Recent Status of Phase I Clinical Trials for Brain Tumors: A Regulatory Science Study of Exploratory Efficacy Endpoints.

BACKGROUND: Appropriate exploratory efficacy data from Phase I trials are vital for subsequent phases. Owing to the uniqueness of brain tumors (BTs), use of different strategies to evaluate efficacy is warranted. We studied exploratory efficacy evaluation in Phase I trials involving BTs. METHODS: Using Clarivate's Cortellis™, 42 Phase I trials of BT interventions conducted from 2020 to 2022 were analyzed for efficacy endpoints, which were set as primary endpoints (PEs) or secondary endpoints (SEs). Additionally, these metrics were compared in two subgroups: trials including only BTs (Group-A) and those including BTs among mixed solid tumors (Group-B). RESULTS: Selected studies included a median of 1.5 PEs (range, 1-6) and 5 SEs (range, 0-19). Efficacy endpoints were included as PEs and SEs in 2 (5%) and 31 (78%) trials, respectively. Among the latter 31 trials that included 94 efficacy endpoints, 24, 22, 20, 9, and 8 reflected overall response rate (ORR), progression-free survival (PFS), overall survival (OS), duration of response (DOR), and disease control rate (DCR), respectively. ORR for BT was determined using various methods; however, the Response Evaluation Criteria in Solid Tumors (RECIST) was used less frequently in Group-A than in Group-B (p = 0.0039). CONCLUSIONS: Recent Phase I trials included efficacy endpoints as SEs, with ORR, PFS, or OS included in ~ 50% trials and DOR or DCR in ~ 25%. No established criteria exist for imaging evaluation of BTs. Phase I trials involving mixed solid tumor cohorts revealed challenges in designing methods to assess the exploratory efficacy of BTs.

Efficacy Endpoints in Phase II Clinical Trials for Meningioma: An Analysis of Recent Clinical Trials.

BACKGROUND: Response Evaluation Criteria in Solid Tumors (RECIST)-based response rates are commonly used as efficacy endpoints in phase II clinical trials for solid tumors. However, no consensus has been reached concerning adequate efficacy endpoints for phase II clinical trials targeting meningioma. Irregularity of lesions after resection, and varying degrees of dysplasia and histologic subtypes make establishing an appropriate efficacy evaluation difficult. METHODS: We analyzed primary efficacy endpoints (PEEs) and background factors from 48 trials retrieved from ClinicalTrials.gov ( https://clinicaltrials.gov/ ) using the search criteria "meningioma," "interventional," "phase II," and "study start 4/1/2001 to 3/31/2021." Primary purpose of the study was efficacy endpoint setting in overall population and three subgroups. RESULTS: Among 45 PEEs set in the 39 trials included; 33 trials with single PEE, and six trials with double PEEs, 17/45 (38%) trials adopted progression-free survival (PFS) rate, 15/45 (33%) trials response rate (seven Macdonald criteria or modified, three RECIST, three volumetric estimation, one RANO criteria, one unknown), 10/45 (22%) PFS, 1/45 (2%) OS, and 2/45 (4%) other endpoints. Although 26 PEEs were time-to-event endpoints, 19 of the 26 PEEs were single-arm studies. CONCLUSIONS: Time-to-event efficacy endpoints were often compared to historical data, and two-dimensional evaluation is more suitable than one-dimensional one. Accumulation of prognostic data is essential to standardize time-to-event efficacy endpoints. Considering the difficulty of setting design for phase II clinical studies targeting meningioma, evaluation might be done with multiple efficacy endpoints.

TMPRSS2 gene polymorphism common in East Asians confers decreased COVID-19 susceptibility.

