Objective-Inflammation is an important component of the response to traumatic brain injury (TBI).... more Objective-Inflammation is an important component of the response to traumatic brain injury (TBI). Progesterone has been shown to inhibit neuroinflammation following (TBI), and may do so through Toll-like receptor (TLR)-mediated pathways. In vitro studies indicate that 1,25dihydroxyvitamin D(3) (VDH) may also modulate the inflammatory response through the TLR4 pathway. We tested the hypothesis PROG and VDH would exert additive and synergistic neuroprotective effects compared with individual treatment by modulating TLR4/NF-κB-mediated inflammation pathways after TBI in rats. Research Design and Methods-Bilateral medial frontal cortical impact injury was induced in young adult Sprague-Dawley rats. Progesterone (i.p., 16 mg/kg body weight) and VDH (1 ug/kg body weight) were injected separately or combined at 1 and 6 h after surgery. Rats were killed 24 h post-surgery and peri-contusional brain tissue harvested for immunostaining and protein measurement. Results-TLR4, phosphorylation of NF-κB,. neuronal loss, and astrocyte activation were significantly reduced with combination treatment after TBI compared to each agent iven individually. Conclusions-At 24h after TBI, combination therapy shows greater efficacy in reducing neuroinflammation compared to progesterone and VDH given separately, and does so by modulating the TLR4/NF-κB signaling pathway.
To date, outcomes for all Phase III clinical trials for traumatic brain injury (TBI) have been ne... more To date, outcomes for all Phase III clinical trials for traumatic brain injury (TBI) have been negative. The recent disappointing results of the Progesterone for the Treatment of Traumatic Brain Injury (ProTECT) and Study of a Neuroprotective Agent, Progesterone, in Severe Traumatic Brain Injury (SyNAPSe) Phase III trials for progesterone in TBI have triggered considerable speculation about the reasons for the negative outcomes of these two studies in particular and for those of all previous Phase III TBI clinical trials in general. Among the factors proposed to explain the ProTECT III and SyNAPSe results, the investigators themselves and others have cited: 1) the pathophysiological complexity of TBI itself; 2) issues with the quality and clinical relevance of the preclinical animal models; 3) insufficiently sensitive clinical endpoints; and 4) inappropriate clinical trial designs and strategies. This paper highlights three critical trial design factors that may have contributed substantially to the negative outcomes: 1) suboptimal doses and treatment durations in the Phase II studies; 2) the strategic decision not to perform Phase IIB studies to optimize these variables before initiating Phase III; and 3) the lack of incorporation of the preclinical and Chinese Phase II results, as well as allometric scaling principles, into the Phase III designs. Given these circumstances and the exceptional pleiotropic potential of progesterone as a TBI (and stroke) therapeutic, we are advocating a return to Phase IIB testing. We advocate the incorporation of dose and schedule optimization focused on lower doses and a longer duration of treatment, combined with the addressing of other potential trial design problems raised by the authors in the recently published trial results.
We investigated whether combinatorial post-injury treatment with progesterone (P4) and vitamin D ... more We investigated whether combinatorial post-injury treatment with progesterone (P4) and vitamin D hormone (VDH) would reduce ischemic injury more effectively than P4 alone in an oxygen glucose deprivation (OGD) model in primary cortical neurons and in a transient middle cerebral artery occlusion (tMCAO) model in rats. In the OGD model, P4 and VDH each showed neuroprotection individually, but combination of the "best" doses did not show substantial efficacy; instead, the lower dose of VDH in combination with P4 was the most effective. In the tMCAO model, P4 and VDH were given alone or in combination at different times post-occlusion for 7 days. In vivo data confirmed the in vitro findings and showed better infarct reduction at day 7 and functional outcomes (at 3, 5 and 7 days post-occlusion) after combinatorial treatment than when either agent was given alone. VDH, but not P4, upregulated heme oxygenase-1, suggesting a pathway for the neuroprotective effects of VDH differing from that of P4. The combination of P4 and VDH activated brain-derived neurotrophic factor and its specific receptor, tyrosine kinase receptor-B. Under specific conditions VDH potentiates P4's neuroprotective efficacy and should be considered as a potential partner of P4 in a low-cost, safe and effective combinatorial treatment for stroke.
