Mass Balance, Metabolic Pathways, Absolute Bioavailability, and Pharmacokinetics of Giredestrant in Healthy Subjects.

Giredestrant is a potent and selective small-molecule estrogen receptor degrader. The objectives of this study were to assess the absolute bioavailability (aBA) of giredestrant and to determine the mass balance, routes of elimination, and metabolite profile of [14C]giredestrant. In part 1 (mass balance), a single 30.8-mg oral dose of [14C]giredestrant (105 µCi) was administered to women of nonchildbearing potential (WNCBP; n = 6). The mean recovery of total radioactivity in excreta was 77.0%, with 68.0% of the dose excreted in feces and 9.04% excreted in urine over a 42-day sample collection period. The majority of the circulating radioactivity (56.8%) in plasma was associated with giredestrant. Giredestrant was extensively metabolized, with giredestrant representing only 20.0% and 1.90% of the dose in feces and urine, respectively. All metabolites in feces resulted from oxidative metabolism and represented 44.7% of the dose. In part 2 (aBA), WNCBP (n = 10) received an oral (30-mg capsule) or intravenous (30-mg solution) dose of giredestrant. The aBA of giredestrant after oral administration was 58.7%. Following the intravenous dose, giredestrant had a plasma clearance and volume of distribution of 5.31 L/h and 266 L, respectively. In summary, giredestrant was well tolerated, rapidly absorbed, and showed moderate oral bioavailability with low recovery of the dose as parent drug in excreta. Oxidative metabolism followed by excretion in feces was identified as the major route of elimination of giredestrant. SIGNIFICANCE STATEMENT: This study provides definitive insight into the absorption, distribution, metabolism, and excretion of giredestrant in humans. The results show that giredestrant exhibits low clearance, a high volume of distribution, and moderate oral bioavailability in humans. In addition, the data show that oxidative metabolism followed by excretion in feces is the primary elimination route of giredestrant in humans. These results will be used to further inform the clinical development of giredestrant.

Phase Ia/b Study of Giredestrant ± Palbociclib and ± Luteinizing Hormone-Releasing Hormone Agonists in Estrogen Receptor-Positive, HER2-Negative, Locally Advanced/Metastatic Breast Cancer.

PURPOSE: Giredestrant is an investigational next-generation, oral, selective estrogen receptor antagonist and degrader for the treatment of estrogen receptor-positive (ER+) breast cancer. We present the primary analysis results of the phase Ia/b GO39932 study (NCT03332797). PATIENTS AND METHODS: Patients with ER+, HER2-negative locally advanced/metastatic breast cancer previously treated with endocrine therapy received single-agent giredestrant (10, 30, 90, or 250 mg), or giredestrant (100 mg) ± palbociclib 125 mg ± luteinizing hormone-releasing hormone (LHRH) agonist. Detailed cardiovascular assessment was conducted with giredestrant 100 mg. Endpoints included safety (primary), pharmacokinetics, pharmacodynamics, and efficacy. RESULTS: As of January 28, 2021, with 175 patients enrolled, no dose-limiting toxicity was observed, and the MTD was not reached. Adverse events (AE) related to giredestrant occurred in 64.9% and 59.4% of patients in the single-agent ± LHRH agonist and giredestrant + palbociclib ± LHRH agonist cohorts, respectively (giredestrant-only-related grade 3/4 AEs were reported in 4.5% of patients across the single-agent cohorts and 3.1% of those with giredestrant + palbociclib). Dose-dependent asymptomatic bradycardia was observed, but no clinically significant changes in cardiac-related outcomes: heart rate, blood pressure, or exercise duration. Clinical benefit was observed in all cohorts (48.6% of patients in the single-agent cohort and 81.3% in the giredestrant + palbociclib ± LHRH agonist cohort), with no clear dose relationship, including in patients with ESR1-mutated tumors. CONCLUSIONS: Giredestrant was well tolerated and clinically active in patients who progressed on prior endocrine therapy. Results warrant further evaluation of giredestrant in randomized trials in early- and late-stage ER+ breast cancer.

Optimizing Early-stage Clinical Pharmacology Evaluation to Accelerate Clinical Development of Giredestrant in Advanced Breast Cancer.

