Journal of Alzheimer’s Disease 13 (2008) 303–321 303 IOS Press Review Article

Colostrinin TM:AnOxidativeStress Modulator for Prevention and Treatment of Age-related Disorders

Istvan Boldogha and Marian L. Kruzelb,∗ aDepartment of Microbiology and Immunology, University of Texas Medical Branch at Galveston, TX 77555, USA bPharmaReview Corporation, Pearland, TX 77584, USA

Communicated by Thamarapu Srikrishnan

Abstract. Colostrum-derived proline-rich polypeptide, also known as ColostrininTM (CLN), has been shown to have a stabilizing effect on cognitive function in Alzheimer’s disease patients. This complex action of CLN could be related to prevention of amyloid- β peptide aggregation, as shown in in vitro studies, and its impact on delicate cassettes of signaling pathways common to cellular redox regulation, proliferation and differentiation. Studies on cultured cells showed that CLN modulates intracellular levels of reactive oxygen species (ROS), via regulation of glutathione metabolism, activity of antioxidant enzymes and mitochondria function. Due to an improvement in senescence-associated mitochondrial dysfunction and a decrease in ROS generation, CLN decelerates the aging processes of both cultured cells and experimental animals. When given orally to mice, CLN increased the lifespan and improved various motor and sensory activities. Although the molecular basis by which CLN exerts its diverse effects are still under investigation, the regulatory effect on the cellular redox state via maintenance of mitochondrial function and modification of ROS-induced cell signaling seem to be of great importance. In this article, we examine experimental data pertinent to the mechanism of action, including a review of CLN’s utility in the maintenance of physiological processes in which oxidative stress has an etiological role.

Keywords: Age-related disorders, Alzheimer’s disease, amyloid-β, immunomodulation, oxidative stress, proline-rich polypeptide

INTRODUCTION amino acid in which the side-chain is cyclized back on to the backbone amide position. This conformation Proline-rich regions of proteins are widely occurring produces a so-called “structural disturbance” and re- in both prokaryotes and eukaryotes. They are frequent- sults in proline-rich proteins and peptides (PRPs) as he- ly found as multiple tandem repeats in proteins of struc- lix and β-sheet breakers [130]. In many ways, it is not tural and functional importance. Despite significant surprising that proline-rich peptides are highly bioac- progress in understanding the structure–function rela- tive molecules. The unique chemical characteristics of tionship in proteins, the biological relevance of proline proline make these peptides responsible for a variety rich regions is still unclear. Proline is a very unusual of biological effects. PRPs have been reported to pos- sess activities ranging from antimicrobial action [83] to prevention of neurodegenerative disorders [48,87]. ∗ Corresponding author: Marian L. Kruzel, Ph.D., PharmaReview Numerous PRPs have been isolated from mammalian Corporation, 2605 Brightlake Way Ln., Pearland, TX 77584, USA. Tel.: +1 713 340 0447; Fax: +1 713 340 0442; E-mail: mlkruzel@ salivary glands and characterized as a “first line de- sbcglobal.net. fense” against the detrimental effects of tannins and

ISSN 1387-2877/08/$17.00  2008 – IOS Press and the authors. All rights reserved 304 I. Boldogh and M.L. Kruzel / ColostrininTM : An Oxidative Stress Modulator for Prevention and Treatment of Age-related Disorders polyphenols in diet [26]. Also, a family of PRP-related CLN obtained by this method appears to be consistent immunomodulators was discovered in the neurosecre- in: 1) molecular size of peptides by SDS PAGE and tory granules of the neurohypophysis [46]. However, high performance liquid chromatography (HPLC); 2) the most extensive studies involving human clinical tri- similar amino acid composition, characterized by high als have used colostrum-derived PRP, also known as content of proline (∼20%; Table 1); and 3) ability to ColostrininTM. The aim of this article is to review data induce cytokines. generated on cultured cells, experimental animals and Further studies utilizing HPLC and mass spec- human trials with regard to the possible mechanism(s) troscopy revealed that CLN is a complex of peptides of action of colostrum-derived PRP. with homology to three protein precursors: annexin, β-casein and a hypothetical β-casein homolog [71]. Remarkably, the sequence of these peptides shows no COLOSTRININTM: STRUCTURE AND significant homology to any specific protein in the cur- FUNCTION rent GenBank database. The synthetic peptides of vari- ous lengths representing the N-terminal sequence of the ColostrininTM (CLN) is found at particularly high CLN peptides are now available for the peptidomimetic concentrations in colostrum, or “first milking” of all studies. mammals. It has long been recognized that breast- The immunotropic activity of CLN was recognized feeding offers a pronounced enhancement of passive by Janusz and colleagues in the early seventies [61,63, immunity and promotes infantile gut immunity [25, 139]. Initially, the immune regulatory effect of CLN 50,68]. Thus, the constituents of colostrum, and, to was demonstrated on the humoral immune response some extent also of mature milk, not only ensure ade- in mice against sheep red blood cells [129]. CLN quate resistance to pathogens by delivery of maternal has been shown to induce maturation and differentia- immunoglobulins and other protective factors, but also tion of murine thymocytes, formation of helper cells play a crucial role in promoting maturation of the im- from PNAhigh thymocytes, and cytotoxic T cells from mune and central nervous system. Milk proteins are of- PNAlow thymocytes [60,141,142]. In addition, it was ten precursors of different biologically active peptides, demonstrated that CLN is a potent inducer of cytokines which are inactive within the sequence of the precursor both in vitro and in vivo [58]. Furthermore, the abil- protein and become active upon physiologically con- ity of CLN to induce IFN-γ was correlated with cell trolled proteolysis. Many colostrum-derived proteins proliferation shown by 3H-thymidine incorporation in- and peptides, such as PRPs and caseinophosphopep- to the DNA [117]. CLN has also been shown as a B tides, reveal multiple bioactivities [29]. Immunotropic cell-trophic mitogen. It activated resting mouse B cells properties of these peptides have been extensively stud- to enter the cell cycle at frequencies comparable with ied, predominantly in animal experimental models [70, those seen for LPS [64]. CLN, when administered in- 140]. traperitoneally, significantly lowered the incidence of CLN was first isolated from ovine colostrum and positive Comb’s reaction in New Zealand black mice. characterized as a complex of low-molecular-weight From a precursor pool of cells, CLN also induced sup- proline-rich polypeptides [61]. CLN is comprised pressor cells that control the development of hemolytic ∼22% of proline, non-polar amino acids, and low anemia [138]. percentage of glycine, alanine, arginine and histidine. There are no tryptophan, or cysteine residues [62]. CLN is not phosphorylated or glycosylated and the hy- COLOSTRININTM: DIVERSE BIOLOGICAL drophobic amino acids constitute ∼50% of the total ACTIVITY AND MECHANISM OF ACTION mass. The amino acid composition of CLN is very sim- ilar in all mammalian species [71,116]. However, due Because oxidative stress has been proposed as to individual variations in colostrums, it is very difficult an etiologic factor in many age-related disorders, it to obtain a consistently identical end-product. A new has become increasingly important to understand the purification method, consisting of alcohol extraction mechanism of action for therapeutic compounds that and membrane filtration, seems to improve the repro- may influence/affect cellular redox homeostasis. Al- ducibility of CLN [72]. More importantly, avoidance though a substantial body of evidence showed that of excessively harsh conditions during purification pre- CLN has diverse immune regulatory functions and serves the peptides’ structure and biological activity. cell/neuroprotective ability, it has been only recent- I. Boldogh and M.L. Kruzel / ColostrininTM : An Oxidative Stress Modulator for Prevention and Treatment of Age-related Disorders 305

