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Biologics Supplement

Progesterone and Vitamin D Hormone as a BiologTreatment of Traumatic Brain Injury in the Aged

Donald G. Stein, PhD, Milos M. Cekic, PhD

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Abstract: There is growing recognition that traumatic brain injury is a highly varcomplex systemic disorder that is refractory to therapies that target individual mecIt is even more complex in elderly persons, in whom frailty, previous comorbiditiemetabolism, and a long history of medication use are likely to complicate the seffects of brain trauma. Progesterone, one of the few neuroprotective agents that hpromise for the treatment of acute brain injury, is now in national and internation3 multicenter trials. New findings show that vitamin D hormone (VDH) and VDH din the aging process (and across the developmental spectrum) may interact with pone and treatment for traumatic brain injury. In this article we review the use of pone and VDH as biologics-based therapies along with recent studies demonstratincombination of progesterone and VDH may promote better functional outcomes thtreatment independently.

PM R 2011;3:S

INTRODUCTION

Traumatic brain injury (TBI) is a very complex, contextual, and dynamic proceFrom the initial insult to the persistent and highly destructive cascade of secondaryto eventual reorganization and recovery [3,4], each phase of the injury involves aset of processes that sometimes overlap. Furthermore, the molecular and physchanges resulting from a trauma to the brain are not spatially limited to the locusand even a well-circumscribed cerebral insult can have distal effects throughout alentire central nervous system (CNS) [5]. These often-subtle alterations in brain cand physiology can persist for years and are associated with complex and sounpredictable behavioral alterations such as posttraumatic stress disorder, mood dor increased mortality from the disability and health complications that can occur lthe initial injury.

In addition to the complex injury process within the CNS, head trauma also hasand extraneuronal effects that need to be considered in the development of therTBI. These processes, which most often are associated with systemic inflammamulti-organ dysfunction, are the actual causes of death in most patients who have sa brain injury [6]. In a recent review, Masel and DeWitt [7] suggest that TBI “sclassified as the beginning of an ongoing, perhaps lifelong process that impactsorgan systems and may be disease causative and accelerative.” Thus TBI should bnot as an isolated or static insult but as an event that dynamically alters the systemsit occurs.

Age and age-associated systemic changes can have additional effects on both surrecovery from severe injury. Although some aspects of the injury may be different,changes in response to brain injury also are likely to occur in children with TBI ordamage. Such changes include alterations in hormonal and drug metabolism, nustatus, immune function, and increased frailty, among others. Thus it is not a gTBIs, or the putative treatments for TBIs, will necessarily behave in the same wayoung children or in older subjects as they do in young adults. This situation iexample of a “contextual” effect. In this review we focus primarily on elderly sub

similar considerations may apply to pediatric TBIs. Elderly patients might require rese

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D.G.S. Department of EmergencEmory University School of MedicClifton Road NE, Suite 5100,30322. Address correspondence-mail: Dstei04@emory.eduDisclosure: 4A, royalties fromof BHR Pharma related to sresearch described in this presBHR; 2A, consultant, BHR. Thearrangement have been reviewproved by Emory University, whthe principal share of any robenefits. In accordance with itinterest policies, the Universityrequired to disclose any similar8B, NIH grants R015R01NS05R01HD61971-02; 7, gift inresearch from H. Allen and Com

M.M.C. Department of EmergenEmory University School of MedicinDisclosure: nothing to disclose

Portions of this article have beepreviously in the article “Traumatiand aging: is a combination ofand Vitamin D hormone a simplecomplex problem?” (Cekic M, Sterotherapeutics 2010;7:81-90) asented with the permission of thepublisher.

Disclosure Key can be found onContents and at www.pmrjourna

archSubmitted for publication October 29, 2010;accepted March 16, 2011.

ademy of Physical Medicine and RehabilitationVol. 3, S100-S110, June 2011

DOI: 10.1016/j.pmrj.2011.03.010

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S101PM&R Vol. 3, Iss. 6S, 2011

directed at the special nature and consequences ofbrain injury and a potentially different approach to tdesigned specifically to address the physiology ofgroup.

