Introduction of biosimilar insulins in Europe.

Regulatory approval of the first biosimilar insulin in Europe, LY2963016 insulin glargine (Abasaglar® ), in 2014 expanded the treatment options available to people with diabetes. As biosimilar insulin products come to market, it is important to recognize that insulin products are biologicals manufactured through complex biotechnology processes, and thus biosimilar insulins cannot be considered identical to their reference products. Strict regulatory guidelines adopted by authorities in Europe, the USA and some other countries help to ensure that efficacy and safety profiles of biosimilar insulins are not meaningfully different from those of the reference products, preventing entry of biological compounds not meeting quality standards and potentially affecting people's glycaemic outcomes. This review explains the concept of biosimilar medicines and outlines regulatory requirements for registration of biosimilar insulins in Europe, which is illustrated by the successful development of LY2963016 insulin glargine and MK-1293 insulin glargine (Lusduna® ). Preclinical and clinical comparative studies of the biosimilar insulin glargine programmes include in vitro bioassays for insulin and insulin-like growth factor 1 receptor binding, assessment of in vitro biological activity, evaluation of pharmacokinetic/pharmacodynamic profiles in phase I studies and assessment of long-term safety and efficacy in phase III studies. The emergence of biosimilar insulins may help broaden access to modern insulins, increase individualized treatment options and reduce costs of insulin therapy.

Treatment escalation options for patients with type 2 diabetes after failure of exenatide twice daily or glimepiride added to metformin: results from the prospective European Exenatide (EUREXA) study.

AIMS: To evaluate third-line thiazolidinedione (TZD) or glimepiride therapy in patients inadequately controlled on metformin + exenatide twice daily, and third-line exenatide twice daily in patients inadequately controlled on metformin + glimepiride. METHODS: In this randomized, open-label, multicentre trial, 144 patients with type 2 diabetes inadequately controlled [glycated haemoglobin (HbA1c) >9% (75 mmol/mol) after 3 months' treatment or >7% (53 mmol/mol) at two consecutive visits 3 months apart, after 6 months' treatment] on metformin + exenatide twice daily were re-randomized to add-on TZD or glimepiride, and 166 patients inadequately controlled on metformin + glimepiride received add-on exenatide twice daily. Changes in HbA1c, body mass index (BMI), lipids, hypoglycaemia and vital signs were evaluated. RESULTS: The median duration of triple therapy was ∼2 years. In patients inadequately controlled on metformin + exenatide twice daily, add-on TZD decreased HbA1c levels significantly better than add-on glimepiride: 130-week difference 0.48% [95% confidence interval (CI) 0.19-0.77] or 5.2 mmol/mol (95% CI 2.1-8.4; p = 0.001), but with significantly increased BMI and systolic blood pressure. The ratio of documented symptomatic (blood glucose ≤70 mg/dl [3.9 mmol/l]) hypoglycaemia rates for add-on glimepiride to add-on TZD was 8.48 (p < 0.0001). Add-on exenatide twice daily after metformin + glimepiride significantly reduced HbA1c levels: mean [standard deviation (s.d.)] change from baseline -0.35 (0.89)% [-3.8 (9.7) mmol/mol] and BMI: mean (s.d.) change from baseline -0.82 (1.9) kg/m(2) at 130 weeks, with a slightly increased rate of documented symptomatic hypoglycaemia from metformin + glimepiride (ratio 1.49). CONCLUSIONS: TZD, but not glimepiride, was an effective and well tolerated third-line therapy in patients without glycaemic control after long-term therapy with metformin + exenatide twice daily. Exenatide twice daily was an effective and well tolerated third-line therapy in patients inadequately controlled on metformin + glimepiride.

Biosimilar insulins: a European perspective.

Biosimilar insulins are likely to enter clinical practice in Europe in the near future. It is important that clinicians are familiar with and understand the concept of biosimilarity and how a biosimilar drug may differ from its reference product. The present article provides an overview of biosimilars, the European regulatory requirements for biosimilars and safety issues. It also summarizes the current biosimilars approved in Europe and the key clinical issues associated with the use of biosimilar insulins.

Role of the eye muscles in thyroid eye disease: identification of the principal autoantigens.

