Non-clinical, quality and environmental impact assessments of cell and gene therapy products: Report on the 5th Asia Partnership Conference of Regenerative Medicine - April 7, 2022.

The 5th Asia Partnership Conference of Regenerative Medicine (APACRM) was held online on April 7, 2022 to promote regulatory harmonization of regenerative medicine products throughout Asia. The recognition of domestic regulatory guidelines within each country and region and the underpinning rationales are important initial steps toward the harmonization of regulations. The 5th APACRM featured open dialog regarding non-clinical, quality and environmental impact assessment settings for cell and gene therapy products through presentations from the industry and panel discussions with regulatory agencies. The latest updates on regenerative medicine fields in each country and region were also introduced. This paper summarizes the proceedings of the 5th APACRM for public dissemination to foster future discussion.

Comparison of guidelines for biological ancillary materials used for the manufacture of gene and cellular therapy products in Asia.

BACKGROUND AIMS: Although biologiocal ancillay materials (AMs) have specific risk associated with their derivations, it plays key role to manufature cell and gene therapy (CGT) products. It is important to understand the regulation relevant to AMs for developers. METHODS: The authors investigated the guidelines and pharmacopeia (hereinafter referred to as "guidelines") for biological AMs used for the manufacture of CGT products in Asia (China, India, Japan, Korea and Taiwan). In addition, the authors benchmarked the relevant guidelines in the United States (US) and European Union (EU). RESULTS AND DISCUSSIONS: The guidelines could be classified into two types based on whether specific AMs are scoped: (i) general guidelines for risk assessment of AMs and (ii) guidelines for specific AMs. The authors compared the risk categories for each type of AM provided in the general guidelines between the US and China and the specific requirements for bovine serum and trypsin in the guidelines of China, Japan, Taiwan, US and EU. The authors further compiled in-depth descriptions of the respective regulations in China, India, Japan, Korea and Taiwan. There is limited availability of some guidelines for specific AMs. Moreover, there are no common requirements established across the surveyed countries and regions. Therefore, the authors suggest a risk assessment approach for AMs with consideration of their biological origin and traceability, production steps applied and ability to control or remove AMs from the final medicinal product over the CGT manufacturing process.

Nonclinical and quality assessment of cell therapy products: Report on the 4th Asia Partnership Conference of Regenerative Medicine, April 15, 2021.

The 4th Asia Partnership Conference of Regenerative Medicine (APACRM) was held online on April 15, 2021, to promote regulatory harmonization of regenerative medicine products throughout Asia. Recognizing domestic regulatory guidelines within each country and region, and their underpinning rationales, is an important initial step toward a convergence of regulations. The 4th APACRM consisted of an open dialog with regulatory agencies regarding nonclinical and quality settings for cell therapy products (CTPs) through industry presentations and panel discussions with regulatory agencies. The latest updates on regenerative medicine fields in each country and region, and specific regulatory schematics in Japan, were also introduced. The objective of this paper is to summarize the proceedings of the 4th APACRM for public dissemination and to foster further discussion in the future.

Gaps between Asian regulations for eligibility of human mesenchymal stromal cells as starting materials of cell therapy products and comparability of mesenchymal stromal cell-based products subject to changes in their manufacturing process.

Working group 2 (WG2) of the Asia Partnership Conference of Regenerative Medicine has discussed eligibility of mesenchymal stromal cells (MSCs) as starting cells for the manufacture of cell therapy products, and comparability before and after changes in their manufacturing process. Asian countries and regions have their own regulations on the quality of starting cells, and these regulations are not harmonized. As cell therapy products are being developed across countries and regions, we propose a risk-based approach based on donor location, window period of virus test, and additional virus tests on the master cell bank to fill the gaps in regulation while controlling the risk of viral contamination. Moreover, a standard procedure of comparability assessment after changes in the manufacturing process of MSC-based products does not exist. The WG2 discussed points of comparability assessment specifically for MSC-based products considering the similarities and differences with parallel assessments for protein and polypeptide products, which are within the scope of the International Council for Harmonization Q5E guideline. We also summarize possible characterization procedures for MSC-based products and report our discussion on stability evaluations under accelerated and stress conditions for comparability assessment of cell therapy products.

Patients with MAC Lung Disease Have a Low Visceral Fat Area and Low Nutrient Intake.