Table 1 Amino acid analysis of CLN Amino Acid Colostrinin Ovine Original Ovine MeOH Bovine MeOH Method∗ Method∗∗ Method∗∗ Asp/Asn 3.42 2.80 5.13 Ser 5.66 5.05 6.94 Glu/Gln 15.48 15.77 17.99 Gly 3.15 3.03 2.95 His 2.54 2.14 3.28 Arg 2.32 3.34 1.62 Thr 5.73 5.30 4.32 Ala 2.78 2.13 3.06 Pro 21.07 22.50 20.79 Tyr 1.36 1.54 0.47 Val 9.27 11.10 8.15 Met 3.33 1.70 0.70 Lys 5.30 4.93 8.15 Ile 3.17 3.42 3.21 Leu 11.04 10.47 10.33 Phe 4.38 4.77 5.41 ∗Janusz et al. [61]. ∗∗Kruzel et al. [71,72]. ly demonstrated that it helps to maintain physiolog- nanomolar levels it can be involved in activation of sig- ical levels of oxygen- and nitrogen-centered reactive nal transduction pathways [28,36,77]. Most important- species [20,135]. As a pleiotropic factor, CLN induces ly, 4-HNE has been shown to be associated with the eti- not only cellular physiological responses but also pre- ology of degenerative disorders such as atherosclerosis, serves redox homeostasis in stressed cells, tissues and Parkinson’s disease, and Alzheimer’s disease (AD) [40, entire organisms (Fig. 1). 114]. Recent studies have investigated the effects of CLN The role of CLN in homeostasis of cellular redox and on 4-HNE-protein adduct formation, generation of signal transduction ROS, glutathione (GSH) metabolism, and on the mod- ification of the signal transduction cascade [17] in Reactive oxygen species (ROS), along with reactive cultured PC12 cells. Results show that treatment of nitrogen species (RNS), are essential secondary mes- PC12 cells with 4-HNE causes c-Jun N-terminal kinase sengers in inter- and intracellular signaling cascades. (JNK) activation (15–30 min post-treatment). How- However, ROS/RNS can also induce cellular patho- ever, when CLN was added prior to 4-HNE treat- logical responses that may lead to diseases conditions ment, JNK activation was not only delayed or re- or senescence processes and apoptosis. The cumula- duced, but was abolished. Additional results show tive production of ROS/RNS through either endoge- that CLN: 1) reduced the levels of 4-HNE-protein nous cellular sources or environmental insults caus- adducts, as shown by fluorescent microscopy and West- es oxidative stress, which is common to carcinogene- ern blot analysis; 2) decreased 4-HNE-induced intracel- sis, inflammation, aging-associated diseases and aging lular levels of ROS, as shown by 2’,7’-dichlorodihydro- processes (Fig. 2), in which levels of oxidative dam- fluorescein-mediated fluorescence; and 3) inhibited 4- age to proteins, lipids and DNA are increased. Ox- HNE-mediated extrusion of GSH via the cytoplasmic idatively damaged proteins are degraded and DNA is membrane into the extracellular milieu as determined repaired; however, oxidation of fatty acids leads to fluorimetrically. the generation of even more potent reactive molecules, It is well-documented that JNK regulates ma- such as 4-hydroxynonenal (4-HNE) and malondialde- jor cellular signaling cascades, along with mitogen- hyde(s) [39]. 4-HNE can react with the nucleophilic activated protein (MAP) kinases, the extracellular sites in DNA, mitochondrial proteins and a variety of signal-regulated kinase, and the p38 MAP kinase [94]. other nucleophiles, including GSH, resulting in cellu- JNKs are activated by a wide variety of stimuli, in- lar stress responses and oxidative stress [36]. While cluding ROS, DNA-damaging agents and inhibitors of micromolar concentrations of 4-HNE are cytotoxic, at protein synthesis, heat or osmotic shock [94]. These 306 I. Boldogh and M.L. Kruzel / ColostrininTM : An Oxidative Stress Modulator for Prevention and Treatment of Age-related Disorders

Fig. 1. CLN’s potential mode of action.

Fig. 2. CLN’s hypothetical mode of action via cellular redox mechanisms. stimuli appear to operate through small GTP-binding proteins such as Ras resulting in sequential activation of various protein kinases. Targets of the JNK sig- nal transduction pathway include the transcription fac- tors ATF2 and c-Jun (binds to the N-terminal region of ATF2 and c-Jun and phosphorylates two sites within the activation domain). These factors are members of the basic leucine zipper group that bind as homo- and heterodimeric complexes to AP-1 and AP-1-like sites in the promoters of several genes, resulting in increased transcriptional activity. Although the biological signif- icance of a CLN-mediated decrease in JNK activation needs to be established further, we can speculate that it may be important, e.g., in the prevention of amyloid- β β Fig. 3. Inhibition of 4-HNE-induced p53 activation by CLN. Cells (A )-induced apoptosis, and extension of lifespan were incubated for 24 h with CLN concentrations as indicated and in cell cultures or animal models, as well as neurite 4-HNE (50 nM) was added. Inset: Cells were lysed and Western outgrowth of neuronal precursor cells [5,8,9,109]. blot analysis was undertaken to determine levels of p53. 4-HNE, There is evidence that p53 lies at the center of a net- 4-hydroxynonenal. work of complex redox interactions [81]. In this net- work, p53 can control the timely production of ROS, kinases or to 4-HNE exposure [81]. Most important- but this activity itself is under the control of changes in ly, CLN showed a potent inhibitory effect on 4-HNE- cellular redox status [81]. Activation of p53 can occur induced activation (Fig. 3). Based on these results, in multiple ways: examples include an increase in ROS CLN modulates the balance between oxidants and an- levels, DNA damage, activations of cell cycle-check tioxidants, thereby also regulates redox-sensitive cel- I. Boldogh and M.L. Kruzel / ColostrininTM : An Oxidative Stress Modulator for Prevention and Treatment of Age-related Disorders 307

