Long-Term Real-World Post-approval Safety Data of Multiple Biosimilars from One Marketing-Authorization Holder After More than 18 Years Since Their First Biosimilar Launch.

BACKGROUND: Biosimilars are additional treatment options that are approved based on robust analytical and clinical comparisons with their reference biologic. At the time of initial approval, the full safety profile of a biosimilar is inferred from the reference biologic. Nonetheless, there are still lingering concerns related to the long-term safety of biosimilars. Therefore, we reviewed the post-approval pharmacovigilance data for eight marketed biosimilars from one Marketing Authorization Holder (MAH) to summarize their safety experience in a real-world setting for up to 18 years since their first biosimilar launch. METHODS: Post-approval cumulative patient exposure and safety experience for eight Sandoz biosimilars [adalimumab (Hyrimoz®), epoetin alfa (Binocrit®), etanercept (Erelzi®), filgrastim (Zarzio®), infliximab (Zessly®), pegfilgrastim (Ziextenzo®), rituximab (Rixathon®), and somatropin (Omnitrope®)] was summarized based on the available pharmacovigilance data from Periodic Safety Update Reports (PSURs) and the corresponding health authority-authored PSUR assessment reports, where available, as of 31 January 2023. Exposure to all biosimilars was calculated in patient treatment days (PTD) except for rituximab, which was expressed in number of patient doses (PD). RESULTS: The combined post-approval cumulative exposure to seven out of the eight marketed Sandoz biosimilars was more than 1.3 billion PTD and for rituximab more than 1.8 million PD. Overall, a critical analysis of the cumulative safety data of all eight Sandoz biosimilar PSURs concluded that the overall benefit-risk profile of each remains favorable and is consistent with the respective reference biologics. CONCLUSIONS: This is one of the largest reviews of post-approval biosimilar pharmacovigilance data to date by one MAH. The real-world experience of all eight marketed Sandoz biosimilars for up to 18 years demonstrates that Sandoz biosimilars can be used as safely as their respective reference biologics. Therefore, patients and healthcare providers can be confident in the clinical benefit and safety of Sandoz biosimilars. It is reasonable to believe that similar conclusions about safety may be reached for other biosimilars developed and approved to the high standards as are already in place by major health authorities such as the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA). The long-term safety of biosimilars demonstrated here provides strong support for the concept of biosimilarity.

Pharmacovigilance of Biologics in a Multisource Environment.

It is important that systems are in place to ensure that appropriate and comprehensive records are kept for use of all medications. It is fundamental to an effective pharmacovigilance system that patient medical records contain sufficient information to identify which medication has been prescribed, when it was administered, and at what dose. The availability of biologics from multiple sponsors has raised questions by some health care providers about the ability of current pharmacovigilance systems to trace specific biologics. In this article, periodic safety update reports were used to assess current postapproval safety monitoring for 3 biosimilars (epoetin alfa, somatropin, and filgrastim) that collectively represented nearly 350 million patient days of treatment worldwide. The reference products have each been marketed for over 10 years, forming a strong baseline of postmarketing safety data against which the safety of biosimilars can be compared. Published data from Denmark were also reviewed as additional evidence of how current pharmacovigilance systems are able to attribute adverse events to particular medicines. Collectively, the data show that spontaneous adverse drug reactions are reported by brand name in the majority of cases and are attributable to a specific medicine. Also discussed are the informational elements critical to monitoring biologics, or indeed any medicine, to ensure the availability of complete information so medicines that a patient has received can be quickly identified should a safety event occur. We support the addition of a single data element, the batch/lot number, to enhance the value of current pharmacovigilance systems. Adoption of 2-D barcodes in the European Union (EU) and standardized numerical identifiers in the United States addresses this need, since they include batch/lot numbers. These identifiers are already being implemented in the United States and the EU to improve patient safety, reduce medication errors, facilitate anticounterfeiting, and enable effective product recalls and adverse event reporting. Importantly, electronic identifiers will ameliorate safety reporting concerns with respect to biosimilars, while concurrently achieving these much broader public health objectives through more complete pharmacovigilance data. DISCLOSURES: This work was funded by Sandoz, a Novartis division. The authors were fully responsible for the content, editorial decisions, and opinions expressed in this article. No author received an honorarium related to the development of this manuscript. Sagi and Cohen are employees of Sandoz, and Woollett is an employee of Avalere Health. Study concept and design were contributed by Sagi and Woollett, along with Cohen. Data were primarily collected by Sagi, along with Woollett, and data interpretation was provided by all the authors. The manuscript was written by Woollett, along with Sagi and Cohen, and revised by Sagi and Cohen, along with Woollett.