OBJECTIVE: This study aimed to examine the nutritional status and nutrient intake of patients with MAC lung disease with a focus on visceral fat area. PATIENTS AND METHODS: Among 116 patients of our hospital with nontuberculous mycobacteriosis who were registered between May 2010 and August 2011, 103 patients with MAC lung disease were included in this study. In all patients, nutritional status and nutrient intake were prospectively examined. RESULTS: Patients were 23 men and 80 women (mean age, 72.3±10.9 years). BMI (kg/m2) at the time of registration was 20.4±2.7 in men and 19.2±2.9 in women. Visceral fat area (cm2) was significantly lower in women (35.7±26.6) than in men (57.5±47.4) (p=0.0111). The comparison with general healthy adults according to age revealed a markedly reduced visceral fat area among patients with MAC lung disease. With respect to nutrient intake, energy adequacy (86.1±15.7%), protein adequacy (82.4±18.2%), lipid adequacy (78.1±21.8%), and carbohydrate adequacy (89.6±19.2%) ratios were all low at the time of registration. BMI was significantly correlated with protein adequacy (p=0.0397) and lipid adequacy (p=0.0214) ratios, while no association was found between visceral fat area and nutrient intake. CONCLUSION: Patients with MAC lung disease had a low visceral fat area and low nutrient intake.

Diffuse intrapulmonary malignant mesothelioma presenting with miliary pulmonary nodules: A case report.

A 67-year-old male with a history of asbestos exposure presented with fever, cough, and dyspnea and was found to have diffuse granular shadowing in both lungs, right pleural effusion, and hilar and mediastinal lymphadenopathy upon chest computed tomography. For definitive diagnosis, a thoracoscopic lung biopsy was performed. Intraoperative findings showed no remarkable macroscopic changes in the visceral and parietal pleura, although a high level of hyaluronic acid in the pleural effusion was noted. Histological findings showed proliferation of atypical cells with round-to-oval nuclei, prominent nucleoli, and eosinophilic cytoplasms. These cells were arranged into sheets or tubules and were located predominantly in the lung parenchyma. Lymphovascular invasion was conspicuous. Immunohistochemically, tumor cells were positive for calretinin, D2-40, and CK5/6, focally positive for Ber-EP4, but negative for WT-1, TTF-1, CEA, and MOC31. Fluorescence in situ hybridization for the tumor suppressor p16 revealed homozygous deletion in the tumor cells. Therefore, we diagnosed the tumor as diffuse intrapulmonary malignant mesothelioma (DIMM). The patient had a poor response to chemotherapy and died 1 year after diagnosis. Although rare, DIMM should be considered when patients present with multiple, tiny intrapulmonary nodules, regardless of macroscopic pleural changes. Furthermore, this is the first report on p16 status in DIMM.

Antiproteinuric effect of pirfenidone in a rat model of anti-glomerular basement membrane glomerulonephritis.

While pirfenidone has been established as an effective anti-fibrosis remedy, whether or not its antifibrotic effect contributes to a reduction of proteinuria remains unclear. We investigated the renoprotective properties of pirfenidone in an anti-glomerular basement membrane (GBM) glomerulonephritis model both prophylactically and therapeutically to determine its profile against proteinuria. In the prophylactic regimen, pirfenidone was treated immediately after anti-serum injection. We observed a significant reduction in the progression of proteinuria (P<0.05) and decline in renal function (P<0.01) and also noted histological improvement in renal injury. These effects appeared to be due to the maintained expression of nephrin and podocin on podocytes as well as the reduced expression of profibrotic factors like transforming growth factor-ß (TGF-ß). The expression of nephrin mRNA was strongly negatively correlated with the amount of urinary protein excretion (R=-0.84, P<0.001), implicating podocyte damage in the outcome of proteinuria (R(2)=0.70). These results suggest that preservation of podocytes with the pirfenidone treatment may have resulted in the decrease of proteinuria. In contrast, when the therapeutic regimen was initiated 2 weeks after nephritis induction, pirfenidone had little effect on the progression of proteinuria, although the decline of renal function and fibrosis were suppressed. Taken together, present findings suggested that pirfenidone prevented the progression of proteinuria only when administered prophylactically but was still able to ameliorate the decline of renal function independent of proteinuria. In conclusion, pirfenidone as a prophylactic regimen reduces proteinuria in anti-GBM nephritis via preservation of podocytes with markedly reduced efficacy when administered as a therapeutic regimen.

Retrospective analysis of nursing and healthcare-associated pneumonia: analysis of adverse prognostic factors and validity of the selection criteria.

