4-Week toxicity study of biosimilar natalizumab in comparison to Tysabri® by repeated intravenous infusion to cynomolgus monkeys.

The objective of the study was to obtain information on the toxicity of biosimilar natalizumab (PB006) in comparison to the reference product Tysabri®. Cynomolgus monkeys (15 males and 15 females), three animals per sex and group were treated with either PB006 or the reference product Tysabri® at dose levels of 3 or 30 mg/kg body weight or placebo by intravenous infusion every other day for a period of 4 weeks. The study is also meant to facilitate ethics committee approval in specific countries for the planned confirmatory efficacy and safety study in patients. Based on study findings, it was concluded that there was no noteworthy difference between the animals treated with PB006 and the animals treated with Tysabri®. There were no noteworthy differences between PB006 and Tysabri® with respect to local tolerance, toxicity and pharmacokinetic profile in cynomolgus monkeys. In conclusion, obtained data are valid and suitable to support entry into clinical studies of PB006 in man.

An update on the animal studies conducted for biosimilar approvals - Regulatory requirement vs actual scenario.

Nonclinical animal studies are considered as an integral part of biosimilar development program to demonstrate similarity and safety. We have compiled, reviewed and summarized animal studies conducted for European Medicines Agency (EMA) and United States Food and Drug Administration (US FDA) submission from 2006 till December 2018. The commonest animal studies conducted included repeat-dose toxicity study along with toxicokinetic, local tolerance and immunogenicity assessments, while the least common included primary pharmacodynamic, pharmacokinetic, safety pharmacology and single-dose toxicity studies. Animal studies were designed based on pharmacology of the drug, disease condition and innovator studies. Studies mostly used EU-sourced reference products as a comparator. For biosimilars approved both in the US and European Union (EU), similar data packages were submitted to these regions. Despite the regulatory guidelines allowing waiver of animal studies based on analytical data, animal studies have been conducted for almost all the approved biosimilars in the US and EU. There is an increasing need to re-assess the relevance of animal studies to support regulatory approval of biosimilars. Stepwise assessment for biosimilarity and conducting animal studies only if required at the right instance based on residual uncertainties may assist in optimizing animal study requirement for biosimilar development.

Nonclinical assessments of the potential biosimilar PF-06439535 and bevacizumab.

Bevacizumab, a recombinant humanized monoclonal antibody targeting vascular endothelial growth factor (VEGF), is approved for treatment of metastatic colorectal cancer, nonsquamous non-small-cell lung cancer, metastatic kidney cancer, and glioblastoma. To support clinical development of the potential bevacizumab biosimilar PF-06439535, nonclinical studies evaluated structural, functional, toxicological, and toxicokinetic similarity to bevacizumab sourced from the European Union (bevacizumab-EU) and United States (bevacizumab-US). Peptide mapping demonstrated the amino acid sequence of PF-06439535 was identical to bevacizumab-EU and bevacizumab-US. Biologic activity, measured via inhibition of VEGF-induced cell proliferation in human umbilical vein endothelial cells and binding to VEGF isoforms, was similar across the three drugs. In vivo similarity was demonstrated in cynomolgus monkeys administered intravenous PF-06439535 or bevacizumab-EU (0 or 10 mg/kg/dose twice weekly for 1 month; total of nine doses). Systemic exposure appeared similar and test article-related effects were limited to physeal dysplasia of the distal femur. The potential for non-target-mediated toxicity of PF-06439535 was evaluated in rats administered intravenous PF-06439535 (15 or 150 mg/kg/dose twice weekly for 15 days; total of five doses). Nonadverse higher liver weights and minimal sinusoidal cell hyperplasia were observed. Collectively, these studies demonstrated similarity of PF-06439535 to bevacizumab, supporting entry into clinical development.

Pharmacokinetic and toxicology comparator testing of biosimilar drugs - Assessing need.

A key element in the development of a biosimilar molecule is the comparability of the biological activity/nonclinical similarity to the innovator drug. Although some regulatory guidelines are encouraging little or no in vivo testing, currently a common practice is to perform at least one toxicology and/or one pharmacokinetic (PK) study to assess if any different findings occur for in-life, clinical pathology and histopathological parameters or in exposure. An exercise was performed in which the results of such testing were evaluated. It was found that 10 PK comparison studies in the cynomolgus monkey across 4 monoclonal (Mab) classes showed similar exposure in all cases. In 17 toxicology comparison studies with 5 Mab classes performed in the same species and in 7 toxicology comparison studies with non-Mab biosimilars in the rat, no new/unexpected findings were seen and drug exposure measurement gave comparable values in all cases. Overall, although this work does not rule out possible utility of some in vivo testing (notably in the form of stand-alone PK testing) to confirm similar exposure between the 2 molecules tested, it is unclear what benefit can be gained from toxicology testing, especially if comparability has been demonstrated from physiochemical and in vitro characterisation.