Although a monotherapy approach may be apfor the handful of diseases known to have specific genvironmental causes, experience shows that sucproach is inadequate for more heterogeneous proceas TBI. Here we suggest that because it is a complex,process, TBI can be addressed best by pleiotropic onation therapy (which, in addition to any pharmtreatment, can certainly include rehabilitation, coand other behavioral approaches contributing to thenation” aspect) and that any such therapy, if itsuccessful in human patients, must take into accoutional contextual factors such as age, gender, and mstatus.

TBI IS A MULTI-ORGAN/SYSTEMICINFLAMMATORY DISORDER

Accumulating evidence now favors the hypothesisflammatory processes throughout the body have a suimpact on morbidity and mortality after a TBI. Abrain and spinal cord injury generally have been tisolated and well-localized insults, experimentalincreasingly suggests that elevated levels of inflacytokines, a well-recognized aspect of the physiolosponse to trauma [8], are the most consistent pmarkers of outcomes in patients with systemic inflaresponse syndrome, sepsis, multi-organ dysfunctdrome (MODS), and multi-organ failure (MOF) [9factors usually cause death, and at the very least, seroften permanent disability after CNS trauma [5,6,1

In many cases, systemic failure seems to be the fiof death after CNS injury. For example, Zygun [6] fodysfunction developed in 89% of patients and failleast one non-neurological organ occurred in 35%tients. Failure of one non-neurological organ sysassociated with a 40% death rate, which increasedwith the failure of 2 systems. Multi-organ pathooccur as a consequence of neurologic injury to t[12,13]. In a study of patients with aneurismal subahemorrhages, mortality was 31% when one orga91% when 2 organs failed, and 100% when 3 or mofailed [14]. The non-neurological systems most fraffected in conjunction with CNS injury seem tcardiovascular and pulmonary systems. The key etthe development of dysfunction in these 2 systemsbly the massive release of cytokines and catecholamoccurs after neurologic injury, which can lead dmyocardial dysfunction [15], severe pulmonary edrespiratory failure [16]. Other mechanisms of fun

importance are inflammation, coagulation, and infection [

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Significant elevations of inflammatory cytokinbeen observed in cerebrospinal fluid, the systemic ci[17,18], and even in the gut [19] after TBI. Given thicant blood flow to the brain under normal conditiodamaged blood�brain barrier after injury, the bratially acts as a filter for the blood, allowing thedissemination of inflammatory mediators away fromof local injury. This phenomenon eventually leadtemic inflammatory response syndrome, which gencharacterized by local and systemic release of inflamcytokines, complement, coagulation and acute phteins (APPs), and immune cell recruitment [10]. Cowith this process is a systemic anti-inflammatory rThe ability to strike a balance between these 2 oforces may determine a patient’s survival. The preinflammation at and near the site of injury doescontribute to local secondary damage—it is probabmechanism in TBI-associated mortality and morbid

For example, in a study of more than 2000Harrison-Felix et al [21] found that patients withinjury who are completing inpatient rehabilitation hexpectancy that is on average 7 years shorter thanpatients without TBI. These patients also are mulikely to die from septicemia, pneumonia, and othertory diseases for up to 1 year after injury than are pathe general population [22]. Even “mild” cases of TBrelated to a greater risk of mortality. Brown et al [2ined death rates in Minnesota for approximately 1tients with mild TBI and found a modest but stasignificant reduction in long-term survival compapatients who did not have a head injury; however, Flcolleagues [24] found a greater risk in adult and otients.

The systemic release of inflammatory cytokinestumor necrosis factor–� (TNF-�), interleukin 1�and especially IL-6 induces the acute phase reactiotemic response to inflammation mainly executed bythat resets a number of homeostatic set points todefense and adaptation [25]. These changes exhibierable variability, from maintaining persistent inflamto promoting adaptive changes. During the acute phaTBI (hours to several days), a number of APPsregulated (eg, C-reactive protein, fibrinogen, and prbin), whereas others are down-regulated (eg, albumdensity lipoproteins, antithrombin III, and proAlthough these changes are presumed to improve sucases of severe injury such as TBI, an increased proppositive to negative APPs has been shown to acceldevelopment of diffuse intravascular coagulation angenerally pathophysiologic effects [10]. Acute inflamalso has been observed to increase levels of whoxidative stress, which is another important med