We investigated the neuroprotective effects of progesterone (P4) treatment in stroke-prone sponta... more We investigated the neuroprotective effects of progesterone (P4) treatment in stroke-prone spontaneously hypertensive rats (SHRSPs) given 60-min transient middle cerebral artery occlusion (tMCAO). The treatment groups were: (1) Wistar-Kyoto (normotensive sham), (2) SHRSP (hypertensive sham), (3) tMCAO SHRSPs (SHRSP + tMCAO), and (4) SHRSP + tMCAO + P4. P4 (8 mg/kg) was administered 1 h after occlusion and then daily for 14 days. We measured cerebral infarction volume, blood pressure and body weight. Behavioral outcomes were analyzed at post-stroke days 3, 9, and 14. To assess morphological protection we measured activation of microglia and astrocytes, oxidative stress, apoptosis, expression of vascular endothelial growth factor (VEGF), an angiogenic marker, and IL-1β, a marker of inflammation, on day 14 post-stroke. There was no effect of P4 on body weight or systolic blood pressure compared to the SHRSP + tMCAO group. However, grip strength and sensory neglect measures in the P4 group were improved compared to SHRSP + tMCAO. In addition, significantly larger infarct volumes were seen in the SHRSP + tMCAO group compared to SHRSP + tMCAO + P4. Increased markers of the injury cascade such as macrophages, activated astrocytes, superoxide anion and apoptotic cells observed in the SHRSP + tMCAO group were significantly decreased by P4. We conclude that, despite hypertensive comorbidity, P4 improves functional outcomes and attenuates stroke infarct in hypertensive rats by reducing superoxide anion expression and by decreasing inflammation and neuronal apoptosis.
Korley et al. recently evaluated the relationship between progesterone treatment and serum levels... more Korley et al. recently evaluated the relationship between progesterone treatment and serum levels of biomarkers of glial and neuronal cell death in the ProTECT III trial for progesterone in the treatment of traumatic brain injury. We found a number of serious problems in this paper that make it difficult to accept the authors' conclusions. We believe caution needs to be observed in interpreting their results to indicate a lack of progesterone efficacy in the treatment of TBI.
Background Severity of illness in COVID-19 is consistently lower in women. Focus on sex as a biol... more Background Severity of illness in COVID-19 is consistently lower in women. Focus on sex as a biologic factor may suggest a potential therapeutic intervention for this disease. We assessed whether adding progesterone to standard of care would improve clinical outcomes of hospitalized men with moderate to severe COVID-19. Research Question Does short-term subcutaneous administration of progesterone safely improve clinical outcome in hypoxemic men hospitalized with COVID-19? Study Design and Methods We conducted a pilot, randomized, open-label, controlled trial of subcutaneous progesterone in men hospitalized with confirmed moderate to severe COVID-19. Patients were randomly assigned to receive standard of care (SOC) plus progesterone (100 mg subcutaneously twice daily for up to five days) or SOC alone. In addition to assessment of safety, the primary outcome was change in clinical status at day 7. Length of hospital stay and number of days on supplemental oxygen were key secondary outcomes. Results Forty-two patients were enrolled from April - August 2020; 22 were randomized to the control group and 20 to the progesterone group. Two patients from the progesterone group withdrew from the study prior to receiving progesterone. There was a 1.5-point overall improvement in median clinical status score on a seven-point ordinal scale from baseline to Day 7 in patients in the progesterone group as compared to controls (95%CI:0.0-2.0; P=0.024). There were no serious adverse events attributable to progesterone. Patients treated with progesterone required 3 fewer days of supplemental oxygen (median of 4.5 vs 7.5 days) and were hospitalized for 2.5 fewer days (median of 7.0 vs 9.5 days) as compared to controls. Interpretation Progesterone at a dose of 100 mg, twice daily by subcutaneous injection in addition to SOC may represent a safe and effective approach for treatment in hypoxemic men with moderate to severe COVID-19.
progress away from the capillary tip depending on the speed with which the amoeba flows into the ... more progress away from the capillary tip depending on the speed with which the amoeba flows into the capillary. This interpretation of the pressure events is certainly not the only plausible one, but because it does satisfy the observations and is consistent with the positive pressure gradient theory of pseudopod extension and retraction, it points out that the cited suction experiments do not constitute a direct test of the hydraulic flow theory but are inconclusive in that regard. Even though the observation that the applied suction '"rarely showed any detectable effect on the streaming pattern except in the immediate vicinity of the capillary orifice" can be interpreted as evidence that the negative pressure gradient is established only in the vicinity of the orifice, since no effect is noticed elsewhere; it would seem that directly testing the theory on this basis awaits the deveopment of a method to measure the internal pressure distribution.
It was demonstrated that magnesium pemoline in DMSO attenuates ECS-produced retrograde amnesia wh... more It was demonstrated that magnesium pemoline in DMSO attenuates ECS-produced retrograde amnesia when administered prior to training in a passive avoidance task. We also observed that although there is RA for the specific step-off response that led to painful footshock, Ss receiving ECS showed an increase in emotionality similar to footshock controls suggesting RA for the specific response but not the general test situation. High doses of pemoline (24 mg/kg) not followed by ECS disrupted performance of the response but not retention. Magnesium hydroxide Mg (01t)2 8 mg/kg administered in DMSO proved to be severely disrupting when followed by ECS indicating an interaction with pemoline since pemoline without magnesium q-ECS and Mg (OH)2 ~-FS had no effect on performance in this type of situation.