Nonclinical pharmacology and toxicology of the first biosimilar insulin glargine drug product (BASAGLAR®/ABASAGLAR®) approved in the European Union.

Basaglar®/Abasaglar® (Lilly insulin glargine [LY IGlar]) is a long-acting human insulin analogue drug product granted marketing authorisation as a biosimilar to Lantus® (Sanofi insulin glargine [SA IGlar]) by the European Medicines Agency. We assessed the similarity of LY IGlar to the reference drug product, European Union-sourced SA IGlar (EU-SA IGlar), using nonclinical in vitro and in vivo studies. No biologically relevant differences were observed for receptor binding affinity at either the insulin or insulin-like growth factor-1 (IGF-1) receptors, or in assays of functional or de novo lipogenic activity. The mitogenic potential of LY IGlar and EU-SA IGlar was similar when tested in both insulin- and IGF-1 receptor dominant cell systems. Repeated subcutaneous daily dosing of rats for 4 weeks with 0, 0.3, 1.0, or 2.0 mg/kg LY IGlar and EU-SA IGlar produced mortalities and clinical signs consistent with severe hypoglycaemia. Glucodynamic profiles of LY IGlar and EU-SA IGlar in satellite animals showed comparable dose-related hypoglycaemia. Severe hypoglycaemia was associated with axonal degeneration of the sciatic nerve; the incidence and severity were low and did not differ between LY IGlar and EU-SA IGlar. These results demonstrated no biologically relevant differences in toxicity between LY IGlar and EU-SA IGlar.

Comparative subcutaneous repeated toxicity study of enoxaparin products in rats.

Enoxaparin is a low-molecular-weight heparin widely used for the prevention and treatment of thromboembolism. With the development of several enoxaparin biosimilars, real medical concerns about their safety and efficacy have been raised. This repeated dose toxicity study consists of preclinical toxicological evaluation of a biosimilar biological version of enoxaparin, the drug product "Enoxa", compared to the enoxaparin reference drug product, "Lovenox". Eighty white Wistar rats were treated with "Enoxa" versus the reference product, using subcutaneous therapeutic and toxic doses, varying from 3.5 to 100 mg/kg/day. Dose levels were adjusted and ultimately fixed at 3.5 and 20 mg/kg/day as therapeutic and toxic doses, respectively. A sodium chloride solution (0.9%) was used as the control, and the comparative study was conducted over periods of 14 and 28 days. Comparable effects were observed with few adverse effects at the administration dose of 20 mg/kg/day, for both enoxaparin biosimilar and reference products. Interestingly, mortality started only at high doses of 40 mg/kg/day and reached 25% at 100 mg/kg/day for both products. These results, as part of the recommended biosimilarity monitoring, demonstrated comparable toxicity profiles of "Enoxa" and "Lovenox" products in rats. Continuing investigation of biosimilarity on humans to confirm safety and efficacy is suggested.

Regulatory forum opinion piece: differences between protein-based biologic products (biotherapeutics) and chemical entities (small molecules) of relevance to the toxicologic pathologist.

With the advances in cell culture methodologies and molecular biology that have occurred over the past several decades, biologics have become as common as small molecules within the portfolios of the pharmaceutical industry. Toxicologic pathologists should be aware of some of the fundamental differences between small molecules and biologics. Effects are not always observed in studies following administration of biologics. When findings are observed, the toxicologic pathologist should initially determine whether the effect(s) are mediated (directly or indirectly) via the intended pharmacology, exaggerated pharmacology, an immune response, and/or off target effects. Following this determination, the toxicologic pathologist should provide an assessment regarding the relevance of the findings to the intended clinical population, usually humans. The toxicologic pathologist may also be asked to assess unusual species and models. Given their broad background in physiology and immunology, toxicologic pathologists are uniquely positioned to provide this input to drug development teams.

Non-clinical pharmacology and toxicology studies of bevacizumab biosimilar LY01008.