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S102 Stein and Cekic BRAIN INJURY, AGING, AND NEUROSTEROIDS

Inflammatory biomarkers also may be useful in pthe severity and outcome of injury [9,27], as weprognosis for recovery [28-31]. Although it is wethat levels of the cytokines TNF-� and IL-1� incresevere injury, the level of IL-6 appears to be the mnostically accurate because it is the chief regulatohepatic acute phase reaction [25] and correlatesdegree of systemic inflammation [9]. Evidence alsothat an increased level of IL-6 in the early period aftis a marker of a high risk of complication and orga[32]. Although considerable effort is being made toprognostic biomarkers for TBI, the issue is complthe fact that so many systemic changes occur in resbrain injury that no single biomarker fully reflects thlying pathology [33]. As with inflammation and otheogies, systemic biomarker activity may be further afthe time when tissue or serum samples are taken, thestress levels, previous health status, other comonutritional history, drug use, gender, and age.

AGING, INFLAMMATION, AND BRAININJURY

Because of the focus on youth-related TBI that occusetting of sports, war, and motor vehicle accidentelderly persons receives less attention, despite thethe Centers for Disease Control and Prevention liinjury as a major cause of morbidity and mortality inolder than 70 years [34]. The incidence of TBI has iby 21% in people older than 65 years during the paseven as it decreased for most other age groups as aimproved primary prevention such as the use of sand safety helmets [35]. Age is an independent premortality resulting from TBI, and in elderly persoceeds that of younger victims by a factor of 2 [36]. Areach their mid to late 70s, the frequency of hospitaattributable to TBI has its most pronounced peakgreatest mortality rates from “moderate” to severe Ting from 60.9% to 86.8% (depending on the study),people 75 to 80 years of age [38].

Effects of Aging in the CNS

Although no overt neuronal loss is observed in the bnormal aging, a variety of changes occur at themedium-scale neuronal networks and individualthat entail more subtle metabolic, structural, andalterations [39,40], all of which have significant eneuronal plasticity and adaptation. Age-related chthe CNS are similar to those that occur in other agand include dysfunction of energy homeostasis, ioxidative damage, protein accumulation, and deoxcleic acid (DNA) damage [39]. As they age, neur

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excitotoxicity [39] because of impairment of mitocmetabolism [41], Ca2� homeostasis [39], and iofunction [42]. Because these processes also are implthe development of injury after trauma, the agingitself is likely to exacerbate the effects of TBI. In oneing report, Wagner and colleagues [43] suggest that odamage loads increase with age but that femaleappear to fare better and have less oxidative stress thmale counterparts.

In addition to these structural and intrinsic chanbrains of aged animals also exhibit gene expressioncharacterized by an increased level of neuroinflamand microglial activation [44-47]. The microglia aremore stimulated in older animals but also appear toan amplified response to inflammatory stimulatiresulting in a general neuroinflammatory “primithough the duration of inflammatory changes is limder normal conditions as the microglia return to anstate upon resolution of immune challenges, in eldjects, the microglia are so sensitive to immune stimthat their activation resolves slowly or not at all. Thtion can result in exacerbation of neurologic diseasestill higher levels of neuroinflammation, with worsecomes in older subjects after TBI secondary to a moand persistent systemic immune response [49].

Effects of Aging on Systemic Inflammand Endocrine Function

In addition to CNS-specific changes, age-related chthe function of the systemic immune system commcur and are implicated in virtually all disease procesciated with advanced age [50]. Aging is associategeneral activation of the inflammatory response, wcause of the chronic antigenic stress on innate imexperienced during a lifetime, becomes the basis forof inflammatory diseases [51] and a reduced ability tan appropriate immune response to antigenic stim[50]. With aging, a decrease in the productioninflammatory hormones also occurs [52], alonggeneral tendency toward production of elevatedof proinflammatory cytokines by peripheral bloonuclear cells [53].