A B

Fig. 4. CLN decreases intracellular ROS levels and oxidative damage to DNA. A, CLN pre-treatment of cells prevents oxidant-induced increases in ROS levels. Cells were grown for five population doublings in media containing 100 ng per ml CLN. Cells were loaded with H2 DCF-DA (final con- centration: 5 µM) and challenged with H2O2 (100 µM), glucose oxidase (GO, 0.2 unit), or Aβ (10 µg per ml). Changes in DCF fluorescence were determined by flow cytometry (FACSAria, Beckton Dickinson). B, CLN decreases the augmentation of 8-oxo-7,8-dihydro-2’-deoxyguanosine levels induced by Aβ (left upper panel). SH-SY5Y cells were grown for five population doublings in media containing 100 ng per ml CLN. Then cell monolayers were treated with polymerized Aβ (10 µg per ml) for 3 hrs, fixed in formalin, and permeabilized by acetone:methanol (1:1). Levels of 8-oxodG were determined by fluorescent confocal imaging, as we previously described [59]. Lower panels show 4’6-diamidino-2-phenylindole dihydrochloride (DAPI)-stained images of nuclei. H2DCF-DA, 2’,7’-Dichlorodihydrofluorescein diacetate; DCF, Dichlorofluorescein; Aβ, amyloid-β. lular signaling that otherwise may lead to pathological neuroblastoma cell) were exposed to H2O2, glucose changes at the cellular and organ level. oxidase (GO), or Aβ (Fig. 4A). In line with this ob- CLN has no direct antioxidant activity, so the basic servation, CLN decreased Aβ-mediated guanine-base question is how is the cellular redox state modulated oxidation in the DNA, as shown by the immunochemi- by CLN? As it was demonstrated, CLN inhibited the cal detection of 8-oxo-7,8-dihydro-2-deoxyguanosine 4-HNE-mediated extrusion of GSH into the extracel- (Fig. 4B). lular milieu, which suggests that CLN might regulate An increase in the intracellular levels of ROS, as levels of the GSH pool by modulating the activity of shown by augmentation of lipid peroxidation, protein cell membrane-associated thiol transporters [20]. Cel- oxidation, and DNA oxidation, is a hallmark of various lular thiol levels and the cellular redox state of thiols disease conditions, including cognitive decline and ag- are critically important molecules in determining the ing. Intracellularly, ROS may be generated by cytoplas- redox status of the intracellular milieu, and therefore mic and membrane-associated oxidases (e.g., NADPH effects of CLN on multiple cellular processes, such as oxidase, diamine oxidase, and xanthine oxidoreduc- cell proliferation, differentiation, and apoptosis are not tase), but the major source of ROS is the mitochondria. surprising. Results from our microarray analyses show Mitochondria are discrete organelles that form a high- altered gene expression profiles of several genes that ly dynamic and interconnected cellular network, and are directly or indirectly involved in the maintenance of their numbers may exceed thousand per cell. The roles the cellular redox state. Examples include mitochon- of mitochondria are well-established in energy gener- drial respiratory complex proteins, cytoplasmic and mi- ation and metabolism, and they are considered to be tochondrial superoxide dismutases, heat shock protein major intracellular sources of ROS. A growing num- 32 (hemeoxygenase-1), and glutathione reductase that ber of neurological diseases are being linked to mito- catalyzes the reduction of glutathione disulfide (GSSG) chondrial dysfunctions described in sarcopenia, AD, to GSH [13]. Huntington’s, and other degenerative diseases [95]. Furthermore, our data show that pre-treatment of Mitochondria-generated ROS play an important role in cells with CLN protects against exogenous oxidants the release of cytochrome c and other pro-apoptotic such as H2O2, and Aβ, or against ROS generated proteins that can trigger caspase activation and apop- enzymatically. CLN did not change redox status of tosis. Importantly, CLN stabilizes mitochondrial func- the redox-sensitive 2’, 7’-dihydro-dichlorofluorescein tion in Aβ-treated human SH-SY5Y and rat PC12 cells (H2DCF) but resulted in decreased H2DCF oxidation, (Fig. 5) and during cellular aging/senescence process- when CLN pre-treated human cells (SH-SY5Y: human es [9]. 308 I. Boldogh and M.L. Kruzel / ColostrininTM : An Oxidative Stress Modulator for Prevention and Treatment of Age-related Disorders

Fig. 6. CLN decreases mutation frequency in cultured cells. A. Il- Fig. 5. Amyloid-β-induced mitochondrial H2O2 production is de- lustration of CLN-mediated decrease in mutation frequency. Arrows creased by CLN. SH-SY5Y and PC12 cells were maintained in me- point to crystal-violet-stained cell colonies mutated at the hypox- dia containing 100 ng CLN per ml. Cells were treated with Aβ, anthine phosphoribosyl-transferase gene locus. B. Chinese hamster and 24 h later mitochondria were isolated. Levels of H2O2 released V79 cells were treated with mutagenic agents in the presence of CLN from intact mitochondria were determined by Amplex red assays, as for 1 h. PBS-washed cells were further incubated in medium contain- we described earlier [10]. Rotenone, 3-NPA (3-nitropropionic acid), ing CLN. Cells (106) then were plated in 6-thioguanine–containing and Antimycin A are inhibitors of respiratory complexes I, II and III, medium. Twelve days later, the cell colonies, consisting of mu- respectively. Catalase was used as a control to degrade H2O2. FC- tated cells were stained for counting. Mutation frequencies were CP (carbonylcyanide-p-trifluoromethoxyphenylhydrazone) is a de- calculated as we previously described [3]. GO: glucose oxidase, a coupler of mitochondrial inner membrane potential. 3-NPA, 3-nitro- ROS-generating enzyme; H2O2: hydrogen peroxide; UV-A: ultravi- propionic acid. ∗∗p = 0.001, ∗∗∗p = 0.0001. olet A; MMS: methyl-methane sulfonate.