LY01008 was a biosimilar of Avastin® developed by Shandong Boan Biotechnology. To support the clinical trial and marketing application of LY01008 as a biosimilar, a series of non-clinical pharmacodynamics (PD), pharmacokinetics (PK), and toxicological studies have been conducted. The PD study results showed that LY01008 had similar pharmacodynamic effects with Avastin in VEGF (vascular endothelial growth factor) binding activity, inhibitory effect on angiogenesis and vascular permeability, and anti-tumor activities in nude mouse models alone or combined with chemotherapeutic agents. PK study showed that LY01008 had similar PK parameters with Avastin at the same doses, and the relative bioavailability of LY01008 was 111.4%. The maximum tolerated dose of LY01008 in the single-dose toxicity study of cynomolgus monkeys was greater than 258 mg/kg. LY01008 had no effects on central nervous system, cardiovascular system and respiratory system in cynomolgus monkeys. LY01008 had no hemolytic effect in vitro and no local irritation in cynomolgus monkeys. The immunogenicity of LY01008 was no higher than that of Avastin in cynomolgus monkeys. In the one-month multiple-dose toxicity study in cynomolgus monkeys, the toxicokinetics profiles of LY01008 was similar with Avastin, the characteristics of the toxic reactions were the same and the extent was similar between LY01008 and Avastin, and no new toxic reactions were observed on LY01008. In conclusion, LY01008 had a good safety profile, and was biosimilar with Avastin in the comparative studies of pharmacodynamics, pharmacokinetics, toxicokinetics and toxicology, which supported the clinical trial and marketing application of LY01008 as a biosimilar of Avastin.

Comparative safety assessments of the biosimilar APZ001 and Erbitux in pre-clinical animal models.

PURPOSE: To evaluate the toxicity of Erbitux as well as its biosimilar APZ001 antibody (APZ001) in pre-clinical animal models including mice, rabbits and cynomolgus monkeys. METHODS: We performed analysis of normal behavior activity, autonomic and non-autonomic nervous functions, nervous-muscle functions, nervous excitability and sensorimotor functions on CD-1 mice. Subsequently, we studied that effects of APZ001 and Erbitux on respiratory system, cardiovascular system and kidney in Cynomolgus monkey models and performed local tolerance experiments on New Zealand rabbits. RESULTS: The comparisons between APZ001 and Erbitux showed no significant differences in mice autonomic nervous system, nervous muscle functions, non-autonomic nervous functions, nervous excitability and sensorimotor functions between treated and untreated group (p>0.05). APZ001 and Erbitux showed negative effect on CD-1 mice in the present of pentobarbital sodium anesthesia (p>0.05). Single administrations of high, medium or low doses of APZ001 did not lead to monkey urine volume alterations (p>0.05). In human tissues, APZ001 and Erbitux showed positive signals in endocardium, lung type II alveolar epithelial cell and surrounding vessels, but showed negative results in kidney and liver tissues. No hemolysis phenomenon and serious side-effects in vessels and muscles were observed in rabbits when administrated with APZ001 and Erbitux respectively. CONCLUSION: The safety comparisons between APZ001 antibody and Erbitux showed that these two antibodies showed highly similarities in mice, rabbits and cynomolgus monkey animal models in consideration of pharmaceutical effects, indicating APZ001 might be a suitable substitute for Erbitux.

Follow-on products for treatment of multiple sclerosis in Latin America: An update.

Both proprietary and non-proprietary medicines are expected to undergo rigorous pre-approval testing and both should meet stringent health authority regulatory requirements related to quality to obtain approval. Non-proprietary (also known as copy or generic) medicines, which base their authorization and use on the proprietary documentation and label, are often viewed as a means to help lower cost and thus increase patient access. If these medicines fail to meet quality standards, such as good manufacturing practice and bioequivalence (in humans), they are then defined as substandard copies and can pose serious risks to patients in terms of safety and efficacy. Availability of this type of compounds is more prevalent in regions where health authorities do not enforce registration regulations as stringent as those of the Food and Drug Administration, European Medicines Agency, or World Health Organization, including preestablished quality standard requirements. This article focuses on non-proprietary medicines for multiple sclerosis, that are not identical to proprietary versions and could thus fail to meet efficacy or have different impact on the safety of patients with multiple sclerosis.

Contribution of animal studies to evaluate the similarity of biosimilars to reference products.

The European Union (EU) was the first region to establish a regulatory framework for biosimilars, in which animal studies are required to confirm similarity to a reference product. However, animal studies described in European public assessment reports (EPARs) or marketing authorization applications (MAAs) did not identify clinically or toxicologically relevant differences despite differences in quality, suggesting that animal studies lack the sensitivity to confirm biosimilarity. Scientific advice provided learning opportunities to evolve existing guidance. Altogether, the data support a step-wise approach to develop biosimilars that focuses on quality and clinical efficacy of biosimilar. This approach might be more effective and does not necessarily require animal studies, which is also reflected in new EU draft guidance.

Structural and functional analyses of biosimilar enoxaparins available in Brazil.