The fulminating inflammatory state associatedcreasing age has been dubbed “inflammaging” [53]. Athis condition is systemic, it also has specific effecthe CNS [48]. Given the importance of inflammatokines in both behavioral modulation and the evotraumatic injury, it is likely that an increased neurmatory cytokine response in elderly patients canneuronal synaptic plasticity, establishing a CNS envipredisposed to long-lasting complications and aability to recover from trauma [48]. The aged brain ap

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S103PM&R Vol. 3, Iss. 6S, 2011

increased immune reactivity and continuous low-lduction of central inflammatory cytokines [54].

Given the apparent correlation between increasiin the elderly and the increase in a chronic level of intory factors in the brain and other organs, it may be nfor the physician, as part of the patient’s physicaltion, to test for levels of these factors and the hormmay be needed to offset the potential for further dein the absence of immediate overt symptoms.

A final factor that must be specifically considereman aging is the decreased activity of a numbesystemic hormones, including thyroid hormonesteroids [56-58], growth hormone (GH) [59], insgrowth factor-I (IGF-I) [59], and possibly 25-hydmin D3 [60-62]. It is important to note that this deendocrine function appears to be associated with thnologic changes discussed previously. Research sugproinflammatory cytokines may down-regulate thelogical responses to a variety of hormones, includinGH, IGF-I, thyroid hormone, and estrogens [63]IL-1�, and IL-6 can modulate the release of GHhormone-releasing hormone, and somatostatin [64reduce concentrations of IGF-I [65] and increase thetrations of glucocorticoids [66] in the serum of hutients. Lower serum IGF-I and dehydroepiandrostefate levels were found in frail compared with nonfraindividuals [67], and an inverse correlation betweeIL-6 and IGF-I was noted only in frail individuals.levels of inflammatory cytokines [68-71] and thement of frailty in older adults [72,73] also have beeated with VDH deficiency.

Aging and VDH Deficiency

Recently VDH deficiency has received quite a bit oversial media attention, partly because an estimatedpeople worldwide are said to exhibit VDH deficinsufficiency [74] and partly because it has been awith autoimmune, inflammatory, and infectious co[75-81], cardiovascular disease [82,83], hypertenatherosclerosis [84], neuromuscular function [85[86], and neurodegenerative diseases [87,88]. In theastern United States, for example, studies demoprevalence of VDH deficiency ranging from 32% inadults [89] to approximately 50% for adolescents anolescents [90-92]. The most dramatic statistics costudies in the elderly, which demonstrate that 40%community-dwelling American and European olderwomen are deficient in VDH. Even higher averagesin the ill and institutionalized patients [93-101].

VDH deficiency appears to be correlated with mafactors, especially those with an inflammatory co[76,77]. This finding is supported by further evid

low levels of serum VDH are associated with elevated levels

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IL-6 [102] and a key marker of frailty in older adultsVDH is known to be a modulator of the cell cycle,function, and calcium homeostasis. As such it maimportant compound not only as an endogenous hbut also in the context of CNS injury, both as a treaits own right and in combination with another agenprogesterone [103].*

PROGESTERONE AS A TREATMENT FORBRAIN INJURY

During the past 30 years, some 50 candidate treatmefor TBI neuroprotection have failed when tested inpatients, and many pharmaceutical companies havtially discontinued the pursuit of acute pharmacoloment for TBI. Despite this pessimism, a large andpreclinical literature, along with the results of 2 rece2 clinical trials [104,105] and a new phase 3 trial,that the hormone progesterone may exert significancial effects in the treatment of TBI.

Preclinical work on progesterone neuroprotecbeen ongoing for more than 20 years. One of the firto demonstrate a beneficial effect of progesteronecame from our laboratory [106]. We tested “pseunant” and normally cycling female rats with largelesions of the frontal cortex on a delayed spatial altlearning task. The pseudopregnant females, whosenous hormone levels were elevated experimentallycal stimulation, performed significantly better inretention of the trained task after injury than did tmal-cycling counterparts. When the brains were exthe pseudopregnant rats also demonstrated substantventriculomegaly, suggesting that traumatic edemaduced by the relatively greater levels of endogenousterone present at the time of surgery and for sevethereafter [107]. We also showed that, in rats, progcan be given up to 24 hours after bilateral cortical instill retain its neuroprotective effects, although the sotreatment is given, the better the outcome [108].

