attenuating the accumulation of tangles of Tau protein. However, the mechanism of action of GHRH antagonists in models of Alzheimer’s disease must be clarified. The beneficial effects of GHRH antagonists on cognition and on the proteo-toxicity must be differentiated. Our work shows that GHRH antagonists exert favorable effects on cognition in animal models of Alzheimer’s disease [33]. However, GHRH and GHRH agonists have also been shown to exert effects on CNS and to improve cognitive function in human subjects with mild cognitive impairment [4,16,86] and in animals. Thus the choice of GHRH analogs (agonist or antagonists) for the desired effect may depend on the degree of cognitive impairment and the antagonists might be indicated in earlier stages of Alzheimer’s disease and even in cases of more severe impairment. Our findings [33,81,82] strongly support the merit of further investigations leading to clinical trials of GHRH antagonists in models of Alzheimer’s disease.

In our most recent studies with other GH-RH antagonists, MIA602 and MIA-606 elicited similar effects as MIA-690 in 5XFAD mice [70]. The synthesis of a new class of GHRH antagonists with even higher GH release inhibitory activity is in progress. These new antagonists of the AVS-RC class which inhibit GH and therefore IGF-1, more powerfully than the MIA class antagonists will be also evaluated on the 5XFAD Alzheimer’s disease model. Other improvements in therapy of AD could depend on advances in nanotechnology. Recent evidence suggests that the blood brain barrier (BBB) is altered in AD [75]. These changes in the BBB could allow alteration of the delivery of therapeutic drugs. The use of approaches based on nanotechnology, such as using nanoparticles loaded with GHRH antagonists that can be targeted to various areas of the brain, is thus actively being pursued to determine feasibility. Agonistic analogs of growth hormone-releasing hormone (GH-RH) could also affect cognitive performance and memory function by direct effects on GHRH receptors in cerebral cortex and other brain regions or through the activation of pituitary GH/hepatic IGF-1 axis [4,16,73,87]. The use of GHRH agonists and antagonists could be also considered for other neurologic diseases and conditions such as posttraumatic stress disorder (PTSD), traumatic brain injury (TBI) and Gulf War Illness (GWI) which profoundly affect many US veterans of the wars such as those in Iraq and Afghanistan [91]. Many patients with these disorders have been reported to show abnormalities in GH secretion, with GH levels being low in many cases. Thus nearly half of US veterans diagnosed with blast concussion show low levels of pituitary hormones, with the deficiency in GH being the most common [91]. More than half the patients with cognitive disorders after TBI were also reported to have growth hormone deficiency [67]. Both classes of GHRH analogs, the agonists and the antagonists are being investigated in animal models of posttraumatic stress disorder (PTSD) and traumatic brain injury (TBI). Speculatively, the initial therapy for PTSD could consist of the use of the GHRH antagonists or other peptide antagonists such as those of Bombesin/Gastrin releasing peptide (vide infra) to induce the extinction of aversive memories. This could be followed by the use of GHRH agonists to improve the cognition. In cases of TBI, our new GHRH agonists of GHRH of MR class, such as MR-490, could be also used to stimulate the regeneration of neural tissues in brain [16]. Another class of peptides may also prove useful. Thus, in response to remarkable CNS effects of various peptides, Bombesin/Gastrin releasing peptide (BN/GRP) antagonist RC-3095, which was synthesized by our group [65], has been shown to cause the extinction of aversive memories in rats [56]. The structure of