The purpose of this investigation was to determine whether sequential (i.e., serial) ablation of ... more The purpose of this investigation was to determine whether sequential (i.e., serial) ablation of the monkey's orbital prefrontal cortex would lead to a reduction in the severity of the behavioral impairment usually associated with one-stage bilateral removal of this tissue. The lateral orbital cortex was ablated in four operations spaced 3 weeks apart or in a one-stage procedure. The monkeys were examined on a visual go-no go differentiation task, spatial delayed-alternation, and object reversal learning. The results reveal no differences between the effects of sequential and one-stage ablations. These findings differ from previous experiments that demonstrated a degree of functional recovery after the sequential removal of a sector of the dorsolateral prefrontal cortex. Since lesion studies with infant monkeys have also demonstrated that functional recovery occurs after early ablation of dorsolateral cortex but not after early removal of orbital frontal cortex, recovery of behavioral functions after infant and sequential lesions may involve similar neural mechanisms. 204
The field of neuroprotection after brain injuries has been littered with failed clinical trials. ... more The field of neuroprotection after brain injuries has been littered with failed clinical trials. Finding a safe and effective treatment for acute traumatic brain injury remains a serious unmet medical need. Repurposing drugs that have been in use for other disorders is receiving increasing attention as a strategy to move candidate drugs more quickly to trial while reducing the very high cost of new drug development. This paper describes our own serendipitous discovery of progesterone's neuroprotective potential, and the strategies we are using in repurposing and developing this hormone for use in brain injuries-applications very different from its classical uses in treating disorders of the reproductive system. We have been screening and testing a novel analog that maintains progesterone's therapeutic properties while overcoming its physiochemical challenges, and testing progesterone in combination treatment with another pleiotropic hormone, vitamin D. Finally, our paper, in the context of the problems and pitfalls we have encountered, surveys some of the factors we found to be critical in the clinical translation of repurposed drugs.
Restorative Neurology and Neuroscience, Mar 6, 2019
Background: In this proof-of-concept paper, we investigated whether combination treatment with pr... more Background: In this proof-of-concept paper, we investigated whether combination treatment with progesterone (P4) and chloroquine (CQ) would reduce ischemic injury more effectively than either agent alone in a transient middle cerebral artery occlusion (tMCAO) model in male rats. Methods: P4 (8 mg/kg) and CQ (25 mg/kg) were given alone or in combination beginning at different times during surgery and for 3 days post-occlusion. Locomotor activity and grip strength were evaluated as measures of impairment and recovery. Infarct size was assessed by TTC staining. Markers of autophagy (LC3 and SQSTM1/p62) and apoptosis (Bcl-2 and Bax) were evaluated with western blotting. Results: At the doses we employed, the combination was not more effective than either drug given separately on measures of grip strength or locomotor activity. However, combination therapy substantially reduced infarct size, and significantly increased Bcl-2 protein levels and suppressed Bax expression. Progesterone decreased the expression of LC3-II 24 h and SQSTM1/p62 after ischemia. Conclusions: Our findings suggest that combination therapy with P4 and CQ is not detrimental and has a small-to-moderate additive neuroprotective effect on ischemic injury in rats without substantively affecting behavioral outcomes. CQ and P4 may help to regulate the expression of both autophagy-related and apoptosis-related proteins.
Background: Repeated mild traumatic brain injuries, such as concussions, may result in cumulative... more Background: Repeated mild traumatic brain injuries, such as concussions, may result in cumulative brain damage, neurodegeneration and other chronic neurological impairments. There are currently no clinically available treatment options known to prevent these consequences. However, growing evidence implicates neuroinflammation and oxidative stress in the pathogenesis of repetitive mild brain injuries; thus, these may represent potential therapeutic targets. Progesterone has been demonstrated to have potent anti-inflammatory and anti-oxidant properties after brain insult; therefore, here, we examined progesterone treatment in rats given repetitive mild brain injuries via the repeated mild fluid percussion injury model. Methods: Male Long-Evans rats were assigned into four groups: sham injury + vehicle treatment, sham injury + progesterone treatment (8 mg/kg/day), repeated mild fluid percussion injuries + vehicle treatment, and repeated mild fluid percussion injuries + progesterone treatment. Rats were administered a total of three injuries, with each injury separated by 5 days. Treatment was initiated 1 h after the first injury, then administered daily for a total of 15 days. Rats underwent behavioural testing at 12-weeks post-treatment to assess cognition, motor function, anxiety and depression. Brains were then dissected for analysis of markers for neuroinflammation and oxidative stress. Ex vivo MRI was conducted in order to examine structural brain damage and white matter integrity. Results: Repeated mild fluid percussion injuries + progesterone treatment rats showed significantly reduced cognitive and sensorimotor deficits compared to their vehicle-treated counterparts at 12-weeks post-treatment. Progesterone treatment significantly attenuated markers of neuroinflammation and oxidative stress in rats given repeated mild fluid percussion injuries, with concomitant reductions in grey and white matter damage as indicated by MRI. Conclusions: These findings implicate neuroinflammation and oxidative stress in the pathophysiological aftermath of mild brain injuries and suggest that progesterone may be a viable treatment option to mitigate these effects and their detrimental consequences.