CLN protects against the spontaneous and induced tations developing spontaneously or induced by ROS, mutations chemical agents, and UV irradiation [5]. Since oxidative damage to DNA is the most common, studies were undertaken to investigate the antimuta- Oxidatively damaged proteins are usually eliminated genic effect of CLN in oxidatively stressed cells. Cellu- by degradation, while damaged DNA is repaired [91]. lar oxidative stress was produced by glucose oxidase, a ROS-, chemical- and physical-agents -mediated DNA known ROS generator [33] or by addition of H 2O2. Da- damage and their incorrect repair lead to DNA se- ta show that when CLN was present during oxidative in- quence alterations, which are implicated in carcinogen- sult of the cells, the mutation frequency decreased sig- esis [49]. Modifications in DNA sequence order al- nificantly (
p = 0.001) (Fig. 6). At present, the mech- so result in altered protein structures and/or activities anism by which CLN exerts its antimutagenic activity (e.g., p53), leading to changes in cell cycle progression, in oxidatively stressed cells is unclear. It is possible and dysregulation of the cellular redox balance, both of that CLN increases the activity of antioxidant enzymes which culminate in the manifestation of pathobiologi- and/or levels of cellular low-molecular-weight antiox- cal processes and diseases [91]. idants, such as glutathione. Another possibility is that Mutational analysis has been an important approach CLN increases the efficacy of DNA base excision re- for testing the genotoxicity of biologically active, natu- pair, the major pathways repairing oxidatively damaged ral, industrial and environmental compounds. A recent- DNA [91]. For example, there is evidence showing ly published study showed that CLN decreased ROS- the importance of redox-dependent, posttranslational induced mutagenicity [5]. In these studies, the pro- modifications of apurimic/apuriminidic endonuclease- tective effect of CLN against spontaneous and induced 1 (APE1/ref-1), a rate-limiting protein in base excision mutations was tested in Chinese hamster V79 cells at repair processes. These modifications of APE1 are the hypoxanthine phosphoribosyl-transferase gene lo- critical for its interaction with members of the repair cus. As described, CLN had no effect on cell prolifer- machinery and its endonuclease activity [67,101]. ation, and plating efficiency, and did not induce the ex- To test further whether CLN was only effective in pression of phosphatidyl serine on the outer cytoplas- suppressing ROS-induced mutagenesis, cells were ir- mic membrane, considered to be an early sign of cel- radiated with ultraviolet A (UVA). UVA radiation- lular toxicity [5]. Moreover, it has been demonstrated induced genomic instability has been linked to the pro- that CLN significantly decreases the frequency of mu- duction of ROS [53,128]. At concentrations of 100 I. Boldogh and M.L. Kruzel / ColostrininTM : An Oxidative Stress Modulator for Prevention and Treatment of Age-related Disorders 309 ng per ml or higher, CLN significantly inhibited UVA- induced mutagenesis. Regardless of the mechanism, these findings have great significance, since UV irra- diation is the primary cause of squamous cell carcino- mas [35] and malignant melanomas [82,111]. The UV doses that were chosen in this study are physiologically relevant because they were equivalent to midday sun exposure for 30 and 100 min [69,90]. These data im- ply that CLN is likely to be suitable for application in humans to decrease UV-induced cellular damage and its well-established carcinogenic effects [82,111]. CLN significantly decreased the mutation frequency induced by the DNA-damaging chemical agents methyl methanesulfonate (MMS) and mitomycin C (MMC). MMS primarily alkylates guanine at the O6-position, which is repaired by O6-methylguanine DNA methyl- transferase [89]. CLN substantially decreased MMS- induced mutation frequency (Fig. 6). The explana- tion may be that MMS-induced genomic damages are primarily due to guanine alkylation and are repaired by O6-methylguanine DNA methyltransferase’s action, which is not redox regulated [89]. These results sug- Fig. 7. CLN decreases eosinophil accumulation and mucin produc- gest that the substantial CLN-mediated decrease in mu- tion induced by the potent ragweed extract. Mice were sensitized tation frequency is independent from CLN’s impact on with alum-mixed ragweed pollen extract (RWE, 150 µg/mouse) on days 0 and 4, and on day 11 were intranasally-challenged with RWE cellular redox in MMS-treated cells. MMC is a DNA (50 µg per animal). At 72 h after challenge, lung sections were pre- cross-linking alkylating agent that,after getting through pared for hematoxylin-eosin and Periodic Acid Shift (PAS) staining. the cell membrane, is activated by NAD(P)H quinone Upper panels show accumulation of eosinolphils in lung sub epithe- oxido-reductase,xanthine dehydrogenase,xanthine ox- lium of mice challenged with RWE and CLN+RWE (left panel). Magnification × 33. Lower panels show PAS-stained mucin produc- idase, cytochrome P450 reductase, and/or cytochrome ing cells in RWE and CLN+RWE (left panel)-challenged lung ep- b5 reductase [30]. By directly interacting with DNA, ithelium of mice. Magnification × 120. Stained sections were pho- MMC induces an array of damage, including DNA tographed using a Photometrix CoolSNAP Fx CCD digital camera inter- and intra-crosslinks, single-strand breaks and mounted on a Nikon Eclipse TE 200 microscope. Arrows: upper pan- el: eosinophils in sub epithelium. Lower panels: mucin-producing double-strand breaks. Taking into consideration that cells. CLN decreases mutation frequency in GO, H2O2 and UV-irradiated cells, the significant decrease in MMC- and recruitment of inflammatory cells [74,124]. A induced mutation frequency by CLN is not surprising. study, which investigated potential allergic responses in In conclusion, these results suggest that the anti- a mouse model to CLN and its impact on allergic sen- mutagenic properties of CLN are mediated by multi- sitization and reactions caused by common allergens, ple but overlapping mechanisms: 1) a decrease in the showed that CLN did not increase IgE/IgG1 levels, or cellular levels of ROS and oxidative damage to DNA; induce cutaneous hypersensitivity reaction, airway in- and/or 2) CLN may increase the efficacy of DNA repair flammation and mucin production [19]. Importantly, via the generation of favorable cell activation signals CLN significantly (p<0.01) decreased IgE/IgG1 pro- and/or cellular responses to DNA damage. duction, eosinophilia, mucin production and airway hy- persensitivity induced by allergenic extracts from rag- CLN decreases allergic inflammation weed pollen grains and house dust mites [19]. Fig- ure 7 shows decreases in eosinophil accumulation and Allergenic molecules are processed via dendritic mucin production in lungs induced by the potent aller- cells and presented as peptides by class II major his- gen ragweed extract. The pollen of short ragweed (A. tocompatibility molecules to allergen-reactive T helper artemisiifolia) is one of the most abundant aeroaller- type 2 (Th2) cells [124]. These interactions orches- gens causing severe seasonal allergic rhinitis, conjunc- trate a cascade of events that lead to IgE synthesis tivitis and airway inflammation in the United States and 310 I. Boldogh and M.L. 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Europe [122,132]. The house dust mite extract was ther indicate the existence of potential mechanisms by isolated from D. pteronyssinus and D. farinae, the most which CLN could modulate a cellular network result- common sources of indoor allergens worldwide [123]. ing in cytokine and chemokine production that decreas- More than 50% of patients with allergies and up to es/prevents inflammatory processes induced by aller- 80% of asthmatic children are sensitized to mite aller- gens. gens [22,34]. It has been shown previously that allergen-bearing CLN induces neurite outgrowth in cultured cells pollens induce airway inflammation in sensitized indi- viduals, and the recruited inflammatory cells produce Neurites are essential for intercellular communica- oxidative stress in the airways [37,86]. Furthermore, tion in the nervous system. Recent studies have shown in addition to their allergenic proteins, pollen grains that CLN induces neuronal differentiation via two in- or their extracts contain NAD(P)H oxidases, which terrelated cellular processes: 1) inhibition of cell pro- increase oxidative stress levels in mucosal epithelial liferation and 2) progression through the stages of neu- cells and the airway epithelium within minutes of ex- rite outgrowth [8]. The induction of neuronal differ- posure [7,16]. This oxidative stress is required for entiation by CLN can be demonstrated in cultures of antigen-mediated robust inflammation in the lower air- normal human epithelial, human neuroblastoma (SH- ways and conjunctiva [7,16]. CLN-mediated decreas- SY-5Y) and rat medullary pheochromocytoma (PC12) es in inflammatory responses to ragweed pollen ex- cells (Fig. 8). Detailed mechanistic studies on CLN- tract may be explained by the fact that CLN-treated mediated neurite outgrowth were carried out using cells have an increased ability to cope with oxida- PC12 cells, developed from a transplantable chromaf- tive stress [5,20,109]. CLN conditioning also de- fin tumor. PC12 cells respond to nerve growth factor creased house dust mite extract-mediated inflammato- (NGF) and other factors with a change in gene expres- ry responses, although it contains proteases to invade sion and/or morphology[84] and are suitable for testing the site of exposure and potentate allergic immune re- biological activity of materials such as CLN. sponses [66,123]. Neurite outgrowth is a tightly regulated cellular pro- Although these studies convincingly show CLN’s cess induced by hormones and neurotrophins such as antiallergenic capacity, the mechanism implicated in NGF both in vivo and in cultured cells. For example, these processes is not known. Whether CLN-induced neurite growth induced by NGF requires the tyrosine immunemodulators (e.g., IF-γ, IL-6, IL-10) have an kinase (TrkA) receptor and a cascade of events through impact on the allergic sensitization processes or the the Ras/MAPK pathway [32,131]. PC12 cells contain manifestations of allergic reactions is the subject of both the TrkA and low-affinity NGF receptors [32,54]. speculation. For example, it may be proposed that CLN Studies on CLN-mediated PC12 differentiation show alters Th2 responses and switches cellular chemokine an anti-proliferative effect, followed by neurite out- and cytokine production towards Th1. Our specula- growth. The percentage of cells showing CLN-induced tion relies on previously published data showing that neurite outgrowth was concentration-dependent in a CLN alters the production of IFN-γ, IFN-α, IL-6 and manner similar to that of NGF, the best characterized IL-10 [134,136]. Th1 cells have been characterized by agonist [80,126]. the production of IL-2 and IFN-γ, whereas Th2 cells Studies show that CLN inhibited cell proliferation secreted IL-4, stimulating IgE production [93]. At this via the p53-p21WAF1 pathway, resulting in an accumu- time, it is not known if CLN mediated any change in lation of cells in the G1 phase of the cell cycle. In- IFN-γ levels in our model system; however, it has been deed, the major step in neurite outgrowth is cell cy- shown that IFN-γ counteracts IgE production in cell cle arrest via p53 stabilization. p53 exists in latent culture systems of both human and mouse origin [43, and antiproliferative forms that differ in their degree 102]. Indeed, CLN significantly (p = 0.001) decreased of post-translational modification, e.g., phosphoryla- serum levels of IgE/IgG1, cutaneous reactions in RWE- tion, acetylation [52,81] and their subcellular distribu- challenged animals and those exposed to house dust tion [112]. Activated p53 alters the transcription of mite extracts [9]. It has also been shown that CLN genes, many of which regulate cell proliferation [52, mediates maturation and differentiation of murine thy- 81,84] and are required for differentiation [4,98]. The mocytes, promotes proliferation of peripheral blood observed stabilization of p53 was due to phosphory- leukocytes, and induces immunomodulators, including lation of p53 at serine 15 that accumulated in the nu- various cytokines [60,62,117,141]. These results fur- clei of CLN-treated PC12 cells. These events oc- I. Boldogh and M.L. Kruzel / ColostrininTM : An Oxidative Stress Modulator for Prevention and Treatment of Age-related Disorders 311