PURPOSE: We describe the clinical pharmacology characterization of giredestrant in a first-in-human study. EXPERIMENTAL DESIGN: This phase Ia/Ib dose-escalation/-expansion study (NCT03332797) evaluated the safety, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity of giredestrant in estrogen receptor-positive HER2-negative locally advanced/metastatic breast cancer. The single-agent dose-escalation stage evaluated giredestrant 10, 30, 90, or 250 mg once daily. The dose-expansion stage evaluated single-agent giredestrant at 30, 100, and 250 mg once daily. Dose-escalation and -expansion phases also evaluated giredestrant 100 mg combined with palbociclib 125 mg. RESULTS: Following single-dose oral administration, giredestrant was rapidly absorbed and generally showed a dose-proportional increase in exposure at doses ranging from 10 to 250 mg. At the 30 mg clinical dose, maximum plasma concentration was 266 ng/mL (50.1%) and area under the concentration-time curve from 0 to 24 hours at steady state was 4,320 ng·hour/mL (59.4%). Minimal giredestrant concentrations were detected in urine, indicating that renal excretion is unlikely to be a major elimination route for giredestrant. Mean concentration of 4beta-hydroxycholesterol showed no apparent increase over time at both the clinical dose (30 mg) and a supratherapeutic dose (90 mg), suggesting that giredestrant may have low CYP3A induction potential in humans. No clinically relevant drug-drug interaction was observed between giredestrant and palbociclib. Giredestrant exposure was not affected by food and was generally consistent between White and Asian patients. CONCLUSIONS: This study illustrates how the integration of clinical pharmacology considerations into early-phase clinical trials can inform the design of pivotal studies and accelerate oncology drug development. SIGNIFICANCE: This work illustrates how comprehensive clinical pharmacology characterization can be integrated into first-in-human studies in oncology. It also demonstrates the value of understanding clinical pharmacology attributes to inform eligibility, concomitant medications, and combination dosing and to directly influence late-stage trial design and accelerate development.

An Open-label Phase I Study of GDC-0927 in Postmenopausal Women with Locally Advanced or Metastatic Estrogen Receptor-Positive Breast Cancer.

PURPOSE: GDC-0927 is a novel, potent, nonsteroidal, orally bioavailable, selective estrogen receptor (ER) degrader that induces tumor regression in ER+ breast cancer xenograft models. PATIENTS AND METHODS: This phase I dose-escalation multicenter study enrolled postmenopausal women with ER+/HER2- metastatic breast cancer to determine the safety, pharmacokinetics, and recommended phase II dose of GDC-0927. Pharmacodynamics was assessed with [18F]-fluoroestradiol (FES) PET scans. RESULTS: Forty-two patients received GDC-0927 once daily. The MTD was not reached. The most common adverse events (AE) regardless of causality were nausea, constipation, diarrhea, arthralgia, fatigue, hot flush, back pain, and vomiting. There were no deaths, grade 4/5 AEs, or treatment-related serious AEs. Two patients experienced grade 2 AEs of special interest of deep vein thrombosis and jugular vein thrombosis, both considered unrelated to GDC-0927. Following dosing, approximately 1.6-fold accumulation was observed, consistent with the observed half-life and dosing frequency. There were no complete or partial responses. Pharmacodynamics was supported by >90% reduction in FES uptake and an approximately 40% reduction in ER expression, suggesting ER degradation is not the mechanistic driver of ER antagonism. Twelve patients (29%) achieved clinical benefit; 17 patients (41%) showed a confirmed best overall response of stable disease. Baseline levels of ER and progesterone receptor protein and mutant ESR1 circulating tumor DNA did not correlate with clinical benefit. CONCLUSIONS: GDC-0927 appeared to be well tolerated with pharmacokinetics supporting once-daily dosing. There was evidence of target engagement and preliminary evidence of antitumor activity in heavily pretreated patients with advanced/metastatic ER+/HER2- breast cancer with and without ESR1 mutations.

The oral selective estrogen receptor degrader GDC-0810 (ARN-810) in postmenopausal women with hormone receptor-positive HER2-negative (HR + /HER2 -) advanced/metastatic breast cancer.