Many publications have now demonstrated the eness of progesterone treatment for TBI [107,109-1more than 170 papers using 22 different injury mod25 laboratories around the world have now repbeneficial effects in attenuating the cytologic, morp

* A recent, apparently controversial report by the Institute of Medicine [103that dietary intake of VDH and calcium is adequate for “most people . . . tobone health.” The report challenges many popular assumptions about vitamin a number of disease conditions including autism, cognitive function, andbut for unexplained reasons ignores the potential role of VDH in cerebral strHowever, the authors do take care to note that “Nonetheless, some sparticularly those who are older and living in institutions or who havpigmentation—may be at increased risk for getting too little vitamin D”

ofauthors also point out that “no central authority has determined which cut-points to use”(in determining sufficiency or deficiency).

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S104 Stein and Cekic BRAIN INJURY, AGING, AND NEUROSTEROIDS

and functional deficits caused by injury to the braicord, and peripheral nerves [107,113-118, 121, 12

Unlike other sex steroids, progesterone is syntholigodendrocytes in the brain, where it modulates thof classical nuclear, membrane-bound, and steroid[123]. Progesterone also can directly affect the exprwater channel proteins (aquaporins) to modulate bgenic and cytotoxic edema [121,124], reduce glutamicity directly, mediate toxic cellular calcium influx bonizing sigma receptors, up-regulate gamma-aminacid A (GABAA) receptors to reduce excitotoxic damreduce apoptosis and necrosis through the up-reguladown-regulation of a variety of genes that controlvival [112,123].

In contrast to the single-receptor, single-mechaproach, we believe it is progesterone’s ability to act oent components of the injury cascade simultaneousexert differential effects over time and place that makeffective than other agents that have been studiedtreatment of TBI. We have shown that, when givencally, progesterone can affect the expression of atgenes thought to play a role in neuroprotection andpair [110]. This pleiotropy, although not fitting neatthe molecular biological paradigm of disease, is lreason for its translational success thus far.

The preclinical data on the neuroprotective efprogesterone after TBI have been confirmed in hutients in a phase 2a, single-center, randomized, douclinical trial for progesterone treatment of moderavere adult TBI sponsored by the National Institute ological Disorders and Stroke [104]. The authors e100 patients enrolled with proxy consent within 11injury and included only those with a postresuscitatgow Coma Scale (GCS) score between 4 and 12. Fopatients assigned to the treatment group, one was asthe placebo control group, and all patients receivednous administration of the test drug or placebo sProxy consent requirements for this study delayed pone treatment for at least 6 hours after injury. Neurofunctional outcomes were evaluated 30 days after thMortality among patients who were given progesteless than half that of control subjects (13.6% vs 30.30-day functional outcomes for moderately injured

† Despite the growing evidence of progesterone’s neuroprotective effects, 2laboratories examining the role of cyclosporin in the treatment of TBI hnegative outcomes [119,120]. In addition, the authors of 2 rec[121a,121b] suggest that, by the authors’ personal, operational definitionot all, preclinical studies testing neuroprotective agents, including progesmeet a number of their criteria for preclinical development of new drugdespite their concerns, given the large number of studies in which theprogesterone, it may be of interest to note that most, if not all, of the criteby Loane and Faden and Gibson et al have been addressed for the sprogesterone, but perhaps to a lesser extent than for most of the other neagents cited in the paper by Loane and Faden. However, even if all theindeed met, no guarantee exists that a completely successful agent for

would be discovered. The review presents a number of important suggestions fodesign of studies in neuroprotection.

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were significantly better than for the placebo groubers of the Data Safety Monitoring Board, appointeNational Institutes of Health (NIH), found no seriouevents attributable to the progesterone treatment.

These initial results were confirmed by a seconddent, randomized, double-blind study from Chinathe authors used a 5-day intramuscular course of twith progesterone in 159 patients with severe TBI.who arrived within 8 hours of injury with a GCS scolower were randomly assigned (1:1) to receive eitheterone or placebo. The primary end point wasOutcome Scale (GOS) measurement 3 months aftejury, but the investigators also used Functional Idence Measure scores and repeated the measures onified Functional Independence Measure and GOS 6after the TBI occurred. Similar beneficial outcome mon morbidity and mortality were observed at bothand 6 months after injury, again without any seriouevents caused by the treatment. The authors alsothat intracranial pressure was lower 72 hours and 7 dtreatment with progesterone [105].