Annals of the New York Academy of Sciences, Jun 1, 1987
MORPHOLOGIC FEATURES OF FETAL FRONTAL TRANSPLANTS During the last several years, there has been a... more MORPHOLOGIC FEATURES OF FETAL FRONTAL TRANSPLANTS During the last several years, there has been a resurgence of interest in the ability of implanted fetal central nervous system (CNS) tissue to promote anatomical restoration and return of function to brain-damaged mammals. ' The parameters under
Tissue plasminogen activator (tPA) is one of the few approved treatments for stroke, but its effe... more Tissue plasminogen activator (tPA) is one of the few approved treatments for stroke, but its effects on the phenotype of microglia/macrophages are poorly understood. One of its side effects is an increase in the inflammatory response leading to neuronal cell damage and death in the ischemic cascade after stroke. Injury-induced activated microglia/macrophages can have dual functions as pro-inflammatory (M1) and anti-inflammatory (M2) factors in brain injury and repair. Recent studies show that progesterone (PROG) is a potent anti-inflammatory agent which affects microglia/macrophage expression after brain injury. We examined the interaction of tPA-induced expression of microglia/macrophage phenotypes and PROG's anti-inflammatory effects. tPA treatment increased the recruitment of microglia/macrophages, the polarity of M1 reactions, the expression of MIP-1α in neurons and capillaries, and the expression of MMP-3 compared to vehicle, and PROG modulated these effects. PROG treatment attenuates tPA-induced inflammatory alterations in brain capillaries and microglia/macrophages both in vivo and in vitro and thus may be a useful adjunct therapy when tPA is given for stroke.
Embryonic cortex from 19-day fetuses was transplanted in a medial frontal cortex wound cavity of ... more Embryonic cortex from 19-day fetuses was transplanted in a medial frontal cortex wound cavity of 105-day-old male rats. Nisslstained tissue revealed little internal laminar organization. Graft sections impregnated by the Loyez method exhibited bands of myelinated fibers surrounding implants as well as long-interconnecting and swirl-like fiber fascicles within the implant. Tissue processed histochemically for acetylcholinesterase and choline acetyltransferase revealed enzyme-positive fibers and cell bodies within the grafts. Cytochrome oxidase histochemistry revealed regional variations in the metabolic activity of the grafts. In summary, although our frontal cortex grafts exhibit many of the morphological features seen in intact frontal cortex, the organization of these components within the implant is dissimilar to normal cortical tissue.
Neonatal stroke is among the top ten causes of childhood death and causes long-term permanent dis... more Neonatal stroke is among the top ten causes of childhood death and causes long-term permanent disability and neurological deficits in survivors. In most cases, recurrent clonic seizure is the only clinical manifestation which worsens the stroke outcomes. A treatment or pediatric stroke is essential, but no candidate intervention is supported by substantiated data. We tested the hypothesis that progesterone (P4) treatment would be beneficial in a neonatal model of stroke. P12 CD1 mice (mixed gender) underwent permanent unilateral right common carotid ligation (pUCCL) or sham surgery (n=10/group). Pups which showed seizure activity during the 1h post-pUCCL were randomly assigned to receive P4 (8 mg/kg) or vehicle injections at 1, 3 and every 24h post-pUCCL for 6 days. We assessed acute behavioral seizures (during the first 4h post-pUCCL), serum pro-inflammatory cytokines (IL-1β, IL-6, TNFα at 6, 24 and 48h) and brain infarction (at day 7 by CV-staining). Cytokine data were analyzed by repeated measures one-way ANOVA followed by LSD and Tukey’s tests for independent comparisons. For seizure and infarct data, a two-tailed unpaired t-test was employed. We observed acute seizures during the first 4h post-pUCCL in the vehicle group (90.8±11.77). P4 treatment significantly ( P <0.05) reduced seizure occurrence (58.4±8.97) by 35% compared to vehicle. Repeated measures ANOVA revealed a significant group effect in IL-1β (F (2,15) =110.706; P <0.001), IL-6 (F (2,15 ) =66.067; P <0.001), and TNFα (F (2,15) =146.263; P <0.001) levels. Serum IL-1β, IL-6 and TNFα were significantly higher ( P <0.001) at 6 and 24h and remained elevated until 48h post-pUCCL in the vehicle group compared to sham. The P4-treated group showed a significant ( P <0.01) decrease in all pro-inflammatory cytokine levels at all time points. Further, pUCCL resulted in severe hemispheric damage (16.88±1.48) as evidenced by cresyl-violet staining at 7 days post-pUCCL. P4 treatment showed a significant ( P <0.05) reduction (4.93±1.1) in infarct volume (~70%) compared to vehicle. Our data demonstrate that P4 reduces acute seizures and brain infarction following neonatal stroke by modulating the inflammatory process, and warrants detailed studies of functional outcomes and mechanism of action.