PURPOSE: GDC-0810 (ARN-810) is a novel, non-steroidal, orally bioavailable, selective estrogen receptor degrader (SERD) that potentially inhibits ligand-dependent and ligand-independent estrogen receptor (ER)-mediated signaling. METHODS: A phase Ia/Ib/IIa dose escalation, combination treatment with palbociclib or a luteinizing hormone-releasing hormone, and expansion study determined the safety, pharmacokinetics, and recommended phase 2 dose (RP2D) of GDC-0810 in postmenopausal women with ER + (HER2 -) locally advanced or metastatic breast cancer (MBC). Baseline plasma ctDNA samples were analyzed to determine the ESR1 mutation status. RESULTS: Patients (N = 152) received GDC-0810 100-800 mg once daily (QD) or 300-400 mg twice daily, in dose escalation, expansion, as single agent or combination treatment. Common adverse events regardless of attribution to study drug were diarrhea, nausea, fatigue, vomiting, and constipation. There was one dose-limiting toxicity during dose escalation. The maximum tolerated dose was not reached. GDC-0810 600 mg QD taken with food was the RP2D. Pharmacokinetics were predictable. FES reduction (> 90%) highlighting pharmacodynamic engagement of ER was observed. Outcomes for the overall population and for patients with tumors harboring ESR1 mutations included partial responses (4% overall; 4% ESR1), stable disease (39% overall; 42% ESR1), non-complete response/non-progressive disease (13% overall; 12% ESR1), progressive disease (40% overall; 38% ESR1), and missing/unevaluable (5% overall; 5% ESR1). Clinical benefit (responses or SD, lasting ≥ 24 weeks) was observed in patients in dose escalation (n = 16, 39%) and expansion (n = 24, 22%). CONCLUSION: GDC-0810 was safe and tolerable with preliminary anti-tumor activity in heavily pretreated patients with ER + advanced/MBC, with/without ESR1 mutations, highlighting the potential for oral SERDs. Clinical Trial and registration date April 4, 2013. NCT01823835 .

Giredestrant reverses progesterone hypersensitivity driven by estrogen receptor mutations in breast cancer.

ESR1 (estrogen receptor 1) hotspot mutations are major contributors to therapeutic resistance in estrogen receptor-positive (ER+) breast cancer. Such mutations confer estrogen independence to ERα, providing a selective advantage in the presence of estrogen-depleting aromatase inhibitors. In addition, ESR1 mutations reduce the potency of tamoxifen and fulvestrant, therapies that bind ERα directly. These limitations, together with additional liabilities, inspired the development of the next generation of ERα-targeted therapeutics, of which giredestrant is a high-potential candidate. Here, we generated Esr1 mutant-expressing mammary gland models and leveraged patient-derived xenografts (PDXs) to investigate the biological properties of the ESR1 mutations and their sensitivity to giredestrant in vivo. In the mouse mammary gland, Esr1 mutations promote hypersensitivity to progesterone, triggering pregnancy-like tissue remodeling and profoundly elevated proliferation. These effects were driven by an altered progesterone transcriptional response and underpinned by gained sites of ERα-PR (progesterone receptor) cobinding at the promoter regions of pro-proliferation genes. PDX experiments showed that the mutant ERα-PR proliferative program is also relevant in human cancer cells. Giredestrant suppressed the mutant ERα-PR proliferation in the mammary gland more so than the standard-of-care agents, tamoxifen and fulvestrant. Giredestrant was also efficacious against the progesterone-stimulated growth of ESR1 mutant PDX models. In addition, giredestrant demonstrated activity against a molecularly characterized ESR1 mutant tumor from a patient enrolled in a phase 1 clinical trial. Together, these data suggest that mutant ERα can collaborate with PR to drive protumorigenic proliferation but remain sensitive to inhibition by giredestrant.

Phase Ib study of the MEK inhibitor cobimetinib (GDC-0973) in combination with the PI3K inhibitor pictilisib (GDC-0941) in patients with advanced solid tumors.