Thyroid-associated ophthalmopathy (TAO) is a progressive orbital disorder associated with Graves' hyperthyroidism and, less often, Hashimoto's thyroiditis in which autoantibodies react with orbital antigens and lead to exophthalmos and eye muscle inflammation. Eye muscle (EM) membrane proteins initially reported as 55 and 64 kd are the best markers of ophthalmopathy. The "64-kd protein" is now shown to be the flavoprotein subunit of mitochondrial succinate dehydrogenase and to have a correct molecular weight of 67 kd. We have cloned a fragment of a novel eye muscle protein, which we call G2s, and sequenced 1.4 kb of the full length cDNA. G2s does not share any significant homologies with other reported proteins. The 5.9 kb G2s mRNA, that corresponds to a protein of approximately 220 kd, is expressed in EM, other skeletal muscle and thyroid, but not in other tissues tested. We have also cloned and sequenced a 63-kd eye muscle protein identified as the calcium binding protein calsequestrin. Antibodies against calsequestrin were found in 40% of patients with active ophthalmopathy, but in 0% of normal subjects. Finally, we have sequenced a 19 amino acid fragment of a 55-kd porcine eye muscle membrane protein that exactly matched porcine and human sarcalumenin, a 160-kd glycoprotein localized in the lumen of the longitudinal sarcoplasmic reticulum of the skeletal muscle fiber where it binds calcium. A 53-kd glycoprotein fragment of the molecule corresponds to the 55-kd protein. In a preliminary study, serum antibodies against purified sarcalumenin were detected in 40% of patients with active TAO of less than 1 year duration, but in no controls tested. We porpose that the primary autoantigen in TAO is G2s, which would also explain the association of ophthalmopathy with thyroid autoimmunity, and that antibodies against the intracellular proteins flavoprotein, calsequestrin, and sarcalumenin are secondary markers of an immune-mediated reaction in eye muscle in patients with thyroid autoimmunity.

T cell interactions with extracellular matrix proteins in patients with thyroid-associated ophthalmopathy.

Although thyroid-associated ophthalmopathy (TAO) is now generally accepted as an autoimmune inflammatory disorder of the extraocular muscles and the orbital connective tissue, its aetiopathogenesis remains poorly understood. Recent data indicate that impaired interactions between T cells and extracellular matrix (ECM) proteins may play an important role in development and maintaining of an inflammatory process. We report here results of the study focusing on interactions between T lymphocytes and collagen-I (Coll-I), collagen-IV (Coll-IV), fibronectin (FN), laminin (LM) in patients with TAO. Using a standard peripheral blood mononuclear cells (PBMC) proliferation assay, we observed a markedly enhanced T cell response to Coll-I in patients with active TAO (mean SI=4.5). The proliferatory response to Coll-I was significantly greater (Wilcoxon test; p < 0.001) than in normal subjects (mean SI=1.88), patients with stable TAO (mean SI=2.05) and patients with thyroid autoimmune diseases (AITD) without ophthalmopathy (mean SI=2.49). PBMC stimulation by Coll-I is likely to be antigen-dependent requiring engagement of the T cell receptor with collagen peptides, rather than mediated via integrins. The percentage of circulating CD29+ (beta1 integrin chain) T cells was not increased in patients with active TAO. Additionally in the assay of costimulation of CD3-mediated proliferation, we found that peripheral blood T cells from patients with TAO and AITD were costimulated only by FN. On the other hand a markedly enhanced costimulation of CD3-mediated proliferative responses by Coll-I, Coll-IV, FN and LM were observed in a retrobulbar T cell line. We conclude that abnormalities in T cell interactions with ECM proteins, especially Coll-I may play a role in the pathogenesis of TAO.

Antibodies against striated muscle, connective tissue and nuclear antigens in patients with thyroid-associated ophthalmopathy: should Graves' disease be considered a collagen disorder?

The identity and subcellular localization of the principal extraocular muscle (EOM) antigens and prevalences of the corresponding serum autoantibodies in thyroid-associated ophthalmopathy (TAO) need to be clarified. We have used porcine eye muscle tissue, which expresses all autoantigens identified in human tissue, as substrate in an indirect immunofluorescence assay. Several different patterns of antibody binding to EOM tissue antigens were observed with sera from patients with TAO namely, membrane, cytoplasmic, interstitial (endomysial) and nuclear. Overall, sera from 75% of patients with TAO contained one or more antibodies reactive with EOM, compared to 32% of patients with Graves' hyperthyroidism, 38% with Hashimoto's thyroiditis, and 16% of normals. All sera which reacted with EOM membrane or cytoplasmic antigens also reacted with the same antigen(s) in other skeletal muscle, but not in the other tissues tested. Sera from 31% of patients with TAO, but only 7% of those with Hashimoto's thyroiditis, and no patient with Graves' hyperthyroidism without evident ophthalmopathy, contained antinuclear antibodies (ANA). The most common nuclear fluorescence pattern was the finely speckled type typically associated with anti-Sm or anti-RNP antibodies. Significant positive correlations in patients with TAO were found between (i) EOM dysfunction and ANA (ii) eye disease of < 1 yr duration and EOM membrane-reactive antibodies and (iii) eye disease of < 1 yr duration and interstitial (endomysial) tissue-reactive antibodies. Although patients with Graves' disease do not usually exhibit other signs or immunologic features of a generalized collagen disorder, the finding of high prevalences of ANA and anti-striated muscle antibodies and, less often, anti-connective tissue antibodies in patients with ophthalmopathy, is consistent with it being a collagen-like disorder of the striated muscle, connective tissue and the thyroid. The reason why the inflammatory process is mainly limited to these tissues is unclear although cross reaction of ANA with tissue specific proteins or increased expression of muscle and connective tissue antigens in the orbit and skin, are possibilities.