COVID-19 has a wide range of clinical presentations, and the susceptibility to SARS-CoV-2 infection and the mortality rate also vary by region and ethnicity. Here, we found that rs12329760 in the TMPRSS2 gene, a missense variant common in East Asian populations, contributes to protection against SARS-CoV-2 infection. TMPRSS2 is a protease responsible for SARS-CoV-2 entry and syncytium formation. rs12329760 (c.478G>A, p. V160M) was associated with a reduced risk of moderate symptoms. The enzymatic activity of Met160-TMPRSS2 was lower than that of Val160-TMPRSS2, and thus the viral entry and the syncytium formation of SARS-CoV-2 were impaired. Collectively, these results indicate that the genetic variation in TMPRSS2, which is common in East Asians, is one of the molecular determinants of COVID-19 susceptibility.

Anti-albuminuric effect of the aldosterone blocker eplerenone in non-diabetic hypertensive patients with albuminuria: a double-blind, randomised, placebo-controlled trial.

BACKGROUND: Renin-angiotensin system inhibitors have renoprotective effects in patients with chronic kidney disease, but most patients treated with these drugs have residual urinary albumin excretion. Some small clinical studies show that mineralocorticoid receptor blockade reduces albuminuria. Our study aimed to examine the beneficial effects of addition of a selective aldosterone antagonist, eplerenone, to renin-angiotensin system inhibitors in hypertensive patients with non-diabetic chronic kidney disease. METHODS: In this double-blind, randomised, placebo-controlled trial, we enrolled hypertensive patients, aged 20­79 years, with albuminuria (urinary albumin-to-creatinine ratio [UACR] in the first morning void urine of 30­599 mg/g), an estimated glomerular filtration rate of 50 mL/min per 1·73 m2 or more, and who had received an angiotensin-converting enzyme inhibitor, an angiotensin receptor blocker, or both, for at least 8 weeks. Participants were from 59 clinics and hospitals in Japan. Eligible patients were randomly assigned (1:1), stratified by baseline characteristics, to either low-dose eplerenone (50 mg/day) or placebo, with continuation of standard antihypertensive treatment to attain therapeutic goals (<130/80 mm Hg) for 52 weeks. We assessed efficacy in all patients who received allocated treatment, provided a baseline and post-treatment urine sample, and remained in follow-up. We assessed safety in all patients who received allocated treatment. The primary efficacy measure was percent change in UACR in the first morning void urine at week 52 from baseline. The trial is registered at the clinical trials registry of University Hospital Medical Information Network (UMIN), trial identification number UMIN000001803. FINDINGS: Between April 1, 2009, and March 31, 2012, we randomly allocated 170 patients to the eplerenone group and 166 patients to the placebo group. In the primary efficacy analysis, mean percent change in UACR from baseline was −17·3% (95% CI −33·65 to −0·94) for 158 patients in the eplerenone group compared with 10·3% (−6·75 to 22·3) for 146 patients in the placebo group (absolute difference −27·6% [­51·15 to −3·96]; p=0·0222). In the safety analyses, 53 (31%) of 169 patients in the eplerenone group had adverse events (five serious), as did 49 (30%) of 163 in the placebo group (seven serious). Although mean serum potassium concentration was higher in the eplerenone group than the placebo group, severe hyperkalaemia (>5·5 mmol/L) was not recorded in either group. INTERPRETATION: Addition of low-dose eplerenone to renin-angiotensin system inhibitors might have renoprotective effects through reduction of albuminuria in hypertensive patients with non-diabetic chronic kidney disease, without serious safety concerns. FUNDING: Pfizer.

[Recent progress and challenges in investigator-driven clinical development of novel drugs and medical devices at the University of Tokyo Hospital].

At the University of Tokyo Hospital, investigator-driven clinical development of novel drugs and medical devices is mainly supported by the Translational Research Center and Clinical Research Support Center. The former supports non-clinical research and the preparation of test materials and the latter supports clinical trials. The Clinical Research Support Center was established in 2010 by the reorganization of the former Clinical Research Center, which was established in 2001. The center adopted International Conference on Harmonisation-Good Clinical Practice (ICH-GCP) as a standard guideline for clinical trials and prepared standard operation procedures and templates for protocols and informed consent documents in 2001 and, thereafter, provided consultation services to researchers for protocol development. In 2010, the service was extended to project management, data management and monitoring to support the credibility of clinical trials. In 2011, The University of Tokyo Hospital was selected by the government as a base for the early and exploratory clinical development of drugs in the fields of psychological and neurological diseases. For this purpose, a phase 1 unit for early phase clinical pharmacology trials is now being built. The center provides training courses for clinical research coordinators and hold seminars for clinical researchers; however, the biggest challenge remains the education and training of medical students who will lead clinical trials in the future.