PROGESTERONE IN A PHASE 3 CLINICTRIAL

Following up on the positive phase 2 clinical data [1a phase 3, 17-center clinical trial for TBI sponsored bnow currently enrolling patients across the UniteThis 1:1 randomized, double-blind controlled trialroll 1200 patients. Treatment must begin within 4injury in patients at least 18 years of age with a Gbetween 12 and 4. After a 1-hour loading dosemg/kg administered intravenously (IV), progesteroncebo is given by IV infusion at 0.5 mg/kg for 72 hthen tapered over the course of an additional 24 ho4-hour window of treatment was designed to optimgesterone’s neuroprotective effects by treating patieTBI as soon as possible. Food and Drug Adminapproval for exemption from informed consent wafor this purpose. The primary study end point will be adichotomy of the GOS at 6 months. The trial will tracity, extent of adverse and serious adverse events, DRating Score, and cognitive, neurologic, and functiocomes. More details can be found at ClinicalTrials.goclinicaltrials.gov/ct2/show/record/NCT00822900).

In addition to the NIH study, BHR, a branch oPharma, a privately held Belgian/French pharmcompany, also recently began enrolling patients inclinical trial called SyNAPSe (http://www.synacom/). Approximately 1800 patients with severe cloTBI (GCS scores of 4-8) in whom treatment can bewithin 8 hours of injury will be enrolled in the study100-120 medical centers in the United States, Wes

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S105PM&R Vol. 3, Iss. 6S, 2011

signed to receive a 5-day continuous IV infusion oterone or placebo in a 1:1 allocation, and folloconducted for 6 months. The GOS is the primary spoint. This global, phase 3, multicenter pivotal trprogesterone infusion for patients with severe TBIconducted in collaboration with the American BraConsortium and the European Brain Injury Consor

We suggest that one of the reasons for increasedsurvival in patients with TBI who are treated withterone may be progesterone’s ability to substanduce systemic acute phase inflammation and thervent MODS and MOF [122,125]. Because, as dpreviously, MODS and MOF frequently are thcauses of death in patients with a TBI, the aprogesterone to modulate the development ofinflammation may be one way that it promotes suthe early stages after brain injury. This idea was suby Chen and colleagues [126], who found that cinjury to the cerebral cortex in rats induced exprIL-1� and TNF-� followed by apoptosis in the imucosa. A 5-day course of treatment with progreduced both the cytokines and cell death in the iThis study shows that an experimental brain intrigger a cascade of inflammation in other tissues,turn can produce serious consequences for braiFor example, gut inflammation could affect nuvariables needed to help with the CNS metabovolved in neuroprotection (see the article by PopoMcTigue [5] for a further discussion of this issue

PROGESTERONE, VITAMIN D, AND AGIN BRAIN INJURY

Progesterone’s effectiveness in treating TBI and thlence of VDH deficiency in the elderly populationquestions: First, would progesterone be as effectivanimals as in young adult animals? Second, woudeficiency affect the outcome of a brain injury and pointerfere with the benefits of progesterone treatmerats? And finally, would a combination of VDH andterone be more effective than either compound alosame aged rat population? We speculated that progwould be effective in treating TBI in old rats but tdeficiency would increase inflammation and decrbenefits of treatment [127]. We also hypothesized tbining VDH with progesterone would improve oBecause the early onset of inflammation is a reliabletic indicator of mortality in human patients with sitrauma, we measured a number of acute inflammateins, cell death, DNA damage, and short-term beindicators of inflammation and secondary damage

and VDH-deficient aged animals after TBI.

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Progesterone and VDH after TBI in ARats

Progesterone was indeed effective in treating TBI inalthough at a higher dose than in young adult animaWe measured levels of inflammatory proteins, ceedema, and behavior during the acute phase of injurhours after TBI) in 20-month-old rats (which is theequivalent” of 60 years of age). Injured animals treaprogesterone beginning within the first hour aftershowed decreased inflammatory markers at all timexamined, indicating a reduction in the acute inflamprocess compared with the old rats given vehicle.observed decreased markers of cell death at all time pwell as decreased edema and improved motor fwhich suggests that the pleiotropic effects of progwere also effective despite the added complexity ofan aged organism.