Purpose We investigated the combination of the MEK inhibitor, cobimetinib, and the pan-PI3K inhibitor, pictilisib, in an open-label, phase Ib study. Experimental Design Patients with advanced solid tumors were enrolled in 3 dose escalation schedules: (1) both agents once-daily for 21-days-on 7-days-off ("21/7"); (2) intermittent cobimetinib and 21/7 pictilisib ("intermittent"); or (3) both agents once-daily for 7-days-on 7-days-off ("7/7"). Starting doses for the 21/7, intermittent, and 7/7 schedules were 20/80, 100/130, and 40/130 mg of cobimetinib/pictilisib, respectively. Nine indication-specific expansion cohorts interrogated the recommended phase II dose and schedule. Results Of 178 enrollees (dose escalation: n = 98), 177 patients were dosed. The maximum tolerated doses for cobimetinib/pictilisib (mg) were 40/100, 125/180, and not reached, for the 21/7, intermittent, and 7/7 schedules, respectively. Six dose-limiting toxicities included grade 3 (G3) elevated lipase, G4 elevated creatine phosphokinase, and G3 events including fatigue concurrent with a serious adverse event (SAE) of diarrhea, decreased appetite, and SAEs of hypersensitivity and dehydration. Common drug-related adverse events included nausea, fatigue, vomiting, decreased appetite, dysgeusia, rash, and stomatitis. Pharmacokinetic parameters of the drugs used in combination were unaltered compared to monotherapy exposures. Confirmed partial responses were observed in patients with BRAF-mutant melanoma (n = 1) and KRAS-mutant endometrioid adenocarcinoma (n = 1). Eighteen patients remained on study ≥6 months. Biomarker data established successful blockade of MAP kinase (MAPK) and PI3K pathways. The metabolic response rate documented by FDG-PET was similar to that observed with cobimetinib monotherapy. Conclusions Cobimetinib and pictilisib combination therapy in patients with solid tumors had limited tolerability and efficacy.

Correction to: Application of a Novel 'Make and Test in Parallel' Strategy to Investigate the Effect of Formulation on the Pharmacokinetics of GDC-0810 in Healthy Subjects.

The Publisher regrets the typesetting mistake of retaining incorrect text in the Figure 1 caption. The correct text for the caption is "Molecular Structure of GDC-0810 NMG Salt". The original article has been corrected.

Application of a Novel 'Make and Test in Parallel' Strategy to Investigate the Effect of Formulation on the Pharmacokinetics of GDC-0810 in Healthy Subjects.

PURPOSE: GDC-0810, administered orally, was used in Phase I and II clinical studies to treat estrogen receptor positive breast cancers. It contains N-methyl-D-glucamine (NMG) salt of GDC-0810 with 10% sodium lauryl sulfate (SLS) as a surfactant and 15% sodium bicarbonate (NaHCO3) as an alkalizing agent to aid dissolution. To improve the processability of the formulation and reduce potential mucosal irritation in future Phase III clinical studies, the salt form and the amount of excipient required further optimization. To achieve this, we employed a novel "Make and Test in Parallel" strategy that facilitated selecting formulation in a rapid timeframe. METHODS: RapidFACT®, a streamlined, data-driven drug product optimization platform was used to bridge Phase I/II and Phase III formulations of GDC-0810. Five prototype formulations, varying in either the form of active pharmaceutical ingredient and/or the levels of the excipients SLS and NaHCO3 were assessed. Uniquely, the specific compositions of formulations manufactured and dosed were selected in real-time from emerging clinical data. RESULTS: The study successfully identified a Phase III formulation with a reduced SLS content, which when administered following a low-fat meal, gave comparable pharmacokinetic exposure to the Phase I/II formulation administered under the same conditions. CONCLUSIONS: Our novel 'Make and Test in Parallel' approach enabled optimization of GDC-0810 formulation in a time- and cost-efficient fashion.

Effect of OATP1B1/1B3 Inhibitor GDC-0810 on the Pharmacokinetics of Pravastatin and Coproporphyrin I/III in Healthy Female Subjects.