Objective-Inflammation is an important component of the response to traumatic brain injury (TBI).... more Objective-Inflammation is an important component of the response to traumatic brain injury (TBI). Progesterone has been shown to inhibit neuroinflammation following (TBI), and may do so through Toll-like receptor (TLR)-mediated pathways. In vitro studies indicate that 1,25dihydroxyvitamin D(3) (VDH) may also modulate the inflammatory response through the TLR4 pathway. We tested the hypothesis PROG and VDH would exert additive and synergistic neuroprotective effects compared with individual treatment by modulating TLR4/NF-κB-mediated inflammation pathways after TBI in rats. Research Design and Methods-Bilateral medial frontal cortical impact injury was induced in young adult Sprague-Dawley rats. Progesterone (i.p., 16 mg/kg body weight) and VDH (1 ug/kg body weight) were injected separately or combined at 1 and 6 h after surgery. Rats were killed 24 h post-surgery and peri-contusional brain tissue harvested for immunostaining and protein measurement. Results-TLR4, phosphorylation of NF-κB,. neuronal loss, and astrocyte activation were significantly reduced with combination treatment after TBI compared to each agent iven individually. Conclusions-At 24h after TBI, combination therapy shows greater efficacy in reducing neuroinflammation compared to progesterone and VDH given separately, and does so by modulating the TLR4/NF-κB signaling pathway.
To date, outcomes for all Phase III clinical trials for traumatic brain injury (TBI) have been ne... more To date, outcomes for all Phase III clinical trials for traumatic brain injury (TBI) have been negative. The recent disappointing results of the Progesterone for the Treatment of Traumatic Brain Injury (ProTECT) and Study of a Neuroprotective Agent, Progesterone, in Severe Traumatic Brain Injury (SyNAPSe) Phase III trials for progesterone in TBI have triggered considerable speculation about the reasons for the negative outcomes of these two studies in particular and for those of all previous Phase III TBI clinical trials in general. Among the factors proposed to explain the ProTECT III and SyNAPSe results, the investigators themselves and others have cited: 1) the pathophysiological complexity of TBI itself; 2) issues with the quality and clinical relevance of the preclinical animal models; 3) insufficiently sensitive clinical endpoints; and 4) inappropriate clinical trial designs and strategies. This paper highlights three critical trial design factors that may have contributed substantially to the negative outcomes: 1) suboptimal doses and treatment durations in the Phase II studies; 2) the strategic decision not to perform Phase IIB studies to optimize these variables before initiating Phase III; and 3) the lack of incorporation of the preclinical and Chinese Phase II results, as well as allometric scaling principles, into the Phase III designs. Given these circumstances and the exceptional pleiotropic potential of progesterone as a TBI (and stroke) therapeutic, we are advocating a return to Phase IIB testing. We advocate the incorporation of dose and schedule optimization focused on lower doses and a longer duration of treatment, combined with the addressing of other potential trial design problems raised by the authors in the recently published trial results.
We investigated whether combinatorial post-injury treatment with progesterone (P4) and vitamin D ... more We investigated whether combinatorial post-injury treatment with progesterone (P4) and vitamin D hormone (VDH) would reduce ischemic injury more effectively than P4 alone in an oxygen glucose deprivation (OGD) model in primary cortical neurons and in a transient middle cerebral artery occlusion (tMCAO) model in rats. In the OGD model, P4 and VDH each showed neuroprotection individually, but combination of the "best" doses did not show substantial efficacy; instead, the lower dose of VDH in combination with P4 was the most effective. In the tMCAO model, P4 and VDH were given alone or in combination at different times post-occlusion for 7 days. In vivo data confirmed the in vitro findings and showed better infarct reduction at day 7 and functional outcomes (at 3, 5 and 7 days post-occlusion) after combinatorial treatment than when either agent was given alone. VDH, but not P4, upregulated heme oxygenase-1, suggesting a pathway for the neuroprotective effects of VDH differing from that of P4. The combination of P4 and VDH activated brain-derived neurotrophic factor and its specific receptor, tyrosine kinase receptor-B. Under specific conditions VDH potentiates P4's neuroprotective efficacy and should be considered as a potential partner of P4 in a low-cost, safe and effective combinatorial treatment for stroke.