When we tested aged rats that were made VDH dwe observed increased inflammation compared wreplete animals in all groups, with or without TBI anwithout progesterone treatment [127]. Our resufirmed findings of previous studies [76,77,129] tgested that VDH deficiency establishes a higher baseof inflammation even before injury, in effect primsystem for an increased immune response after Televated acute-phase response correlated with incredeath and DNA damage, indicating a more severe seinjury process after injury even with progesterone tr

Both TNF-� and IL-6 were significantly increaseddeficiency, and both cytokines demonstrated an inbetween deficiency and treatment. Although mosvariability in TNF-� was accounted for by injury, thIL-6 was primarily affected by VDH deficiency, acorresponds to other independent data connectlevels with VDH deficiency, frailty, and inflam[73,102,130,131]. The elevation in IL-6 was most eour data comparing VDH-deficient and VDH-normaafter TBI and progesterone treatment. Although moother cytokines were elevated 2- or 3-fold in VDH-animals, IL-6 was increased nearly 5-fold by 72 hothe injury occurred, suggesting that IL-6 may be thecytokine involved in the detrimental effects of VDciency after TBI.

Combination Therapy in TBI

Combination therapy is a relatively novel idea in thbrain injury, but it is a well-established pharmacoproach [4] in other disease processes such as HIV/Atuberculosis. Most drug development still focuses onual agents and is targeted toward a few specific mecat most. As discussed previously, this approach maappropriate in the setting of TBI. Rationales for co

drugs include targeting multiple divergent mechanisms and

kinetiThis idBI [131) dru, wherouslyed tome effis casetmentto ovinatort mayf huml monin injueffect

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S106 Stein and Cekic BRAIN INJURY, AGING, AND NEUROSTEROIDS

overcoming single-drug limitations such as receptorpharmacology, and signaling pathways [132,133].has recently gained ground in the treatment of T“Polytherapy” approaches can proceed in 2 ways: (can be designed for target promiscuity or pleiotropysingle agent affects multiple pathways simultanesequentially or both; or (2) drugs can be designadministered in combination, which achieves the saof targeting multiple processes, but separately in thhybrid approach, which may be wise for the treaTBI, is to combine pleiotropic drugs themselvescome the limitations of monotherapy. A combapproach to treatment is not only reasonable buessential given the complexity and heterogeneity oTBI and the fact that, of the various preclinicatherapy drugs that have shown promise for braneuroprotection, all have been shown to be inwhen taken to clinical trial [134].

Combining Progesterone and VDH in

Given our results with progesterone and VDH defiaged rats, we speculated that acute correction of Vciency with VDH would restore the efficacy of progCombining progesterone and VDH, which are pldrugs that work primarily through intracellular rtranscription factors, also merits serious consideratoutside the context of VDH-deficiency as meeting thfor combinatorial therapies [4]. Because both progand VDH are pleiotropic and multifunctional compis difficult to predict specific mechanisms of interacboth agents could be used to target cell death afterexample, as one of the many mechanisms by whicinjury evolves.

It is worth emphasizing here that VDH is in fvitamin but rather a secosteroid hormone with tcholesterol backbone as other sex steroids (such aterone) and has its own class of nuclear steroid recepsignaling mechanisms [135,136]. Like progesteronea pleiotropic agent with important implications for topment and treatment of systemic disease. Recentlypalan and colleagues [137] examined VDH receptoin lymphoblastoid cells after treatment with VDfound more than 2700 gene positions with VDHsignaling gene transcription and approximately 2that changed their level of expression in responseMany of these genes (like progesterone) are directcated in modulating the immune/inflammatory resinjury and neurodegenerative diseases.

Importantly, progesterone complements VDH’sflammatory effects not only in the brain but in othaffected by the inflammatory cascade that often accoTBI or stroke. To reiterate, one of the hormone’s ma

fits and probably one of the main reasons it is so effective

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a systemic treatment for serious injury is because itthe inflammatory cascade not only in the brain buttissues such as gut, heart, spleen, and thymus thatsubject to cytokine damage [138-143].