Fig. 8. CLN induces cell differentiation. Human primary epithelial cells, human neuroblastoma (SH-SY-5Y) and pheochromocytoma (PC12) cells were cultured in their well-defined culture media [6,8] and then treated with 100 ng per ml CLN. Neurite outgrowths were visualized microscopically and photographed with a Photometrix CoolSNAP Fx camera mounted on a NIKON Eclipse TE 200 UV microscope. curred in parallel with an increase in p21WAF1 level and from senescence-resistant (MDFR) and senescence- cell cycle arrest. PC12 cells transfected with inhibito- prone (MDFSP) mice. In studies by Bacsi and col- ry oligonucleotides specific for p21WAF1 knocked out leagues [9], MDF cultures were continuously passaged CLN-induced p21WAF1 expression, while p53 activa- at ambient oxygen concentration until they reached a tion was not altered. These inhibitory oligonucleotides crisis in growth, and showed morphological charac- significantly decreased p53-p21WAF1 activation and teristics of senescence. Cells in the state of replica- the percentage of cells showing neurite outgrowth in tive senescence exhibited a low saturation density, and both CLN and NGF-treated cultures. These results an increased population-doubling time, along with in- strongly suggest that CLN induced a cascade of events creasing number of enlarged and polyploid cells. These in PC12 cells similar to that of neurtrophins. changes occurred in cultures of MDFSP at 10–12 pop- CLN, like NGF, induces GAP-43 expression in PC12 ulation doubling levels (PDL), much earlier than that cells during differentiation processes [8]. GAP-43 is of MDFR cells (16–19 PDL). When CLN was added a marker of neuronal differentiation whose expression (250 ng per ml) into the growth medium, the lifespans is restricted to the nervous system during development were increased in both cell cultures. The MDFSP cell’s and regeneration [12]. GAP-43 expression requires lifespan increased significantly (p = 0.001) from 11–12 Ras-dependent integrated signaling through extracel- PDLs to 15–17 PDLs. CLN has increased the lifespan lular signal-regulated kinase, JNK, and MAP [88,103, of MDFR cultures from 15–16 to 20–21 PDLs. Such 133]. Neurite outgrowth of PC12 cells induced by an effect of CLN on PDL of MDFSP cells was concen- CLN showed kinetics similar to that caused by NGF. tration dependent [9]. The most remarkable observa- This raised the possibility that CLN may interact with tion was that CLN significantly (p = 0.001) decreased the NGF receptor, TrkA and utilize a cell activation spontaneous immortalization frequency. pathway similar to that of NGF. The neurite outgrowth As described by Bacsi and his colleagues [9], at induced by combination of NGF and CLN was only PDL4 and PDL7 there was no significant difference in slightly higher than that caused by NGF or CLN alone, ROS levels between MDFSP and MDFR cells. Com- suggesting that the cell surface receptor(s) may not be pared to cells at PDL4, MDFSP cells at PDL9 showed different [8]. Taken together, it may be proposed that a two-fold increase in ROS levels while, at the stage CLN via cell surface receptor(s) mediates a wide spec- of replicative senescence (PDL 12), MDFSP cells there trum of activities similar to those induced by hormones was a greater then three-fold increase in ROS levels. and neurotrophins leading to neurite outgrowth. ROS levels in MDFR cells gradually increased from PDL12 and reached a greater then two-fold increase CLN increases the lifespan of cultured cells by at PDL16. Most importantly, in MDFSP and MDFR preventing mitochondrial dysfunction cultures that were maintained in a medium containing CLN, ROS levels were significantly lower, even at a Increased ROS generation and mitochondrial dys- stage of replicative senescence, when compared to cul- function contribute to aging and neurodegenerativedis- tures that contained no CLN in their media. eases, as shown in human studies, in transgenic rodent Oxidative damage to mitochondrial respiratory com- models and cell culture studies [11,23,24,38]. To study plexes and other molecular components leads to a whether CLN delays the onset of replicative senes- vicious cycle of sustained ROS generation, and in- cence, murine diploid fibroblast cells (MDF) were used creasing mitochondrial damage, that adversely affects 312 I. Boldogh and M.L. Kruzel / ColostrininTM : An Oxidative Stress Modulator for Prevention and Treatment of Age-related Disorders