Developed as an oral anticancer drug to treat estrogen receptor-positive breast cancer, GDC-0810 was shown to be a potent inhibitor of organic anion-transporting polypeptide 1B1 and 1B3 (OATP1B1/1B3) from an in vitro assay. A clinical study was conducted to assess the drug-drug interaction potential between GDC-0810 and pravastatin, which is a relatively selective and sensitive OATP1B1/1B3 substrate. Fifteen healthy female subjects of non-childbearing potential were enrolled in the study. On day 1 in period 1, a single 10-mg dose of pravastatin was administered to all subjects. Following a 4-day washout period, 600 mg of GDC-0810 was administered once daily on days 5 through 8 in period 2 to achieve steady-state concentrations. On day 7, a single dose of 10-mg pravastatin was coadministered with the 600-mg GDC-0810 dose. Concentrations of pravastatin (periods 1 and 2) and GDC-0810 (period 2 only) were quantified in blood samples and subsequently used to calculate the pharmacokinetics (PK) parameters. The pravastatin mean maximal concentration and area under the curve values were approximately 20% and 41% higher, respectively, following pravastatin coadministration with GDC-0810 compared to pravastatin alone. Based on the magnitude of change in this drug-drug interaction study, dose adjustments for pravastatin (and other OATP1B1/1B3 substrates) were not considered necessary when administered with GDC-0810. Retrospectively, the endogenous biomarkers of OATP1B1/1B3, coproporphyrin I and III, were also measured and showed changes comparable to those of pravastatin, indicating their utility in detecting weak inhibition of OATP1B1/1B3 in the clinical setting.

Absorption, Metabolism, Excretion, and the Contribution of Intestinal Metabolism to the Oral Disposition of [14C]Cobimetinib, a MEK Inhibitor, in Humans.

The pharmacokinetics, metabolism, and excretion of cobimetinib, a MEK inhibitor, were characterized in healthy male subjects (n = 6) following a single 20 mg (200 µCi) oral dose. Unchanged cobimetinib and M16 (glycine conjugate of hydrolyzed cobimetinib) were the major circulating species, accounting for 20.5% and 18.3% of the drug-related material in plasma up to 48 hours postdose, respectively. Other circulating metabolites were minor, accounting for less than 10% of drug-related material in plasma. The total recovery of the administered radioactivity was 94.3% (±1.6%, S.D.) with 76.5% (±2.3%) in feces and 17.8% (±2.5%) in urine. Metabolite profiling indicated that cobimetinib had been extensively metabolized with only 1.6% and 6.6% of the dose remaining as unchanged drug in urine and feces, respectively. In vitro phenotyping experiments indicated that CYP3A4 was predominantly responsible for metabolizing cobimetinib. From this study, we concluded that cobimetinib had been well absorbed (fraction absorbed, Fa = 0.88). Given this good absorption and the previously determined low hepatic clearance, the systemic exposures were lower than expected (bioavailability, F = 0.28). We hypothesized that intestinal metabolism had strongly attenuated the oral bioavailability of cobimetinib. Supporting this hypothesis, the fraction escaping gut wall elimination (Fg) was estimated to be 0.37 based on F and Fa from this study and the fraction escaping hepatic elimination (Fh) from the absolute bioavailability study (F = Fa × Fh × Fg). Physiologically based pharmacokinetics modeling also showed that intestinal clearance had to be included to adequately describe the oral profile. These collective data suggested that cobimetinib was well absorbed following oral administration and extensively metabolized with intestinal first-pass metabolism contributing to its disposition.

Role of endogenous Schwann cells in tissue repair after spinal cord injury.

Schwann cells are glial cells of peripheral nervous system, responsible for axonal myelination and ensheathing, as well as tissue repair following a peripheral nervous system injury. They are one of several cell types that are widely studied and most commonly used for cell transplantation to treat spinal cord injury, due to their intrinsic characteristics including the ability to secrete a variety of neurotrophic factors. This mini review summarizes the recent findings of endogenous Schwann cells after spinal cord injury and discusses their role in tissue repair and axonal regeneration. After spinal cord injury, numerous endogenous Schwann cells migrate into the lesion site from the nerve roots, involving in the construction of newly formed repaired tissue and axonal myelination. These invading Schwann cells also can move a long distance away from the injury site both rostrally and caudally. In addition, Schwann cells can be induced to migrate by minimal insults (such as scar ablation) within the spinal cord and integrate with astrocytes under certain circumstances. More importantly, the host Schwann cells can be induced to migrate into spinal cord by transplantation of different cell types, such as exogenous Schwann cells, olfactory ensheathing cells, and bone marrow-derived stromal stem cells. Migration of endogenous Schwann cells following spinal cord injury is a common natural phenomenon found both in animal and human, and the myelination by Schwann cells has been examined effective in signal conduction electrophysiologically. Therefore, if the inherent properties of endogenous Schwann cells could be developed and utilized, it would offer a new avenue for the restoration of injured spinal cord.