Trk and cAMP-dependent survival activity of adenosine A(2A) agonist CGS21680 on rat motoneurons in culture.

The survival activity of adenosine A(2A) agonist CGS21680 on motoneurons in culture through the transactivation of neurotrophin receptor TrkB has been reported previously; however, since adenosine A(2A) receptor belongs to a Gs-protein-coupled receptor, we investigated the involvement of the cAMP pathway in the survival activity of CGS21680 using purified motoneurons in culture. CGS21680 alone showed only small survival activity, but the activity was significantly enhanced by the addition of a phosphodiesterase inhibitor, IBMX. This survival activity was partially inhibited by a protein kinase A inhibitor H89 or a neurotrophin receptor tyrosine kinase inhibitor K252a, and was completely inhibited by their combination. These results indicate that the survival activity of CGS21680 on motoneurons is exerted by the mixed effect of the adenylate cyclase-cAMP-PKA pathway and transactivation of Trk neurotrophin receptor. Under conditions in which the maximum survival of motoneurons was supported by sufficient concentrations of brain-derived neurotrophic factor (BDNF), a TrkB ligand, the addition of 100µM AMPA for 3 days led to significant cell death. Treatment with CGS21680 and IBMX protected motoneurons from the toxicity of AMPA, further supporting the presence of a TrkB-independent pathway of CGS21680 activity and suggesting a novel therapeutic approach to motoneuron diseases such as amyotrophic lateral sclerosis.

Rationale and design of the Eplerenone combination Versus conventional Agents to Lower blood pressure on Urinary Antialbuminuric Treatment Effect (EVALUATE) trial: a double-blinded randomized placebo-controlled trial to evaluate the antialbuminuric effects of an aldosterone blocker in hypertensive patients with albuminuria.

Although inhibitors of the renin-angiotensin system are effective as first-line antihypertensive drugs in hypertensive patients with chronic kidney disease, they cannot completely prevent the progression of renal injury. Many animal studies, including our own, and a few human studies suggest that mineralocorticoid receptor blockade could inhibit the ongoing renal damage in chronic kidney disease. Thus, we designed this double-blinded, randomized, placebo-controlled trial to evaluate the antialbuminuric effect of a low dose (50 mg day(-1)) of the mineralocorticoid receptor antagonist eplerenone. The study subjects will include 340 hypertensive patients (blood pressure: 130-180/80-100 mm Hg) with albuminuria (urinary albumin/creatinine ratio: 30-600 mg g(-1) in the first morning void urine), who are treated with an inhibitor of the renin-angiotensin system. Other classes of antihypertensive drugs may be added as needed to achieve the target blood pressure (<130/80 mm Hg). The primary study end point is the change in the urinary albumin/creatinine ratio after a 1-year study period. This trial is expected to show whether a low dose of mineralocorticoid receptor antagonists can exert an antialbuminuric effect in patients with chronic kidney disease.

Survival-promoting activity of pituitary adenylate cyclase-activating polypeptide in the presence of phosphodiesterase inhibitors on rat motoneurons in culture: cAMP-protein kinase A-mediated survival.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to be neurotrophic or neuroprotective in various neurons in culture. It is expressed in spinal motoneurons in vivo and its expression is increased markedly after axotomy, suggesting a neuroprotective role via an autocrine mechanism. However, neurotrophic activity of PACAP has not been reported for motoneurons. In the present study, we investigated the effects of PACAP on rat motoneurons in culture. In the presence of a phosphodiesterase inhibitor, PACAP showed significant neurotrophic activity at concentrations as low as 0.01 nM. Previously, we found that glutamate was excitotoxic to motoneurons even in the presence of brain-derived neurotrophic factor, which is neurotrophic for motoneurons. PACAP with a phosphodiesterase inhibitor protected motoneurons against this excitotoxicity. The activity of PACAP was inhibited by the protein kinase A inhibitor N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide dihydrochloride, as was the case with the activity of forskolin, suggesting downstream involvement of a cAMP-protein kinase A signaling pathway. The present results may suggest a physiological role of PACAP in vivo, and implicate the PACAP-cAMP signaling pathway for the possible therapeutic target of amyotrophic lateral sclerosis as glutamate excitotoxicity was suggested in sporadic amyotrophic lateral sclerosis.