SP R A MDF and MDF cells, because the higher oxida- tive stress observed in the accelerated senescence-prone strains of mice are associated with mitochondrial dys- functions [55]. Indeed, in-depth investigations us- ing microscopic imaging (Fig. 9A) and biochemical assays showed that mitochondria are the source of ROS in these cells [9]. Further, mitochondria from cells reaching the senescence stage generated signif- icantly higher amounts of ROS than did those ac- tively replicating MDF cells (Fig. 9B). Remarkably, mitochondria from cells maintained in the presence of CLN generated significantly less ROS (Fig. 9A). The CLN-mediated decrease in mitochondrial ROS levels (Fig. 9B) is in agreement with its ability to lower levels of oxidative stress [17,18] and extend lifespan [9]. An increase in the lifespan of diploid cul- tured cells over-expressing mitochondrial antioxidant enzymes was previously documented [137]. Based on observations described above and shown in Fig. 9, we speculate that CLN increases mitochondrial GSH levels B and/or activity of antioxidant enzymes thus improving mitochondrial function and delaying the development of an increased oxidative state and cellular senescence processes. Those approaches that aimed to improve mitochon- drial dysfunction and/or lower oxidative stress levels in age-associated diseases have attributed a great im- portance to CLN. Oxidative damage to mitochondrial respiratory complexes and other inner membrane pro- teins lead to a vicious cycle of increasing mitochondrial damage and sustained ROS generation, which adverse- Fig. 9. Intra-mitochondrial oxidative stress levels in CLN-treated cells. A, Senescent MDF cells in a thermo-controlled microscopic ly affects tissue function in vital organs and the cen- chamber were loaded with 2 µM dihydroethidium (H2Et; Molecular tral nervous system [11,41,56,76,96,113]. The precise Probes, Eugene, OR) for 10 min, and excess probe was removed by mechanism by which CLN delays senescent process- washing cells with PBS. MitoTracker Red (Molecular Probes, Eu- gene, OR), a cell-permeable fluorescent probe that accumulates in es is unknown; however, these data suggest that CLN mitochondria, was used to stain mitochondria at a final concentra- may be used in preventive and therapeutic approach- tion of 50 nM. Fluorescent images were captured by a Photometrix es to delay the onset of symptoms of age-associated Cool-SNAP Fx digital camera mounted on a NIKON Eclipse TE 200 diseases that have been shown to be associated with UV microscope. The microscopic images were superimposed by us- ing MetamorphTM Version7.0r software (Universal Imaging, Down- mitochondrial dysfunction. ingtown, Pennsylvania). The MitoTracker-mediated image overlaps with the dihydroethidium/superoxide fluorescence after superimpo- CLN protects against amyloid-β-induced apoptosis in sition of images showing that the sites of oxidative stress are the mi- neuroblastoma cells tochondria. B, CLN decreases age-associated oxidative stress levels. Parallel cultures of PDL-3, PDL-10 and PDL-16 MDF cells in a ther- mo-controlled microscopic chamber were loaded with 2 µMH2Et Amyloidosis is a neuronal disease caused by aber- for 10 min, and excess probe was removed by washing cells with rant protein folding in which insoluble globular pro- PBS. Fluorescent images were captured and fluorescent intensities teins are deposited in the brain tissues, leading to tis- determined using MetamorphTM Version 7.0r software (Universal Imaging, Downingtown, Pennsylvania). FLI, fluorescence intensity; sue damage and disease [51]. Epidemiological studies PDL, population doubling level; A.U, arbitrary units. have shown that the alterations in the metabolism of the amyloid-β protein precursor (AβPP) and the formation functions that cause a loss of ATP-generating capac- of Aβ plaques are the main cause of neuronal death ity [76,113]. These observations may be valid for in AD [47]. These plaques promote oxidative stress- I. Boldogh and M.L. Kruzel / ColostrininTM : An Oxidative Stress Modulator for Prevention and Treatment of Age-related Disorders 313 induced injury in the brains of AD patients. More- Whether CLN inhibits fibril formation by Aβ and al- over, Aβ plaques have been shown to instigate cells so decreases the toxicity of soluble Aβ monomers and to produce excessive quantities of free radicals, which oligomers in vivo are yet to be assessed. Inhibition of cause further damage to brain cells [75,104,125]. The fibril formation may lead to increased levels of soluble successful development of clinical protocols using an- Aβ monomers/oligomers that are thought to be impor- tioxidants for AD solely depends on the availability of tant effectors of synapse loss and neuronal injury [127]. products that are safe and have a proven mechanism of CLN has been shown to have a stabilizing effect on action. cognitive functions, including long-term memory en- Schuster and his colleagues reported that CLN hancement in AD patients [78], and therefore it may be prevented the aggregation of Aβ peptide (1–40) in proposed that CLN not only inhibits fibril formation, vitro [109]. The impact of CLN on fibril formation but decreases the toxicity of Aβ monomers/oligomers was monitored by optical and electron microscopy. in vivo. The microscopic investigations showed that Aβ (1–40) peptides-formed fibrils (25 µm in length) after 24–48 hours of incubation. The presence of 250 nM CLN COLOSTRININTM: EFFECTS ON LEARNING, β completely abolished the fibril formation. A (1–42) MEMORY AND LIFESPAN IN ANIMAL peptides grow into dense fibers when examined at the MODELS 20th day. In the presence of CLN, however, the fibrils are much shorter and less dense. More importantly, the In the late nineties, the in vivo and in vitro im- addition of CLN as late as the 17th day can dissolve munomodulatory properties of CLN were linked to neu- the preformed fibrils. These observations were com- roprotective activity in humans and animals. In par- pared to the effects of CLN on the neurotoxic activity of Aβ peptides in cell culture model (SHSY-5Y). The ticular, the effects of CLN on spatial learning and in- Aβ peptides were pre-incubated with CLN at various cidental memory in rats of two age groups, 13-month- length of times and SHSY-5Y cells were treated for up old (aged) and 3-month-old (young), were spectacu- µ to 4 days [109]. The cytotoxic effect was monitored by lar [99]. CLN at low doses (4 g per rat) facilitates the trypan blue exclusion. The results showed that 24–48 acquisition of spatial learning of aged (13 months old), hrs treatment resulted in toxicity of 10–50 µMofAβ but not young (3-month-old) rats. The statistically sig- peptides. Pre-incubation of 0.0025–0.25 µM of CLN nificant difference between placebo-treated and CLN- with 25 µMofAβ peptides led to near-complete abo- treated animals suggests that CLN improves the ability lition of cytotoxicity. Importantly, low doses of CLN of aged animals subjected to the Morris Water Maze (e.g., 2.5 nM) attained cytotoxic protection similar to test to find safety on a platform in the tank. Detailed those seen at the highest doses (e.g., 0.25 µM). The analysis of swimming behavior indicated that the aged time course for the appearance of Aβ fibrils coincides placebo-treated rats were impaired in the latter stages with that for cytotoxicity, and the reduction of fibrils of of spatial learning. Treatment with CLN resulted in a Aβ peptides by CLN is concomitant with the reduction significantly (p = 0.05) higher precision in the animals of the cytotoxic effects of Aβ on SHSY-5Y neuroblas- ability to find the platform as revealed by the higher toma cells. These studies suggest that the neuroprotec- number of direct hits. The fact that aged CLN-treated tive effects exerted by CLN are related to the decrease animals were not impaired suggests that CLN improved in Aβ fibrils [109]. the ability of aged subjects to find the platform more The impact of CLN on fibril formation was mea- effectively [99]. In similar studies, treatment with one sured by the thioflavin T (ThT) method [21]. The as- of the constituent peptides of CLN (called nonapep- say measures changes in fluorescence intensity of ThT tide: Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro) substan- upon binding to amyloid fibrils. Unbound ThT does tially, but not significantly affected the acquisition of not fluoresce. Consequently, it can be used to moni- spatial learning and memory retrieval in the Morris Wa- tor the effect of drugs in their capacity to inhibit Aβ ter Maze test [100]. The difference in cognitive effects aggregation. The enhanced fluorescence is easily ob- between CLN and the CLN-derived nonapeptide may served using this spectroscopic assay, and is common- be explained by a possible modification of its chemi- ly used to monitor fibril formation over time. It has cal structure when it is isolated or by the absence of been demonstrated that CLN effectively decreases the other peptides also required for mediation of the highly aggregation of Aβ and fibril formation [109] in vitro. complex cognitive activities. 314 I. Boldogh and M.L. Kruzel / ColostrininTM : An Oxidative Stress Modulator for Prevention and Treatment of Age-related Disorders