We investigated the neuroprotective effects of progesterone (P4) treatment in stroke-prone sponta... more We investigated the neuroprotective effects of progesterone (P4) treatment in stroke-prone spontaneously hypertensive rats (SHRSPs) given 60-min transient middle cerebral artery occlusion (tMCAO). The treatment groups were: (1) Wistar-Kyoto (normotensive sham), (2) SHRSP (hypertensive sham), (3) tMCAO SHRSPs (SHRSP + tMCAO), and (4) SHRSP + tMCAO + P4. P4 (8 mg/kg) was administered 1 h after occlusion and then daily for 14 days. We measured cerebral infarction volume, blood pressure and body weight. Behavioral outcomes were analyzed at post-stroke days 3, 9, and 14. To assess morphological protection we measured activation of microglia and astrocytes, oxidative stress, apoptosis, expression of vascular endothelial growth factor (VEGF), an angiogenic marker, and IL-1β, a marker of inflammation, on day 14 post-stroke. There was no effect of P4 on body weight or systolic blood pressure compared to the SHRSP + tMCAO group. However, grip strength and sensory neglect measures in the P4 group were improved compared to SHRSP + tMCAO. In addition, significantly larger infarct volumes were seen in the SHRSP + tMCAO group compared to SHRSP + tMCAO + P4. Increased markers of the injury cascade such as macrophages, activated astrocytes, superoxide anion and apoptotic cells observed in the SHRSP + tMCAO group were significantly decreased by P4. We conclude that, despite hypertensive comorbidity, P4 improves functional outcomes and attenuates stroke infarct in hypertensive rats by reducing superoxide anion expression and by decreasing inflammation and neuronal apoptosis.
Korley et al. recently evaluated the relationship between progesterone treatment and serum levels... more Korley et al. recently evaluated the relationship between progesterone treatment and serum levels of biomarkers of glial and neuronal cell death in the ProTECT III trial for progesterone in the treatment of traumatic brain injury. We found a number of serious problems in this paper that make it difficult to accept the authors' conclusions. We believe caution needs to be observed in interpreting their results to indicate a lack of progesterone efficacy in the treatment of TBI.
Background Severity of illness in COVID-19 is consistently lower in women. Focus on sex as a biol... more Background Severity of illness in COVID-19 is consistently lower in women. Focus on sex as a biologic factor may suggest a potential therapeutic intervention for this disease. We assessed whether adding progesterone to standard of care would improve clinical outcomes of hospitalized men with moderate to severe COVID-19. Research Question Does short-term subcutaneous administration of progesterone safely improve clinical outcome in hypoxemic men hospitalized with COVID-19? Study Design and Methods We conducted a pilot, randomized, open-label, controlled trial of subcutaneous progesterone in men hospitalized with confirmed moderate to severe COVID-19. Patients were randomly assigned to receive standard of care (SOC) plus progesterone (100 mg subcutaneously twice daily for up to five days) or SOC alone. In addition to assessment of safety, the primary outcome was change in clinical status at day 7. Length of hospital stay and number of days on supplemental oxygen were key secondary outcomes. Results Forty-two patients were enrolled from April - August 2020; 22 were randomized to the control group and 20 to the progesterone group. Two patients from the progesterone group withdrew from the study prior to receiving progesterone. There was a 1.5-point overall improvement in median clinical status score on a seven-point ordinal scale from baseline to Day 7 in patients in the progesterone group as compared to controls (95%CI:0.0-2.0; P=0.024). There were no serious adverse events attributable to progesterone. Patients treated with progesterone required 3 fewer days of supplemental oxygen (median of 4.5 vs 7.5 days) and were hospitalized for 2.5 fewer days (median of 7.0 vs 9.5 days) as compared to controls. Interpretation Progesterone at a dose of 100 mg, twice daily by subcutaneous injection in addition to SOC may represent a safe and effective approach for treatment in hypoxemic men with moderate to severe COVID-19.
progress away from the capillary tip depending on the speed with which the amoeba flows into the ... more progress away from the capillary tip depending on the speed with which the amoeba flows into the capillary. This interpretation of the pressure events is certainly not the only plausible one, but because it does satisfy the observations and is consistent with the positive pressure gradient theory of pseudopod extension and retraction, it points out that the cited suction experiments do not constitute a direct test of the hydraulic flow theory but are inconclusive in that regard. Even though the observation that the applied suction '"rarely showed any detectable effect on the streaming pattern except in the immediate vicinity of the capillary orifice" can be interpreted as evidence that the negative pressure gradient is established only in the vicinity of the orifice, since no effect is noticed elsewhere; it would seem that directly testing the theory on this basis awaits the deveopment of a method to measure the internal pressure distribution.
It was demonstrated that magnesium pemoline in DMSO attenuates ECS-produced retrograde amnesia wh... more It was demonstrated that magnesium pemoline in DMSO attenuates ECS-produced retrograde amnesia when administered prior to training in a passive avoidance task. We also observed that although there is RA for the specific step-off response that led to painful footshock, Ss receiving ECS showed an increase in emotionality similar to footshock controls suggesting RA for the specific response but not the general test situation. High doses of pemoline (24 mg/kg) not followed by ECS disrupted performance of the response but not retention. Magnesium hydroxide Mg (01t)2 8 mg/kg administered in DMSO proved to be severely disrupting when followed by ECS indicating an interaction with pemoline since pemoline without magnesium q-ECS and Mg (OH)2 ~-FS had no effect on performance in this type of situation.