Evidence also suggests that, like progesterone, Vministered after a brain injury can have neuropeffects, although a number of its actions involvepathways from those of progesterone [144]. Both pone and VDH are natural hormones that are synthboth men and women, and both can act as ageproduce other metabolites in the brain with theirtinct modes of action in CNS repair. These propertiin the context of the clinical trial results showing vprogesterone safety and efficacy in TBI, makes the tprogesterone and VDH in a preclinical combinedcompelling approach to consider for use in a clinespecially for elderly persons.

Perhaps similar consideration should be given tofor vitamin D supplementation in pediatric TBI treatsafety and efficacy trial for progesterone in pediaunder the auspices of the Pediatric Emergency CareResearch Network (PECARN) is beginning to takGrowing evidence suggests that many children cVDH insufficiency or even deficiency, and this condiaffect long-term disease outcome and possibly the eness of neuroprotective treatments such as progesteearly brain injury [145,146].

Progesterone/VDH Combination in ARats with TBI

We have demonstrated the effectiveness of a combiprogesterone and VDH in both in vitro and in vivo mour laboratory. Combining VDH and progesterone wtive in vitro in increasing the survival of primaryneurons under 24 hours of excitatory glutamate cAlthough VDH alone and progesterone alone both pbeneficial effects on neuronal cell survival, the comof VDH and progesterone produced significantly mroprotection than either compound given alone andbest individual doses [147]. We used glutamateexcitotoxicity as our primary outcome measure breliably triggers cell death in models of TBI, stroke, ancord injury.

Interestingly, when we applied the same treatmcept in vivo to VDH-deficient aged rats with largecontusions of the frontal cortex, the only treatmreduced inflammatory marker proteins was the comof progesterone and VDH (5 mg/kg in a single dopared with vehicle or either compound given alocombination treatment was also the only one thattially improved behavioral outcomes. If these coapply to human patients, the clinical implications

as important: a combination treatment of progesterone and

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S107PM&R Vol. 3, Iss. 6S, 2011

VDH administered to elderly patients with TBI shprove survival compared with progesterone giventhe same population. Because few, if any, better altexist, a clinical investigation would appear to be a restep to take.

Briefly summarized, we believe that: (1) progeseffective in reducing acute inflammation, a key indsurvival in human patients and in aged rats with bra(2) D deficiency increases acute-phase inflammaattenuates the benefits of progesterone treatment inwith TBI, which suggests that such a deficiency ccrease mortality and worsen outcome in TBI surviv(3) a combination of progesterone and VDH partiallythe effects of D deficiency and reduces post-TBI acutmation in old rats.

CONCLUSION

Should our results translate to the clinic, the implithe research discussed here is that a simple vitamintion combined with progesterone could help sahuman lives. Extrapolating from the animal data tthat D deficiency enhances inflammatory activity evintact rats, and because vitamin D insufficiency appeprevalent in so many elderly and frail patients,rehabilitation physicians to screen routinely for vlevels in their patients, especially those who havenced a stroke or TBI. Our animal data also can besuggest that ignoring D deficiency or insufficiencytime, exacerbate the injury, probably reduce the bphysical and cognitive therapy, and delay or prevtional recovery.

As we noted previously, the reduction in systemimation resulting from combined VDH and progtreatment may be one of the key mechanisms bprogesterone and VDH, in combination or alone, cnificantly increase survival after TBI in human patthough eventually better alternatives to the treatmeproposing may be found, it is becoming increasinthat no single receptor, gene, or mechanism can acthe complex cascade of focal and systemic toxic evduced by a brain injury, whether it is in children, aelderly persons. It is highly unlikely that a single ageable to trigger all the neuroprotective and neuroplasrequired for the damaged brain to reorganize to coptraumatic injury, degenerative disorder, or seriouDespite the evidence that progesterone and its memight work well, their effectiveness has not yet beensively demonstrated. Individual or combination therpleiotropic agents capable of addressing multiorgantion is becoming more acceptable (and more approwe begin to understand the complex systems andinvolved in the mediation of brain repair and fu

recovery.

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