Evaluation of cystine transport in cultured human kidney cells and establishment of cystinuria type I phenotype by antisense technology.

Cystinuria is a rare hereditary disease resulting in recurrent stone formation and the need for repeated invasive interventions. So far, two responsible genes have been identified which encode the two transporters, rBAT and b(0,+)AT forming a heterodimer to transport cystine in proximal tubular cells (PTC) and whose defect results in increased excretion of cystine. A human cell line mimicing the phenotype of cystinuria in vitro is yet to be developed. Human kidney (HK)-2 is a PTC line derived from normal HK. After determining the presence of rBAT gene by RT-PCR and Western blot analysis, radioactively labeled cystine (S(35)) was used to evaluate the functional presence of the amino acid transport in HK-2 cells when cultured in vitro. To achieve a cystinuria type I phenotype in HK-2 cells, the rBAT gene was silenced using antisense oligonucleotides complimentary to human rBAT mRNA. The reduced transport activity of cystine was then determined by radiolabeled cystine uptake measurements. RT-PCR and Western blot confirmed the expression of the rBAT gene in HK-2 cells. Considerable transport of the radio labeled cystine was observed in HK-2 cells and was linearly dependent on the incubation time with the amino acid. The cystine transport in rBAT knockdown cells after incubation with antisense oligonucleotides was significantly lower compared to control (0.76 vs. 0.98%; P=0.0008), proving a transient knock-down of the rBAT gene. This study demonstrates the presence of the b(0,+) amino acid transport system in human proximal tubular HK-2 cells when cultured in vitro. Inhibition of this transport system is possible by using antisense technology. A permanent inhibition of the cystine transport, based on our model, would be useful for the development and evaluation gene therapeutic approaches.

Impact of hypoxia and hypercapnia on calcium oxalate toxicity in renal epithelial and interstitial cells.

Although there is an ongoing controversy about the primary site of calcium oxalate stone (CaOx) formation, there is some evidence for extratubular crystallization. However, the mechanisms leading to such interstitial calcifications are not clear. Anatomical studies have demonstrated a close association between the renal vasculature and renal tubules. It has been hypothesized that disorders of the vasculature may contribute to renal stone formation. The exceptional papillary environment with low oxygen and high carbon dioxide is of interest in this context and its impact on CaOx toxicity to renal cells has to be evaluated. LLC-PK1, Madin-Darby canine kidney (MDCK), human umbilical vein endothelial (HUVEC) and fibroblast cell lines were exposed to hypoxia (3% O2) alone, hypercapnia combined with hypoxia (3% O2, 18% CO2) or standard culture conditions (20% O2) for 72 h. Cell survival rates were determined microscopically after 4 h of incubation with CaOx at final concentrations of 1, 2 and 4 mM. DAPI staining and western blot were used to evaluate the induction of apoptosis. We confirmed that CaOx leads to concentration-dependent effects on the viability of the cell lines. HUVECs were most vulnerable to CaOx among the four cell lines. Incubation under hypoxia alone had no impact on CaOx toxicity to any of the cell lines in terms of survival. However, under combined hypoxic and hypercapnic conditions, all cell lines displayed a significant reduction of cell survival compared to room air incubation. Again, this effect was most pronounced for HUVECs. The induction of apoptosis could not be demonstrated in any experimental setting. Combined hypoxia and hypercapnia clearly aggravate CaOx toxicity to renal cell lines. As we could not demonstrate the induction of apoptosis, this effect may be a result of toxic necrosis. Especially the CaOx effect on interstitial cell lines might be of interest in the chronic ischemic papillary environment. An increased toxicity may lead to recurrent stone formation, and vice versa, diseases of the vasculature, like arteriosclerosis, may further promote stone formation by induction of local ischemia. This issue has to be clarified by further studies.