Protein kinase C-mediated protection of motoneurons from excitotoxicity.

Amyotrophic lateral sclerosis (ALS) is a progressive motoneuron disease, whose pathogenesis remains unclear. Phorbol 12-myristate 13-acetate (PMA) has been shown to be neurotrophic and neuroprotective in several types of neurons in culture. However, the survival effect of PMA on motoneurons has not been reported. Therefore, we investigated this using purified rat motoneurons. PMA showed a small but significant neurotrophic activity, which was inhibited by GF109203X, a protein kinase C (PKC) inhibitor. Under the conditions in which the survival of motoneurons is supported by brain-derived neurotrophic factor (BDNF), excitotoxicity was induced by glutamate. PMA markedly protected motoneurons against excitotoxicity. The up-regulation of PKC in the spinal cord of ALS patients was previously reported, therefore, the present results indicate that PKC activation might be involved in neuroprotection.

Smad4 silencing in pancreatic cancer cell lines using stable RNA interference and gene expression profiles induced by transforming growth factor-beta.

The transforming growth factor-beta (TGF-beta)-Smad signaling pathway inhibits the growth of human epithelial cells and plays a role in tumor suppression. The Smad4 gene is mutated or deleted in 50% of pancreatic cancers. In this study, we succeeded in establishing Smad4 knockdown (S4KD) pancreatic cancer cell lines using the stable RNA interference (RNAi) method. Smad4 protein expression was reduced dramatically and TGF-beta-Smad signaling was markedly inhibited in the S4KD cell lines. The S4KD and control cells were stimulated with TGF-beta and analysed using a cDNA microarray that contained 3756 genes, in order to screen for target molecules downstream of TGF-beta. The microarray analysis revealed that 187 S4KD genes and 155 genes in the control cells were regulated immediately upon TGF-beta stimulation. Quantitative RT-PCR analysis on several of these genes produced results that corroborated the outcome of the microarray analysis. Most of the genes in the S4KD and control cells identified by the array differed, which suggests signaling pathways that differ according to Smad4 status. Of the identified genes, 246 have not been reported previously as genes that lie downstream of TGF-beta. Genes that are involved in cell proliferation, adhesion, and motility were found to be regulated differentially with respect to S4KD and control cells. Cell migration induced by TGF-beta was inhibited in the S4KD cells, which might be associated with a different regulation of integrin beta7. The knock down of a specific gene using stable RNAi appears to be a promising tool for analysing endogenous gene function.

The upregulated expression of sonic hedgehog in motor neurons after rat facial nerve axotomy.

Nerve injury leads to the induction of a large number of genes to repair the damage and to restore synaptic transmission. We have attempted to identify molecules whose mRNA expression is altered in response to facial nerve axotomy. Here we report that facial nerve axotomy upregulates Sonic hedgehog (Shh) and its receptor Smoothened (Smo) in facial motor neurons of adult rats, whereas facial nerve axotomy does not upregulate mRNA of Shh or Smo in neonatal rats. We tested whether overexpression of Shh in facial motor neurons of axotomized neonatal rats may promote neuronal survival. Adenovirus-mediated overexpression of Shh, but not that of beta-galactosidase, transiently rescues axotomy-induced neuronal cell death for 3-5 d after axotomy. Finally, the pharmacological inhibitor of Shh signaling, cyclopamine, induces motor neuron death in adult rats after axotomy. These results suggest that Shh plays a regulatory role in nerve injury.