The purpose of this investigation was to determine whether sequential (i.e., serial) ablation of ... more The purpose of this investigation was to determine whether sequential (i.e., serial) ablation of the monkey's orbital prefrontal cortex would lead to a reduction in the severity of the behavioral impairment usually associated with one-stage bilateral removal of this tissue. The lateral orbital cortex was ablated in four operations spaced 3 weeks apart or in a one-stage procedure. The monkeys were examined on a visual go-no go differentiation task, spatial delayed-alternation, and object reversal learning. The results reveal no differences between the effects of sequential and one-stage ablations. These findings differ from previous experiments that demonstrated a degree of functional recovery after the sequential removal of a sector of the dorsolateral prefrontal cortex. Since lesion studies with infant monkeys have also demonstrated that functional recovery occurs after early ablation of dorsolateral cortex but not after early removal of orbital frontal cortex, recovery of behavioral functions after infant and sequential lesions may involve similar neural mechanisms. 204
The field of neuroprotection after brain injuries has been littered with failed clinical trials. ... more The field of neuroprotection after brain injuries has been littered with failed clinical trials. Finding a safe and effective treatment for acute traumatic brain injury remains a serious unmet medical need. Repurposing drugs that have been in use for other disorders is receiving increasing attention as a strategy to move candidate drugs more quickly to trial while reducing the very high cost of new drug development. This paper describes our own serendipitous discovery of progesterone's neuroprotective potential, and the strategies we are using in repurposing and developing this hormone for use in brain injuries-applications very different from its classical uses in treating disorders of the reproductive system. We have been screening and testing a novel analog that maintains progesterone's therapeutic properties while overcoming its physiochemical challenges, and testing progesterone in combination treatment with another pleiotropic hormone, vitamin D. Finally, our paper, in the context of the problems and pitfalls we have encountered, surveys some of the factors we found to be critical in the clinical translation of repurposed drugs.
Restorative Neurology and Neuroscience, Mar 6, 2019
Background: In this proof-of-concept paper, we investigated whether combination treatment with pr... more Background: In this proof-of-concept paper, we investigated whether combination treatment with progesterone (P4) and chloroquine (CQ) would reduce ischemic injury more effectively than either agent alone in a transient middle cerebral artery occlusion (tMCAO) model in male rats. Methods: P4 (8 mg/kg) and CQ (25 mg/kg) were given alone or in combination beginning at different times during surgery and for 3 days post-occlusion. Locomotor activity and grip strength were evaluated as measures of impairment and recovery. Infarct size was assessed by TTC staining. Markers of autophagy (LC3 and SQSTM1/p62) and apoptosis (Bcl-2 and Bax) were evaluated with western blotting. Results: At the doses we employed, the combination was not more effective than either drug given separately on measures of grip strength or locomotor activity. However, combination therapy substantially reduced infarct size, and significantly increased Bcl-2 protein levels and suppressed Bax expression. Progesterone decreased the expression of LC3-II 24 h and SQSTM1/p62 after ischemia. Conclusions: Our findings suggest that combination therapy with P4 and CQ is not detrimental and has a small-to-moderate additive neuroprotective effect on ischemic injury in rats without substantively affecting behavioral outcomes. CQ and P4 may help to regulate the expression of both autophagy-related and apoptosis-related proteins.
Background: Repeated mild traumatic brain injuries, such as concussions, may result in cumulative... more Background: Repeated mild traumatic brain injuries, such as concussions, may result in cumulative brain damage, neurodegeneration and other chronic neurological impairments. There are currently no clinically available treatment options known to prevent these consequences. However, growing evidence implicates neuroinflammation and oxidative stress in the pathogenesis of repetitive mild brain injuries; thus, these may represent potential therapeutic targets. Progesterone has been demonstrated to have potent anti-inflammatory and anti-oxidant properties after brain insult; therefore, here, we examined progesterone treatment in rats given repetitive mild brain injuries via the repeated mild fluid percussion injury model. Methods: Male Long-Evans rats were assigned into four groups: sham injury + vehicle treatment, sham injury + progesterone treatment (8 mg/kg/day), repeated mild fluid percussion injuries + vehicle treatment, and repeated mild fluid percussion injuries + progesterone treatment. Rats were administered a total of three injuries, with each injury separated by 5 days. Treatment was initiated 1 h after the first injury, then administered daily for a total of 15 days. Rats underwent behavioural testing at 12-weeks post-treatment to assess cognition, motor function, anxiety and depression. Brains were then dissected for analysis of markers for neuroinflammation and oxidative stress. Ex vivo MRI was conducted in order to examine structural brain damage and white matter integrity. Results: Repeated mild fluid percussion injuries + progesterone treatment rats showed significantly reduced cognitive and sensorimotor deficits compared to their vehicle-treated counterparts at 12-weeks post-treatment. Progesterone treatment significantly attenuated markers of neuroinflammation and oxidative stress in rats given repeated mild fluid percussion injuries, with concomitant reductions in grey and white matter damage as indicated by MRI. Conclusions: These findings implicate neuroinflammation and oxidative stress in the pathophysiological aftermath of mild brain injuries and suggest that progesterone may be a viable treatment option to mitigate these effects and their detrimental consequences.