Cytotoxicity of cisplatin in bladder cancer is significantly enhanced by application of bcl-2 antisense oligonucleotides.

BACKGROUND: The aim of our study was to examine the effects of the combined application of cisplatin and bcl-2 antisense oligonucleotide on human bladder cancer cell lines to determine the possible synergistic effects in cytotoxicity and to estimate its potential value for subsequent in vivo trials. MATERIALS AND METHODS: Human bladder cancer cell lines (UM-UC 3, RT 112, T24/83 and HT 1197) were treated with bcl-2 antisense oligonucleotide, cisplatin, or a combination of both and incubated for 48 h under standard conditions. Cell survival was determined using a Neubauer haemocytometer or standard MTT assay. BCL-2 expression was verified using western blotting. RESULTS: The combined treatment resulted in significant lower cell survival rates compared to individual treatment. Additionally, there was a decrease in cell survival rate with an increase in cisplatin concentration in combined treatment that was not observed in cisplatin mono treatment. CONCLUSIONS: For the combined treatment with oligonucleotides and cisplatin a synergistic effect can be strongly suggested. Therefore, further investigations and in vivo trials have to be done to determine the possible benefits for clinical applications.

Impact of holmium:YAG and neodymium:YAG lasers on the efficacy of DNA delivery in transitional cell carcinoma.

New approaches in the treatment of transitional cell carcinoma (TCC) are using gene therapy to influence the disease at the genetic level. Technical advances in genomics, the availability of tissue-specific gene promoters and other developments have made this approach more realistic. Transporting the gene into the target cell is still the major problem. Several transfection techniques have been introduced. Transfection of naked DNA is one of the simplest to perform but transfection rates have been very poor. We investigated the influence of laser energy on transfection efficacy in urothelial cancer cells in vitro with two types of medical lasers. A suspension of human transitional cancer cells (UM-UC3; 3.5 million cells/ml) was mixed with 200 microg of plasmid DNA (pEGFP-N1). Two types of laser energy, neodymium:YAG (Nd:YAG) and holmium:YAG (Ho:YAG), were applied to the cell suspension in different energy settings. Twenty four hours after treatment, transfection rates were measured with FACS analysis. Energy setting parameters that determine the efficacy of laser were investigated. The significance of different transfection rates was estimated with the student's t-test. We demonstrated that the Nd:YAG laser was not suitable for achieving significant transfection of the reporter gene to the cells. In contrast, the Ho:YAG laser produced satisfactory transfection rates. There was an increase in transfection with increasing frequency of laser pulses, from 16% with 2 Hz up to 40% with 10 Hz (p < 0.0005). Pulse frequency was therefore stabilised at 10 Hz. Pulse energy (mJ) showed the same dependency: a transfection rate of 18.3% was achieved with 1,000 mJ and 53.8% with 2,000 mJ (p > 0.0005). Additionally, we investigated the impact of total pulse number (imp) with different pulse energies. At 1,000 mJ, a transfection rate of 18.3% was estimated with 200 imp and 48.56% with 750 imp, (p < 0.0005). At 2,000 mJ, a transfection rate of 53.8% was achieved with 200 imp and 58.26% with 500 imp. The optimal laser setting observed in this experiment was 10 Hz, 2,000 mJ and 500 imp. This study indicates that the efficacy of naked DNA delivery into TCC in vitro is improvable by application of Ho:YAG laser energy. The Nd:YAG laser did not increase transfection rates in our model. Our results with the Ho:YAG laser are encouraging for further studies to optimise DNA delivery. As TCC tissue is relatively easy to access, this method could become an effective and minimally invasive procedure in urothelial cancer treatment.

The influence of oxalate on renal epithelial and interstitial cells.