Interaction of the hepatitis B virus X protein (HBx) with heat shock protein 60 enhances HBx-mediated apoptosis.

Understanding the function of the hepatitis B virus X protein (HBx) is fundamental to elucidating the underlying mechanisms of hepatitis and hepatocarcinogenesis caused by hepatitis B virus (HBV) infection. We identified heat shock protein 60 (Hsp60) as a novel cellular target of HBx by the combination of affinity purification and mass spectrometry. Physical interaction between HBx and Hsp60 was confirmed by standard immunoprecipitation and immunoblot methods. Analysis of HBx deletion constructs showed that amino acids 88-117 of HBx were responsible for the binding to Hsp60. Confocal laser microscopy demonstrated that HBx and Hsp60 colocalized in mitochondria. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP end labeling (TUNEL) revealed that the introduction of Hsp60 into cells facilitated HBx-induced apoptosis. These findings suggest the importance of the molecular chaperon protein Hsp60 to the function of HBV viral proteins.

Analysis of information submitted by clinical trial sponsors regarding the safety of investigational drugs.

During performance of clinical trials in medical institutions, information regarding the safety of investigational drugs is submitted by trial sponsors according to guidelines for good clinical practice. In the present study, reports of clinical trials conducted at the University of Tokyo Hospital were examined, focusing on the safety information provided to the Institutional Review Board (IRB). Two hundred two reports (52 protocols) of safety information were submitted to the IRB by clinical trial sponsors between April 2000 and March 2001, of which 185 contained a total of 3021 cases of adverse events. Of those, 194 reports were judged by clinical investigators/physicians not to be associated with any significant problems and the trials were continued. For 157 of those 194 reports, it was considered unnecessary to inform the test subjects of the report contents, including the adverse events. The decision of whether or not the test subjects should be informed of such contents tended to depend on the causal relationship between the adverse events and drug intake, as well as the predictability of the adverse events. For 8 of those 194 reports, the IRB recommended that the clinical investigators/ physicians provide information to the test subjects and/or submit detailed information on the status of these subjects to the IRB. From these results, we suggest that establishment of a system to unify and evaluate drug safety information is necessary to provide safe and efficient clinical trials.

Proteomic analysis of the TGF-beta signaling pathway in pancreatic carcinoma cells using stable RNA interference to silence Smad4 expression.

Smad4 is a tumor-suppressor gene that is lost or mutated in 50% of pancreatic carcinomas. Smad4 is also an intracellular transmitter of transforming growth factor-beta (TGF-beta) signals. Although its tumor-suppressor function is presumed to reside in its capacity to mediate TGF-beta-induced growth inhibition, there seems to be a Smad4-independent TGF-beta signaling pathway. Here, we succeeded in establishing Smad4 knockdown (S4KD) pancreatic cancer cell lines using stable RNA interference. Smad4 protein expression and TGF-beta-Smad4 signaling were impaired in S4KD cells, and we compared the proteomic changes with TGF-beta stimulation using two-dimensional gel electrophoresis (2-DE) and mass spectrometry. We identified five proteins that were up-regulated and seven proteins that were down-regulated; 10 of them were novel targets for TGF-beta. These proteins function in processes such as cytoskeletal regulation, cell cycle, and oxidative stress. Introducing siRNA-mediated gene silencing into proteomics revealed a novel TGF-beta signal pathway that did not involve Smad4.

Smad4-independent regulation of p21/WAF1 by transforming growth factor-beta.

The transforming growth factor-beta (TGF-beta)-Smad signaling pathway inhibits the growth of human epithelial cells and plays a role in tumor suppression. The Smad4 gene is mutated or deleted in 50% of pancreatic cancers. In this study, the Smad4-null pancreatic cancer cell line BxPC-3 was transfected with either the Smad4 expression vector or the empty vector and incubated in the presence or absence of TGF-beta. The cells were analysed using a cDNA microarray, which included 2280 named genes to screen for target genes regulated by TGF-beta in either a Smad4-dependent or -independent manner. The microarray and subsequent quantitative RT-PCR analysis demonstrated that the Smad4-independent and -dependent signaling pathways driven by TGF-beta upregulated only one of the 2280 genes, respectively, suggesting that Smad4-independent signaling downstream of TGF-beta might be as widespread as Smad4-dependent signaling. In this study, we demonstrated that the cyclin-dependent kinase inhibitor p21/WAF1, which has been considered the major effector of the Smad-dependent growth inhibitory signal of TGF-beta, is upregulated in a Smad4-independent manner. The upregulation occurs through Smad2/3-dependent transcriptional activation of the p21/WAF1 promoter region. These results suggest a novel mechanism of gene regulation, that is, a novel signal mediator other than Smad4.