Somatosensory evoked potentials can be recorded on the midline of the skull with subdermal electrodes in non-sedated rats elicited by magnetic stimulation of the tibial nerve.

Somatosensory evoked potentials (SSEPs) are a sensitive quantitative measure of conduction in somatosensory pathways of the central nervous system and are increasingly used in both clinical trials and animal experiments. SSEPs can be recorded in non-sedated rodents by magnetic stimulation (MS) of peripheral nerves. To overcome some disadvantages caused by using anesthesia and implanted recording electrodes, we used subdermal needle electrodes located on the midline of the skull to successfully record SSEPs in non-sedated rats, elicited by stimulating the tibial nerve with a magnetic stimulator. The wave form contains a typical P1 peak and N1 peak. Although there is a variation of P1 latency, N1 latency, and P1-N1 amplitude between right side and left side, it was not statistically significant. In addition, there is a significantly positive relationship between P1-N1 amplitude and MS strength, suggesting that the increase in magnetic stimulating strength resulted in the increase in P1-N1 amplitude. Results in the present study demonstrate that our modified method is a reliable and feasible paradigm for recording SSEPs in non-sedated rats.

Tail nerve electrical stimulation combined with scar ablation and neural transplantation promotes locomotor recovery in rats with chronically contused spinal cord.

To date, few treatment strategies applying cellular transplantation to the chronically injured spinal cord have yielded significant functional improvement in animal experiments. Here we report that significant improvement of locomotor function was achieved in rats with chronic spinal cord injury (SCI) by the application of combination treatments with tail nerve electrical stimulation (TANES), which can activate the central pattern generator, inducing active weight-supported stepping. Contusion injury (25 mm) to spinal cord T10 was produced by using the NYU impactor device in female, adult Long-Evans rats. Rats in 2 of 4 groups with SCI received basic treatments (scar ablation followed by transplantation of lamina propria of olfactory mucosa and cultured olfactory ensheathing cells into the lesion cavity) 6 weeks after SCI. Rats both with and without basic treatments were subjected to TANES one week after secondary surgery or 7 weeks after SCI. Sixteen weeks after secondary surgery or 22 weeks after SCI rats in two groups receiving TANES significantly improved their functional recovery compared with those without TANES, when evaluated with BBB open field rating scale (p<0.01). Among them, however, rats with basic treatments performed better than those without basic treatments. TANES may contribute to the activity-dependent plasticity below the injury level, which is critical for functional recovery. Additionally, TANES may promote axonal regeneration, including those from supraspinal level. Since TANES demonstrated considerable potential for achieving improvement of functional recovery in rat model, it would suggest a new strategy for chronic SCI.

Scar ablation combined with LP/OEC transplantation promotes anatomical recovery and P0-positive myelination in chronically contused spinal cord of rats.

We have successfully removed an existing glial scar in chronically contused rat spinal cord using a rose Bengal-based phototoxic method. The purpose of this study is to examine if scar ablation benefits the anatomical recovery by cell/tissue transplantation, and thus provides a more permissive physical and biochemical environment for axonal growth, which may lead to functional recovery. Immediately after scar ablation, we transplanted lamina propria (LP) of the olfactory mucosa alone or in combination with cultured olfactory ensheathing cells (OEC) into the lesion cavity 6 weeks after contusion injury (NYU impactor device, 25 mm height setting) at spinal cord segment T10 of adult female Long-Evans rats. Sixteen weeks after scar ablation and transplantation, we found that the initial repaired tissue significantly expanded, companied by remarkable reduction or disappearance of the lesion cavity and integration of repaired tissue with the spared tissue, thus resulting in histological repair of damaged cord tissue at the injury epicenter. Glial scar reformation was effectively prevented after ablation due to the tissue repair. In addition, at the injury epicenter P0 (myelin glycoprotein P-zero)-positive myelination formed by Schwann cells, which are known to myelinate regenerating and demyelinated axons, were significantly increased in number compared with the control animals. However, when evaluated with BBB open-field scale a significant improvement of locomotor function was not observed in this study; the possible reasons were discussed.