Most renal stones in humans are composed of calcium oxalate. An increase in urinary oxalate levels has been shown to result in renal epithelial cell injury and crystal retention. However, the underlying mechanisms are unclear. Although the localization of primary stone formation and the associated cells playing the pivotal role in stone formation are still unknown, renal epithelial cells and interstitial cells seem to be involved in this process. The aim of this study was to evaluate the effects of oxalate on distinct renal epithelial and endothelial cells as well as fibroblasts. The first part focused on the toxicity of oxalate on the cells and a potential time- and dose-dependency. In the second part, renal epithelial cells were cultured in a two-compartment model to examine the vulnerability of the tubular or basolateral side to oxalate. LLCPK1, MDCK, renal fibroblast and endothelial cell lines were cultured under standard conditions. In part 1, cells were grown in standard culture flasks until confluent layers were achieved. Sodium oxalate was delivered at final concentrations of 1, 2 and 4 mM to either the apical or basolateral side (plain medium was delivered to the contralateral side). Cell survival was assessed microscopically by trypan blue staining after 1, 2 and 4 h. The influence of oxalate on proliferation and apoptosis induction was also investigated. In the second part, MDCK and LLCPK1 cells were grown in 6-well plates until confluent layers were achieved. Sodium oxalate at the above concentrations was applied, to either the apical or basolateral side and plain medium was delivered to the opposite side. The same protocol was then followed as in part 1. Part 1: sodium oxalate led to a time- and concentration-dependent decline in cell survival that was comparable in LLCPK1 and MDCK. Non-tubular cell lines like fibroblasts and endothelial cells were significantly more vulnerable to oxalate. These observations were reflected by significant impairment to cell proliferation. We could not demonstrate an induction of apoptosis in any cell line. Part 2: both cell lines were more vulnerable to oxalate on the basolateral side. This effect was more pronounced in MDCK cells at high oxalate concentrations (4 mM). Cells are apparently more resistant on the apical (tubular) side. Our results show that sodium oxalate has a negative effect on the growth and survival of renal epithelial cells and, to a greater extent, also fibroblasts and endothelial cells. We could not demonstrate any induction of apoptotic processes which implies a direct induction of cell necrosis. The finding of interstitial calcification and the proximity of tubules, vessels and interstitial cells make involvement of non-tubular renal cells in tissue calcification processes possible. Renal epithelial cells are apparently more vulnerable to oxalate on their basolateral side. Therefore, calcification processes within the interstitium may exert pronounced toxic effects to these cells, leading to inflammation and necrosis. These observations further support the idea of the interstitium as a site of primary stone formation.

In vitro and ex vivo gene delivery into proximal tubular cells by means of laser energy--a potential approach for curing cystinuria?

Cystinuria is the cause of 1-2% of stones observed in adults and about 10% of those occurring in children. Recurrent stone formation and multiple operations cause considerable morbidity. We investigated the transfection efficiency of naked plasmid DNA in porcine kidney cells by applying holmium laser (Ho:YAG) energy in vitro as well as ex vivo in a porcine kidney papilla model. In the in vitro experiments, naked plasmid DNA was added to LLC-PK1 cells suspended in a medium and Ho:YAG laser applied with varying pulses. The transfection efficiency was measured by the expression of EGFP reporter gene in the cells by FACScan analysis and fluorescence microscopy. Ex vivo, papilla from porcine kidney was excised and naked plasmid DNA was added to the tissue in the medium. The laser was then applied and the cryosectioned tissue observed under fluorescence microscope. The efficiency of transfection in vitro significantly improved with the increase in impulses (P<0.01). Transfection at 50 impulses averaged 0.7+/-0.3%, at 200 impulses 28.3+/-7.7%, and at 500 impulses 36.1+/-3.1%. The cell mortality rate increased with higher pulse rate up to 70%. Ex vivo trials showed transfection in extended regions of the tissue and also in the peripheral layers of the papilla. Our study indicates that the transfection of benign kidney cells by Ho:YAG is a promising new gene transfer strategy. The ex vivo trials showed that peripheral renal tissue layers are susceptible to transfection by Ho:YAG applied from the papillary surface.