Items of concern associated with source document verification of clinical trials for new drugs.

In the present study, we analyzed concerns of the sponsors of clinical trials regarding source document verification (SDV) procedures performed at the University of Tokyo Hospital during April 1999 and March 2001, with special focus on the differences in description between the source document and case report form (CRF). Of 132 SDV procedures (78 protocols, 496 cases), the sponsors had problematic concerns with 348 cases (70.2%) totalling 693 items, which consisted of description inconsistencies between the source documents and the CRF (41.4%), lack of description in the CRF (39.8%), and lack of description in the source documents (8.8%). The most frequently found inconsistencies between the source documents and CRF were concerning items regarding observations, laboratory examinations, and compliance, which were associated with misdescription of clinical data and/or items for evaluation in the CRF. It was also revealed that the frequent lack of description in the CRF was associated with patient history and/or complications, adverse events, and concomitant drugs and/or therapy. In contrast, the frequent lack of description in the source documents was associated with items concerning patient background, observations, and informed consent. Further, we found that submission of a report of deviation from the protocols was required for 4.0% of the claims. These results suggest the necessity of better data management during the practice of clinical trials for the purpose of maintaining the quality of clinical trials.

Adenoviral gene transfer of GDNF, BDNF and TGF beta 2, but not CNTF, cardiotrophin-1 or IGF1, protects injured adult motoneurons after facial nerve avulsion.

We examined neuroprotective effects of recombinant adenoviral vectors encoding glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT1), insulin-like growth factor-1 (IGF1), and transforming growth factor-beta2 (TGFbeta2) on lesioned adult rat facial motoneurons. The right facial nerves of adult Fischer 344 male rats were avulsed and removed from the stylomastoid foramen, and adenoviral vectors were injected into the facial canal. Animals avulsed and treated with adenovirus encoding GDNF, BDNF, CNTF, CT1, IGF1 and TGFbeta2 showed intense immunolabeling for these factors in lesioned facial motoneurons, respectively, indicating adenoviral induction of the neurotrophic factors in these neurons. The treatment with adenovirus encoding GDNF, BDNF, or TGFbeta2 after avulsion significantly prevented the loss of lesioned facial motoneurons, improved choline acetyltransferase immunoreactivity and prevented the induction of nitric oxide synthase activity in these neurons. The treatment with adenovirus encoding CNTF, CT1 or IGF1, however, failed to protect these neurons after avulsion. These results indicate that the gene transfer of GDNF and BDNF and TGFbeta2 but not CNTF, CT1 or IGF1 may prevent the degeneration of motoneurons in adult humans with motoneuron injury and motor neuron diseases.

Survival-promoting activity of nimodipine and nifedipine in rat motoneurons: implications of an intrinsic calcium toxicity in motoneurons.

L-type calcium channel antagonists, nimodipine and nifedipine, were tested for effects on the survival of purified rat motoneurons in culture. They showed significant activity, with maximum survival at 30 microm after 3 days in culture as high as 75%, which was comparable to the maximum effect obtained with brain-derived neurotrophic factor, a potent neurotrophic factor for rat motoneurons. It was also found that depolarizing conditions with a high potassium concentration (30 mm) were toxic to motoneurons. This toxicity was blocked by co-treatment with nimodipine. These results implicate a pre-existing calcium burden through calcium channels in motoneurons; they may offer further insights into understanding the selective death of motoneurons and have therapeutic implications in amyotrophic lateral screlosis.