ing/memory capacities [15]. Improved neurological performance correlated well with the decreased levels of oxidative stress markers measured in various organs. In particular, it has been shown that CLN decreased lev- els of 8-oxo-7,8-dihydro-2’-deoxyguanosine in DNA and significantly lowered oxidative damage to proteins in brain and liver [15]. The results of these comprehen- sive studies are in agreement with investigations that describe extended lifespan in cultured cells [9]. We propose that at a cellular level CLN binds to not-yet- identified membrane receptor(s) and modulate cellular signaling and the cellular redox state via mitochondrial Fig. 10. CLN extends of lifespan of mice. Mice were fed with CLN metabolic processes, resulting in improved motor and (via drinking water, 1 or 0.1 µg per kg per day) from 3 months of age for their entire lifespan. Control mice received the same amounts of sensory activities and prolonged lifespans. colostrum or the solvent (PBS). CLN-fed group: male mice (n = 40) Stewart and Banks have shown that CLN derived and female mice (n = 45) were used to construct the survival curves. from ovine or bovine sources exhibits potency as a Colostrum-fed group: male mice (n = 41); female mice (n = 40), cognitive enhancer of avoidance training on the weak solvent-fed group: male mice (n = 37), female mice (n = 34). A control group of animals (female: n = 27; male n = 31) were left aversive stimulus of 10% methyl antranilate in new- untreated. ly hatched chicks [119]. CLN’s efficacy on improv- ing memory for the weak aversant showed a dose– To have a more comprehensive understanding of the response curve with potency over a 100-fold range. It effect of CLN on experimental animals, the lifespan brings the retention of memory up to the level normally of New Zealand black mice was determined [138]. In seen with 100% methyl antranilate [85]. The memo- these studies parallel groups of mice were treated with ry enhancement by CLN for the weak aversant (10% 10 ng, 100 ng, and 1000 ng of CLN twice a week be- methyl antranilate) is comparable to that shown previ- ginning on day 165 of their life. Importantly, the lifes- ously in the chick passive avoidance model [85,106] pans of these mice were increased significantly (p = where memory was improved with compounds which 0.01) when treated with 100 ng of CLN, compared to stimulated corticosterone action [85]. The cognitive mock (saline)-treated animals. Administration of 10 ng enhancing effect of CLN was independent of whether and 1000 ng of CLN only substantially increased their it was intracranially or intraperitoneally administered. lifespan. The senescence-accelerated mouse is a useful The likely explanation for this finding is that in the animal model to study aging or age-associated disor- newly hatched chicks, the blood-brain barrier is incom- ders due to its inherited aging phenotype [121]. In a plete, thus allowing rapid access of CLN to the brain. recently conducted study, the effects of the oral admin- In mammals where there is a notable blood brain barri- istration of CLN on the lifespan and on various behav- er, CLN can exert a cognitive effect even when injected ior characteristics in senescence-accelerated mice were intra-peritoneally. These data are supported by a study investigated (Fig. 10). Results show that CLN admin- on a rat model [100]. It has been demonstrated that istration via drinking water significantly increased the CLN injected intraperitoneally in older animals (of 13 lifespan of these mice [15]. months of age) at a dose of 4 µg per rat facilitated the Groups of CLN-fed mice were also tested for their acquisition of spatial learning. At this concentration, it ability to perform an age-sensitive battery of tests for also improved incidental learning. CLN also has effi- cognitive and motor function. A swim maze task was cacy in the treatment of humans suffering from mild or employed to measure the ability of the mice to learn and moderate AD [14]. Although the precise mechanism remember the location of a hidden platform. This task of CLN’s action in enhancing memory is unclear, one is dependent on cortical and hippocampal functions. In possibility is that this may involve second step in a two- addition, a set of psychomotor tests was used to evalu- stage memory process [105]. The first does not require ate different dimensions of age-associated loss, includ- de novo synthetic processes and decays at some 6–8 h ing spontaneous locomotion, coordinated running, bal- post-training, whereas the second stage requires a cas- ance, muscle strength, sensory reactivity, and reaction cade of synthetic events which ensure that memory is time. Data in Fig. 11 show that CLN administration to formed as a long-term store. The weak (10% methyl mice improves age-associated locomotion, and learn- antranilate) avoidance training task fails to stimulate I. Boldogh and M.L. Kruzel / ColostrininTM : An Oxidative Stress Modulator for Prevention and Treatment of Age-related Disorders 315 the second wave of events and thus memory is lost after of AD. Although memantine is less toxic than other some 6–8 h. CLN may exert an effect by acting upon potential drugs, its effectiveness in AD is still under the transmitter/receptor systems which are known to continuous evaluation. Inflammation surrounding Aβ be rapidly up-regulated on weak avoidance learning in plaques is thought to be a key factor in the progres- the chick, influenced by N-methyl-D-aspartate recep- sion of AD dementia. Therefore nonsteroidal anti- tor agonists [118]. If so, then the cascade of process- inflammatory drugs have been tested in various preven- es involved in long-term memory formation including tive protocols [57,120]. In general, nonsteroidal anti- protein synthesis are initiated and, hence, the second inflammatory drugs show some neuroprotective effect, phase of memory formation also occurs and at 24 h the but the overall benefit for AD patients is not signif- results of memory retention following treatment with icant [2,107]. Also, evidence is growing that Gink- 10% methyl antranilate is similar to the test seen with go biloba extract improves cognitive functions in AD 100% methyl antranilate. These data show a clear en- patients [97]. Although the overall benefit of Ginkgo hancing effect of CLN on training for a weak, aver- therapy is accepted, there have been some side effects, sive task, and demonstrate its widespread efficacy as a including coma, bleeding, and seizures [42,45,92]. In cognitive enhancer in other animal species and humans. another approach, scientists have been exploring the possibilities of a vaccine-based therapy for AD. Vac- cination of transgenic mice against human Aβ, a con- COLOSTRININTM: CLINICAL UTILITY IN stituent of senile plaques in AD, led to clearance of this ALZHEIMER’S DISEASE protein from the brain [108]. However, in human trials, some subjects developed autoimmune encephalitis that Dementia is a brain disorder that seriously affects a prompted termination of the trial [27]. person’s ability to carry out daily activities. One of Evidence continues to accumulate that protection the most common forms of dementia, known as AD, against increased levels of intracellular ROS may pro- involves the frontal lobe of the brain and affects such vide the best approach for the prevention of cognitive cognitive functions as language, thought processes and decline in elderly patients. Oxidative stress is one of memory [65,115]. The hallmarks of AD pathology are the earliest events of AD, and may prove to be an im- the neuritic plaques, microscopic foci of extracellular portant mediator in the onset, progression and patho- Aβ deposition, and the neurofibrillary tangles, intracel- genesis of the disease. The sources of ROS-mediated lular fibrils composed of hyperphosphorylatedTau pro- damage appear to be multi-faceted in AD, with inter- tein. There is substantial evidence showing that both actions between abnormal mitochondria, NADPH oxi- Aβ deposits and Tau protein fibrils are the products of dase, transition metals, and other factors. misfolded proteins largely involved in increased pro- Recently, significant progress has been made in test- duction of ROS in brain [1,73,110]. Although there is ing the clinical utility of CLN. In the first observational major progress in understanding pathogenesis of AD clinical trials, volunteers were treated with low doses of dementia, the preventive and therapeutic measures still CLN (100 and 200 µg/day/patient) for three weeks. As have to be explored. a result of the treatment, volunteers developed a tran- There are a number of drugs presently employed to sient tolerance to CLN, as measured by the induction of alleviate the effects of AD dementia or delay its pro- IFN-γ and TNF-α in the whole blood cultures [58]. It gression; however, the treatments are symptomatic and is worth mentioning that CLN tolerance has been docu- there is no cure to reverse AD pathology. A family of mented in only two volunteers. No statistical data were cholinesterase inhibitor drugs (tacrine, donepezil, met- obtained, and, more importantly, the two volunteers re- rifonate, rivastigmine and galantamine),which increase sponded differently. Interestingly, these initial findings the availability of acetylcholine in central synapses has were used for the development of a clinical protocol for been approved by the FDA for the treatment of AD a study in AD. Hence the protocol for the subsequent dementia since the 1990s. However, these drugs only interventional study included a 2 weeks hiatus between offer short-term benefits during the early stages of AD. 3 weeks long sessions of CLN treatment [78,79]. Ini- Moreover acetylcholinesterase inhibitors cause signif- tially 46 AD patients were recruited into the study. Pa- icant side effects, including nausea, anorexia, vomit- tients were randomly assigned for treatment with CLN ing, and diarrhea [31]. Recently, memantine, a non- (100 µg per dose, every other day); commercially avail- competitive antagonist of glutamateric NMDA recep- able bioorganic selenium (100 µg per dose, every other tors has been approved for treatment of advanced stages day) or placebo tablets. Each patient received 10 cycles 316 I. Boldogh and M.L. Kruzel / ColostrininTM : An Oxidative Stress Modulator for Prevention and Treatment of Age-related Disorders