Histological repair of damaged spinal cord tissue from chronic contusion injury of rat: a LM observation.

The spinal cord has an intrinsic, limited ability of spontaneous repair; the endogenous repair of damaged tissue starts a few days after spinal cord injury (SCI). To date, however, detailed observation in histology at the injury site has not been well documented. In the present study we analyzed the histological structure of the repaired tissue from injury site of rats 6 or 14 weeks after contusion injury (NYU impactor device, 25 mm height setting) on T10, and rats 8 weeks after transplantation of lamina propria (LP) or acellular lamina propria. We found that the initial repaired tissue can be histologically divided into three different zones, i.e., fibrotic, cellular and axonal. The fibrotic zone consists of invading connective tissue, while the cellular zone is composed of invading, densely compacted Schwann cells. Schwann cells migrate from dorsal roots laterally toward and merge underneath the fibrotic zone, forming the U-shape shell of the cellular zone. The major component of the axonal zone is regenerating axons. Schwann cells myelinate regenerating axons in all three zones. In rats with combination treatments including scar ablation and LP transplantation, both cellular and axonal zones significantly expand in size, resulting in the disappearance of the lesion cavity and the integration of repaired tissue with spared tissue. Olfactory ensheathing cells from transplanted LP may promote the expansion of the cellular and axonal zones through stimulating host Schwann cells, indirectly contributing to tissue repair and axonal regeneration. The ependyma-derived cells may be directly involved in tissue repair, but not contribute to the formation of myelin sheaths.

Extensive scarring induced by chronic intrathecal tubing augmented cord tissue damage and worsened functional recovery after rat spinal cord injury.

Intrathecal infusion has been widely used to directly deliver drugs or neurotrophins to a lesion site following spinal cord injury. Evidence shows that intrathecal infusion is efficient for 7 days but is markedly reduced after 14 days, due to time dependent occlusion. In addition, extensive fibrotic scarring is commonly observed with intrathecal infusion. These anomalies need to be clearly elucidated in histology. In the present study, all adult Long-Evans rats received a 25 mm contusion injury on spinal cord T10 produced using the NYU impactor device. Immediately after injury, catheter tubing with an outer diameter of 0.38 mm was inserted through a small dural opening at L3 into the subdural space with the tubing tip positioned near the injury site. The tubing was connected to an Alzet mini pump, which was filled with saline solution and was placed subcutaneously. Injured rats without tubing served as control. Rats were behaviorally tested for 6 weeks using the BBB locomotor rating scale and histologically assessed for tissue scarring. Six weeks later, we found that the intrathecal tubing caused extensive scarring and inflammation, related to neutrophils, macrophages and plasma cells. The tubing's tip was occluded by scar tissue and inflammatory cells. The scar tissue surrounding the tubing consists of 20-70 layers of fibroblasts and densely compacted collagen fibers, seriously compressing and damaging the cord tissue. BBB scores of rats with intrathecal tubing were significantly lower than control rats (p<0.01) from 2 weeks after injury, implying serious impairment of functional recovery caused by the scarring.

Tail nerve electrical stimulation induces body weight-supported stepping in rats with spinal cord injury.

Walking or stepping has been considered the result from the activation of the central pattern generator (CPG). In most patients with spinal cord injury (SCI) the CPG is undamaged. To date, there are no noninvasive approaches for activating the CPG. Recently we developed a noninvasive technique, tail nerve electrical stimulation (TANES), which can induce positive hind limb movement of SCI rats. The purpose of this study is to introduce the novel technique and examine the effect of TANES on CPG activation. A 25 mm contusion injury was produced at spinal cord T10 of female, adult Long-Evans rats by using the NYU impactor device. Rats received TANES ( approximately 40 mA at 4 kHz) 7 weeks after injury. During TANES all injured rats demonstrated active body weight-supported stepping of hind limbs with left-right alternation and occasional front-hind coordination, resulting in significant, temporary increase in BBB scores (p<0.01). However, there is no response to TANES from rats with L2 transection, consistent with other reports that the CPG may be located at L1-2. S1 transection negatively implies the key role of TANES in CPG activation. The TANES not only renders paralyzed rats with a technique-induced ability to walk via activating CPG, but also is likely to be used for locomotor training. It has more beneficial effects for physical training over other training paradigms including treadmill training and invasive functional electrical stimulation. Therefore the TANES may have considerable potential for achieving improvement of functional recovery in animal models and a similar method may be suggested for human study.