BACKGROUND: Nursing and healthcare-associated pneumonia (NHCAP) is a relatively new condition that was recently defined by the Japanese Respiratory Society. Previous reports and guidelines have not thoroughly investigated the adverse prognostic factors and validity of the selection criteria for NHCAP. The purpose of this research was to clarify the adverse prognostic factors of NHCAP and investigate the validity of the selection criteria with respect to patient deaths. METHODS: We retrospectively analyzed 418 patients with pneumonia who were admitted to our hospital between January 2009 and December 2011. RESULTS: We analyzed 215 (51.4%) cases of community-acquired pneumonia (CAP) and 203 (48.6%) cases of NHCAP. NHCAP patients were generally older and had poorer performance status (PS), more complications, and higher levels of mortality than CAP patients. In both groups, the most common causative pathogen was Streptococcus pneumoniae. A multivariate analysis of NHCAP revealed that age ≥ 80 years, oxygen saturation (SpO2) ≤ 90%, and methicillin-resistant Staphylococcus aureus (MRSA) infection to be independent factors associated with mortality. Of the NHCAP selection criteria, a PS ≥ 3 and a hospitalization history within the past 90 days were adverse prognostic factors in the broad community-acquired pneumonia category (CAP+NHCAP), according to a multivariate analysis. Univariate analysis revealed that admission to an extended care facility or nursing home was associated with death. CONCLUSIONS: Our results demonstrated that age ≥ 80 years, SpO2 ≤ 90%, and MRSA infection were adverse prognostic factors for NHCAP patients. Furthermore, we confirmed the validity of the NHCAP selection criteria.

Antifibrotic effects of pirfenidone in rat proximal tubular epithelial cells.

OBJECTIVE: Renal fibrosis is a common cause of renal dysfunction with chronic kidney disease. We previously investigated the renoprotective effects of the antifibrotic agent pirfenidone in a rat model of subtotal nephrectomy. Here, we further evaluated the antifibrotic effects of pirfenidone in rat proximal tubular epithelial cells. METHODS: NRK52E cells were incubated in a medium containing either transforming growth factor (TGF)-ß1 (3 ng/mL) or platelet-derived growth factor (PDGF)-BB (5 Ang/mL) or both, with or without pirfenidone (0.1-1 mmol/L), for 24 h to assess mRNA expression, for 48 h to assess protein production, and for 1 h or various time (5-120 min) to assess phosphorylation of signal kinase. RESULTS: TGF-ß1, a key mediator in renal fibrosis, induced increases in the mRNA expression of various profibrotic factors and extracellular matrix, including plasminogen activator inhibitor type 1 (PAI-1), fibronectin, type 1 collagen, and connective tissue growth factor (CTGF)-increases which pirfenidone significantly inhibited. Specifically, pirfenidone potently inhibited TGF-ß1-induced increases in the mRNA expression and protein secretion of PAI-1, an effect mediated, at least in part, via the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling. Further, PDGF-BB, which has been implicated in renal interstitial fibrosis, potently activated PAI-1 expression under TGF-ß1 stimulation, and pirfenidone significantly inhibited TGF-ß1- and PDGF-BB-induced increases in PAI-1 expression. CONCLUSIONS: Taken together, these results suggest that TGF-ß1 closely correlates with renal fibrosis in cooperation with several fibrosis-promoting molecules, such as PAI-1 and PDGF, in rat proximal tubular epithelial cells, and pirfenidone inhibits TGF-ß1-induced fibrosis cascade and will therefore likely exert antifibrotic effects under pathological conditions.

Anti-herpes virus activity of polyether antibiotic CP-44161 in vivo.

In the previous study, we discovered a polyether antibiotic CP-44161, which was reported earlier as an anticoccidal agent, as an anti-varicella zoster virus compound. In this study, we demonstrated that CP-44161 had a very strong and broad anti-herpes virus activities against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in vitro. To determine the antiviral activity of CP-44161 in vivo, we examined its effect on the cutaneous HSV-2 infection model in Balb/c mice. CP-44161 showed inhibitory effect on lesion development as well as acyclovir (ACV) when the treatment was started from day 3. Meanwhile, in case the start of treatment was delayed until day 4, when ACV was no longer effective, the effectiveness of CP-44161 still remained. In this model, CP-44161 also showed inhibitory effect on the proliferation of HSV-2 DNA in dorsal root ganglia. This is the first article to report that polyether antibiotics can be effective on viral infection in vivo.