Fig. 11. Effect of CLN on learning and memory in young, middle-aged and old animals. The length of the path taken to reach the hidden platform was analyzed to assess the efficiency with which the mice located the platform independently of their speed of swimming [44]. ∗Significant difference compared to control (solvent-fed) animals (individual comparison within three-way ANOVA). of treatment during the year of the clinical trial (3 weeks rameters were evaluated at baseline between weeks 15 of treatment and a 2 weeks hiatus). The clinical benefits and 30. Two separate statistical analyses were under- were assessed by psychiatrists who were blinded to the taken, a full sample analysis, in which all missing val- type of treatment. Eight of the 15 AD patients treated ues were replaced with the worst result observed and a with CLN improved and the disease had stabilized in 7 valid for-efficacy analysis, in which those patients who others. In contrast, none of the 31 patients from the se- had serious protocol violations were excluded. This lenium or placebo groups improved. Most importantly, resulted in 14 patients being excluded from the valid in addition to significant improvements in disease, no efficacy-analysis. The full-sample analysis at week serious side effects were reported during this study. In 15 showed a significant (p = 0.02) stabilizing effect a subsequent trial, 33 patients with mild to moderately of CLN on cognitive functions in the AD assessment, severe AD symptoms were enrolled for long-term CLN scale-cognitive test and on instrumental activities of treatment (16–28 months) and evaluated by the Mini- daily living. The overall patient response was also in Mental-State Examination (MMSE) scale [78]. Again, favor of the active treatment (p = 0.03). Patients grad- the results showed that CLN given orally (100 µg per ed as mild on entry also showed a superior (p = 0.01) every other day) resulted in stabilization of the disease response, as measured by the AD assessment scale, with a trend of improvement. The adverse reactions cognitive test compared with more advanced cases. Ev- (including anxiety, logorrhea, and insomnia) were re- idence from this study indicates an early, beneficial ef- markably mild. fect on cognitive symptoms and daily function. Again, The largest interventional study with CLN in AD was based on the current knowledge of CLN safety and tox- recently reported in the Journal of Alzheimer’s Dis- icity, it seems reasonable to revise the original clinical ease [14]. The study was designed to confirm or negate protocol and consider a different drug regimen. In the findings from earlier trials demonstrating that CLN has context of this potential re-evaluation of the original potential for the treatment of mild or moderate AD. One protocol, it is worth mentioning a recent presentation hundred and five patients were recruited from six psy- entitled “Colostrinin increases the lifespan and neuro- chiatric centers in Poland. The trial consisted of a 15 logical performance in senescence accelerated mouse weeks double-blind phase comparing CLN with place- model” [15]. In this study the beneficial effect was bo, followed by a second 15 weeks open-labeled phase demonstrated for mice treated with CLN daily, at a dose when all patients received CLN. The dosage of CLN equivalent to that used in human study for the entire was 100 µg on alternate days for three weeks, followed lifespan. Thus, it seems apparent that the use of CLN by a 2 weeks drug-free period. This cycle was repeat- is safe and no toxicity is associated with daily low dose ed three times for each phase. The primary outcome administration of CLN for a prolong period of time. measures used were an AD assessment scale-cognitive portion and clinical global impression of change. Sec- ondary outcome measures were instrumental activities CONCLUSION of daily living (MMSE): the AD assessment scale, non- cognitive test and overall patient response. The main Oxidative stress is believed to be one of the leading outcome measures were assessed at week 15 when ac- contributor to the aging processes and to age-associated tive treatment was compared with placebo, but all pa- diseases, such as AD, the most frequent form of demen- I. Boldogh and M.L. Kruzel / ColostrininTM : An Oxidative Stress Modulator for Prevention and Treatment of Age-related Disorders 317 tia among the elderly. Oxidative damage occurs early [3] T. Albrecht, M.P. Fons, C.Z. Deng and I. Boldogh, Increased the in brain of AD patients, before the onset of plaque frequency of specific locus mutation following human cy- pathology, precedes Aβ deposition, and is character- tomegalovirus infection, Virology 230 (1997), 48–61. [4] R. Aloni-Grinstein, I. Zan-Bar, I. Alboum, N. Goldfinger and ized by such neuropathologicalhallmarks as extracellu- V. Rotter, Wild type p53 functions as a control protein in lar Aβ plaques and intracellular neurofibrillary tangles, the differentiation pathway of the B-cell lineage, Oncogene composed of the abnormally hyperphosphorylatedpro- 8 (1993), 3297–3305. β [5] A. Bacsi, L. Aguilera-Aguirre, P. German, M.L. Kruzel and tein, tau. Tau and A , as well as mitochondrial dys- I. Boldogh, Colostrinin decreases spontaneous and induced function, exhibit synergistic effects leading to deficient mutation frequencies at the hprt locus in Chinese hamster energy metabolism and accelerated neurodegeneration. V79 cells, J Exp Ther Oncol 5 (2006), 249–259. When given orally, CLN has been shown to have a sta- [6] A. Bacsi, B.K. Choudhury, N. Dharajiya, S. Sur and I. Boldogh, Subpollen particles: carriers of allergenic proteins bilizing effect on cognitive functions in improving the and oxidases, J Allergy Clin Immunol 118 (2006), 844–850. conditions of patients suffering from mild and moder- [7] A. Bacsi, N. Dharajiya, B.K. Choudhury, S. Sur and I. ate AD. In line with these findings, CLN resulted in Boldogh, Effect of pollen-mediated oxidative stress on im- a 100-fold enhancement of retention of memory and mediate hypersensitivity reactions and late-phase inflamma- tion in allergic conjunctivitis, J Allergy Clin Immunol 116 learning and improved various motor and sensory ac- (2005), 836–843. tivities in animal models. These observations, support- [8] A. Bacsi, G.J. Stanton, T.K. Hughes, M. Kruze and I. ed by findings in cell culture models including mod- Boldogh, Colostrinin-driven neurite outgrowth requires p53 ulation of cellular redox, prevention of mitochondri- activation in PC12 cells, Cell Mol Neurobiol 25 (2005), 1123–1139. al dysfunction in oxidatively-stressed cells, as well as [9] A. Bacsi, M. Woodberry, M.L. Kruzel and I. Boldogh, induction of cell differentiation/neurite outgrowth, are Colostrinin delays the onset of proliferative senescence of similar to those induced by hormones and neurotropins. diploid murine fibroblast cells, Neuropeptides 41 (2007), 93– Peptides in CLN bind to as-yet-unidentified membrane 101. [10] A. Bacsi, M. Woodberry, W. Widger, J. Papaconstantinou, receptor(s), and induce signaling pathways that appear S. Mitra, J.W. Peterson and I. 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Holmgren, Thiol-based mecha- central nervous system. nisms of the thioredoxin and glutaredoxin systems: implica- tions for diseases in the cardiovascular system, Am J Physiol Heart Circ Physiol 292 (2007), H1227–H1236. [14] A. Bilikiewicz and W. Gaus, Colostrinin (a naturally occur- ACKNOWLEDGEMENT ring, proline-rich, polypeptide mixture) in the treatment of Alzheimer’s disease, J Alzheimers Dis 6 (2004), 17–26. This work was supported by ReGen Therapeutics, [15] I. Boldogh, A. Bacsi, L. Agulera-Aguirre, P. German and M. TM Plc, London, England. Dr. Kruzel serves as a consul- Kruzel, Colostrinin Increases the Lifespan and Neuro- logical Performance of Mice, Neurodegener Dis 4 (Suppl 1) tant to ReGen Therapeutics, Plc. We are grateful to (2007), 264. Mardelle Susman for scientific and editorial advice and [16] I. Boldogh, A. Bacsi, B.K. Choudhury, N. Dharajiya, R. corrections made in the manuscript. Alam, T.K. Hazra, S. Mitra, R.M. Goldblum and S. 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