Biosimilar enoxaparins have been available for clinical use in Brazil since 2009. Although their use has reduced costs of treatment expenses, their implementation still raises some concerns about efficiency, safety, regularity and reproducibility of batches. We undertook structural and functional analyses on over 90 batches of pharmaceutical-active ingredient, and 330 ones of the final products of biosimilar enoxaparins available in the Brazilian market between 2009 and 2014. Besides a nationwide-scale analysis, we have also employed methods that go beyond those recommended by the standard pharmacopeias. We have used high-resolution 2D NMR, detailed assessment of the anticoagulant and antithrombotic properties, check of side effects in experimental animals after continuous administration, and analyses of individual composing oligosaccharides. The 1D 1H NMR spectra of all batches of biosimilar enoxaparins are fairly coincident, and the resultant average spectrum is quite identical to that from the original drug. This structural equality was also assured by highly resolved 2D NMR spectra. The anticoagulant activity, determined by diverse assays and the in vivo antithrombotic and bleeding effects of the biosimilar version were confirmed as equal as of the parental enoxaparins. Structure and function of the composing oligosaccharides were identical in both enoxaparin types. No side effect was observed after continuous subcutaneous administration to rats for 30 days at the dose of 2 mg kg⁻¹ body weight. Biosimilar enoxaparins available in Brazil fulfilled the requirement of the five items defined by FDA-USA for approval of this type of drug.

Safety and biosimilarity of ior(®) EPOCIM compared with Eprex(®) based on toxicologic, pharmacodynamic, and pharmacokinetic studies in the Sprague-Dawley rat.

This study examined the safety, pharmacodynamic (PD), and pharmacokinetic (PK) biosimilarity of the human recombinant erythropoietin (EPO) products ior(®) EPOCIM and Eprex(®) following a 28-day repeated intravenous dose administration in male and female Sprague-Dawley rats with a 14-day recovery period. Safety profiling was based on clinical observations, clinical pathology, and pathology findings for control rats dosed with vehicle and rats dosed either with 30, 300, and 600 I.U./kg of ior(®) EPOCIM or 600 I.U. of Eprex(®) . Adverse findings for both ior(®) EPOCIM and Eprex(®) were similar and were a consequence of thrombotic events (ulcerative skin lesions, swollen hock joints/lameness, stomach ulcers) and decreased body weight gains, all known adverse reactions to this class of drug in rats. With the exception of stomach ulcers, all other adverse findings were fully reversible. Neither drug stimulated the production of antidrug antibodies. As expected, ior(®) EPOCIM and Eprex(®) both increased reticulocyte, red blood cell, hemoglobin, and hematocrit levels in rats. The PK of EPO following dosing with ior(®) EPOCIM was well behaved and consistent with the literature. The results of this study imply that ior(®) EPOCIM and Eprex(®) had safety profiles, PD responses, and toxicokinetic profiles that were biosimilar.

Comparative safety assessments of the biosimilar APZ001 and Erbitux in pre-clinical animal models

Abstract Purpose: To evaluate the toxicity of Erbitux as well as its biosimilar APZ001 antibody (APZ001) in pre-clinical animal models including mice, rabbits and cynomolgus monkeys. Methods: We performed analysis of normal behavior activity, autonomic and non-autonomic nervous functions, nervous-muscle functions, nervous excitability and sensorimotor functions on CD-1 mice. Subsequently, we studied that effects of APZ001 and Erbitux on respiratory system, cardiovascular system and kidney in Cynomolgus monkey models and performed local tolerance experiments on New Zealand rabbits. Results: The comparisons between APZ001 and Erbitux showed no significant differences in mice autonomic nervous system, nervous muscle functions, non-autonomic nervous functions, nervous excitability and sensorimotor functions between treated and untreated group (p>0.05). APZ001 and Erbitux showed negative effect on CD-1 mice in the present of pentobarbital sodium anesthesia (p>0.05). Single administrations of high, medium or low doses of APZ001 did not lead to monkey urine volume alterations (p>0.05). In human tissues, APZ001 and Erbitux showed positive signals in endocardium, lung type II alveolar epithelial cell and surrounding vessels, but showed negative results in kidney and liver tissues. No hemolysis phenomenon and serious side-effects in vessels and muscles were observed in rabbits when administrated with APZ001 and Erbitux respectively. Conclusion: The safety comparisons between APZ001 antibody and Erbitux showed that these two antibodies showed highly similarities in mice, rabbits and cynomolgus monkey animal models in consideration of pharmaceutical effects, indicating APZ001 might be a suitable substitute for Erbitux.