Annals of the New York Academy of Sciences, Jun 1, 1987
MORPHOLOGIC FEATURES OF FETAL FRONTAL TRANSPLANTS During the last several years, there has been a... more MORPHOLOGIC FEATURES OF FETAL FRONTAL TRANSPLANTS During the last several years, there has been a resurgence of interest in the ability of implanted fetal central nervous system (CNS) tissue to promote anatomical restoration and return of function to brain-damaged mammals. ' The parameters under
Tissue plasminogen activator (tPA) is one of the few approved treatments for stroke, but its effe... more Tissue plasminogen activator (tPA) is one of the few approved treatments for stroke, but its effects on the phenotype of microglia/macrophages are poorly understood. One of its side effects is an increase in the inflammatory response leading to neuronal cell damage and death in the ischemic cascade after stroke. Injury-induced activated microglia/macrophages can have dual functions as pro-inflammatory (M1) and anti-inflammatory (M2) factors in brain injury and repair. Recent studies show that progesterone (PROG) is a potent anti-inflammatory agent which affects microglia/macrophage expression after brain injury. We examined the interaction of tPA-induced expression of microglia/macrophage phenotypes and PROG's anti-inflammatory effects. tPA treatment increased the recruitment of microglia/macrophages, the polarity of M1 reactions, the expression of MIP-1α in neurons and capillaries, and the expression of MMP-3 compared to vehicle, and PROG modulated these effects. PROG treatment attenuates tPA-induced inflammatory alterations in brain capillaries and microglia/macrophages both in vivo and in vitro and thus may be a useful adjunct therapy when tPA is given for stroke.
Embryonic cortex from 19-day fetuses was transplanted in a medial frontal cortex wound cavity of ... more Embryonic cortex from 19-day fetuses was transplanted in a medial frontal cortex wound cavity of 105-day-old male rats. Nisslstained tissue revealed little internal laminar organization. Graft sections impregnated by the Loyez method exhibited bands of myelinated fibers surrounding implants as well as long-interconnecting and swirl-like fiber fascicles within the implant. Tissue processed histochemically for acetylcholinesterase and choline acetyltransferase revealed enzyme-positive fibers and cell bodies within the grafts. Cytochrome oxidase histochemistry revealed regional variations in the metabolic activity of the grafts. In summary, although our frontal cortex grafts exhibit many of the morphological features seen in intact frontal cortex, the organization of these components within the implant is dissimilar to normal cortical tissue.
Neonatal stroke is among the top ten causes of childhood death and causes long-term permanent dis... more Neonatal stroke is among the top ten causes of childhood death and causes long-term permanent disability and neurological deficits in survivors. In most cases, recurrent clonic seizure is the only clinical manifestation which worsens the stroke outcomes. A treatment or pediatric stroke is essential, but no candidate intervention is supported by substantiated data. We tested the hypothesis that progesterone (P4) treatment would be beneficial in a neonatal model of stroke. P12 CD1 mice (mixed gender) underwent permanent unilateral right common carotid ligation (pUCCL) or sham surgery (n=10/group). Pups which showed seizure activity during the 1h post-pUCCL were randomly assigned to receive P4 (8 mg/kg) or vehicle injections at 1, 3 and every 24h post-pUCCL for 6 days. We assessed acute behavioral seizures (during the first 4h post-pUCCL), serum pro-inflammatory cytokines (IL-1β, IL-6, TNFα at 6, 24 and 48h) and brain infarction (at day 7 by CV-staining). Cytokine data were analyzed by repeated measures one-way ANOVA followed by LSD and Tukey’s tests for independent comparisons. For seizure and infarct data, a two-tailed unpaired t-test was employed. We observed acute seizures during the first 4h post-pUCCL in the vehicle group (90.8±11.77). P4 treatment significantly ( P <0.05) reduced seizure occurrence (58.4±8.97) by 35% compared to vehicle. Repeated measures ANOVA revealed a significant group effect in IL-1β (F (2,15) =110.706; P <0.001), IL-6 (F (2,15 ) =66.067; P <0.001), and TNFα (F (2,15) =146.263; P <0.001) levels. Serum IL-1β, IL-6 and TNFα were significantly higher ( P <0.001) at 6 and 24h and remained elevated until 48h post-pUCCL in the vehicle group compared to sham. The P4-treated group showed a significant ( P <0.01) decrease in all pro-inflammatory cytokine levels at all time points. Further, pUCCL resulted in severe hemispheric damage (16.88±1.48) as evidenced by cresyl-violet staining at 7 days post-pUCCL. P4 treatment showed a significant ( P <0.05) reduction (4.93±1.1) in infarct volume (~70%) compared to vehicle. Our data demonstrate that P4 reduces acute seizures and brain infarction following neonatal stroke by modulating the inflammatory process, and warrants detailed studies of functional outcomes and mechanism of action.
Uploads
Papers by Donald Stein