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this analog [D-Tpi6 ,Leu13 (CH2 NH)-Leu14 ]BN(6-14) is completely different from that of GHRH analogs indicating that multiple classes of peptides can affect behavior and induce extinction of memory. These findings support the merit of further studies aimed at neuro-psychiatric applications of such analogs for the treatment of patients with fear-related disorders. A still more powerful BN/GRP antagonist than RC-3095, designated RC-3940-II, with the structure [Hca6 ,Leu13 (CH2 N-Tac14 ]BN(6-14) which was also synthesized in our laboratory [17,66] is available for experimental studies. Analogs of GHRH were initially conceived for other clinical applications, GHRH antagonists mainly for cancer treatment [72] and acromegaly and GHRH agonists for stimulation of the pituitary in pediatric patients with growth deficiencies and later for geriatric disorders [16]. The results obtained with the group of Dr. J. Hare indicate that our GHRH agonist, JI-38, can stimulate the proliferation c-kit+ cardiac precursor cells [35] and cardiac stem cells [34]. Cardiac stem cells isolated from mouse, rat and pig express receptors for GHRH. JI-38 and our new agonists, MR-356 and MR409, stimulated their proliferation and survival [23]. JI-34, another agonist of GHRH synthesized by us was also shown to augment the viability of mesenchymal stem cells derived from mouse bone marrow and sustained their transplantation [57]. These findings may have profound implications and we can no longer ignore the many other possible clinical applications of GHRH analogs, the list of which is continuously growing [16,33] and which could include neurodegenerative diseases like Alzheimer’s disease, brain injuries and disorders of US veterans such as TBI, PTSD and GWI. Collectively these developments suggest that analogs of GHRH may find a variety of uses in the therapeutic armamentarium.

[4]

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[9]

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[13] [14]

[15] [16]

Acknowledgement [17]

Experimental studies of Dr. Andrew V. Schally’s group cited in this article were carried out at the VA Medical Center in New Orleans and Tulane University Medical School, New Orleans, La and Miami VA Medical Center and the University of Miami School of Medicine, Miami FL and were supported by the Medical Research Service of the Veterans Affairs Department and by various NIH grants. Experimental studies with the group of Dr. J. Hare at the University of Miami, School of Medicine were supported by various NIH grants. The work with the group of Dr. W. Banks and Dr. J. Morley at the VA Hospitals, St. Louis, MO and Seattle, WA and University of St. Louis School of Medicine, University of Washington School of Medicine in Seattle, was supported by grants from VA and NIH. Clinical studies in Mexico with Dr. Carlos Gual were carried out at the Instituto Nacional de Ciencias Medicas y Nutricion, in Mexico City. Clinical studies in Mexico with Dr. David Gonzalez Barcena and Dr. Arturo Zarate were performed at the Hospitals of the Instituto Nacional de Seguro Social in Mexico City, Mexico. Clinical studies with Prof. Hall and Dr. Michael Besser were performed at the Department of Medicine, Royal Victoria Infirmary, Newcastle upon Tyne and The Medical Professorial Unit and Department of Chemical Pathology, St. Barholomew’s Hospital, London, England; and with Dr. M.O. Thorner at the Middlesex Hospital, London, England. Experimental studies with Prof. Gyula Telegdy were performed at the University of Szeged, Szeged, Hungary.

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Endogenous opioids and feeding behavior: A decade of further progress (2004-2014). A Festschrift to Dr. Abba Kastin.

Abba J. Kastin, the Zeitgeist, and the inception of Peptides.

A Historical Tale of Two Lymphomas: Part I: Hodgkin lymphoma.

Abba Kastin: The melanocyte stimulating hormone story and the future of the proteophathies.

A tale of two approaches.

Endocrine and metabolic diseases. Treatment of thyroid disease: I.

Recollections of my young days: -the pleasure of creation-.

Successful treatment of accessory breast cancer with endocrine therapy.

Association of linear IgA bullous disease with ulcerative colitis: a case of successful treatment with infliximab.

Successful antiviral treatment after 6years of chronic progressive neurological disease attributed to VZV brain infection.

Hand manifestations of neurological disease: some alternatives to consider.

Higher burden of migraine compared to other neurological conditions: results from a cross-sectional study.

Nerve growth factor and some inherited neurological conditions.

Eales' disease with neurological involvement. Part 2. Pathology and pathogenesis.

Diverticular disease: three studies. Part I--Relation to other disorders and fibre intake.

Roderick K. Clayton: a life, and some personal recollections.

Children's expectations and recollections of discomfort associated with dental treatment.

DQ polymorphism with Alzheimer's disease.

Wilson's disease and other neurological copper disorders.

Delirium tremens. Some clinical features. Part I.

Ethnopharmacological Approaches for Therapy of Jaundice: Part I.

[New approaches for the treatment of neurological diseases].

Some aspects of interactivity between endocrine and immune systems required for successful reproduction.

Retromer in Alzheimer disease, Parkinson disease and other neurological disorders.