Pharmacological characterization of the competitive GLUK5 receptor antagonist decahydroisoquinoline LY466195 in vitro and in vivo.

The excitatory neurotransmitter glutamate has been implicated in both migraine and persistent pain. The identification of the kainate receptor GLU(K5) in dorsal root ganglia, the dorsal horn, and trigeminal ganglia makes it a target of interest for these indications. We examined the in vitro and in vivo pharmacology of the competitive GLU(K5)-selective kainate receptor antagonist LY466195 [(3S,4aR,6S,8aR)-6-[[(2S)-2-carboxy-4,4-difluoro-1-pyrrolidinyl]-methyl]decahydro-3-isoquinolinecarboxylic acid)], the most potent GLU(K5) antagonist described to date. Comparisons were made to the competitive GLU(K5)/alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor antagonist LY293558 [(3S,4aR,6R,8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl]-decahydroisoquinoline-3-carboxylic acid], other decahydroisoquinoline GLU(K5) receptor antagonists, and the noncompetitive AMPA receptor antagonist LY300168 [1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodi-azepine]. When characterized electrophysiologically in rat dorsal root ganglion neurons, LY466195 antagonized kainate (30 microM)-induced currents with an IC50 value of 0.045 +/- 0.011 microM. In HEK293 cells transfected with GLU(K5), GLU(K2)/GLU(K5), or GLU(K5)/GLU(K6) receptors, LY466195 produced IC50 values of 0.08 +/- 0.02, 0.34 +/- 0.17, and 0.07 +/- 0.02 microM, respectively. LY466195 was efficacious in a dural plasma protein extravasation (PPE) model of migraine with an ID100 value of 100 microg/kg i.v. LY466195 was also efficacious in the c-fos migraine model, with a dose of 1 microg/kg i.v. significantly reducing the number of Fos-positive cells in the rat nucleus caudalis after electrical stimulation of the trigeminal ganglion. Furthermore, LY466195 showed no contractile activity in the rabbit saphenous vein in vitro. The diethyl ester prodrug of LY466195 was also efficacious in the same PPE and c-fos models after oral administration at doses of 10 and 100 microg/kg, respectively while having no N-methyl-D-aspartate antagonist-like behavioral effects at oral doses up to 100 mg/kg.

LY503430, a novel alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor potentiator with functional, neuroprotective and neurotrophic effects in rodent models of Parkinson's disease.

Glutamate is the major excitatory transmitter in the brain. Recent developments in the molecular biology and pharmacology of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) subtype of glutamate receptors have led to the discovery of selective, potent, and systemically active AMPA receptor potentiators. These molecules enhance synaptic transmission and play important roles in plasticity and cognitive processes. In the present study, we first characterized a novel AMPA receptor potentiator, (R)-4'-[1-fluoro-1-methyl-2-(propane-2-sulfonylamino)-ethyl]-biphenyl-4-carboxylic acid methylamide (LY503430), on recombinant human GLUA1-4 and native preparations in vitro and then evaluated the potential neuroprotective effects of the molecule in rodent models of Parkinson's disease. Results indicated that submicromolar concentrations of LY503430 selectively enhanced glutamate-induced calcium influx into human embryonic kidney 293 cells transfected with human GLUA1, GLUA2, GLUA3, or GLUA4 AMPA receptors. The molecule also potentiated AMPA-mediated responses in native cortical, hippocampal, and substantia nigra neurons. We also report here that LY503430 provided dose-dependent functional and histological protection in animal models of Parkinson's disease. The neurotoxicity after unilateral infusion of 6-hydroxydopamine into either the substantia nigra or the striatum of rats and that after systemic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice were reduced. Interestingly, LY503430 also had neurotrophic actions on functional and histological outcomes when treatment was delayed until well after (6 or 14 days) the lesion was established. LY503430 also produced some increase in brain-derived neurotrophic factor in the substantia nigra and a dose-dependent increases in growth associated protein-43 (GAP-43) expression in the striatum. Therefore, we propose that AMPA receptor potentiators offer the potential of a new disease modifying therapy for Parkinson's disease.