Serum antibodies reactive with eye muscle antigens and the TSH receptor in a euthyroid subject who developed ophthalmopathy and Graves' hyperthyroidism.

Serum antibodies reactive with eye muscle autoantigens, in particular a 64-kDa protein that is also expressed in the thyroid, and the TSH receptor, are associated with the ophthalmopathy that occurs in about 50% of patients with Graves' hyperthyroidism. We have had the opportunity to study a euthyroid, apparently normal, 35-year-old woman with a family history of thyroid autoimmunity and "colitis" but no clinical or biochemical evidence for thyroid disease or ophthalmopathy, who developed Graves' hyperthyroidism and ophthalmopathy together 18 months later. Serum taken when the patient was first seen was positive for antibodies reactive with (i) 9 different eye muscle proteins ranging in size from 15 to 130 kDa, notably those of 64, 55, and 50 kDa, by immunoblotting with eye muscle membranes, (ii) eye muscle and Müller's muscle cell membrane antigens in antibody-dependent cell-mediated cytotoxicity (ADCC), (iii) an eye muscle cytoplasmic antigen in indirect immunofluorescence, and (iv) the TSH receptor as measured in a radioreceptor binding inhibition assay. When she developed Graves' disease, serum concentration of antibodies to the 55-kDa protein had decreased from +2 to +/-, those reactive with other eye muscle antigens had not changed significantly, and TSH receptor antibodies had increased 3-fold. This case report suggests that antibodies reactive with eye muscle antigens and the TSH receptor are markers of the ophthalmopathy and able to predict its development in predisposed subjects. The significance of these findings needs to be confirmed in a prospective study of first-degree relatives of patients with thyroid-associated ophthalmopathy and patients with Graves' hyperthyroidism without eye signs.

Antibody-dependent cell-mediated cytotoxicity against orbital target cells in thyroid-associated ophthalmopathy and related disorders; close relationship between serum cytotoxic antibodies and parameters of eye muscle dysfunction.

We have carried out tests for antibody-dependent cell-mediated cytotoxicity (ADCC) against extra ocular muscle (EOM), Müller's muscle, orbital fibroblasts and skeletal muscle in patients with thyroid-associated ophthalmopathy (TAO) and related eye disorders. Cytotoxicity was measured as lactate dehydrogenase (LDH) release and results expressed as % cytotoxicity. Tests were positive, with EOM cells, in 65% of patients with TAO, 75% with ocular myopathy, a variant of TAO in which periorbital inflammation is minimal, 50% with euthyroid Graves' disease defined as ophthalmopathy associated with subclinical thyroiditis and in 50% of patients with stable lid lag and retraction but no other signs of progressive ophthalmopathy, but in only 13% of patients with Graves' hyperthyroidism without ophthalmopathy, 10% with Hashimoto's thyroiditis and 14% of patients with other thyroid disorders. Tests were positive, with Müller's muscle cells, in 40% of patients with TAO, 25% with ocular myopathy, 40% with euthyroid Graves' disease, 44% with lid lag, 19% with Graves'hyperthyroidism, 50% with Hashimoto's thyroiditis and in 37.5% of patients with other thyroid disorders. When skeletal muscle cells were used as target, tests were positive in 13% of patients with TAO, 31% with lid lag, 25% with Graves' hyperthyroidism and in 29% of patients with Hashimoto's thyroiditis, but in no patient with euthyroid Graves' disease or other thyroid disorders. Tests were negative in all patients and normals tested when EOM-derived fibroblasts were used as targets in ADCC. A significant positive correlation between % cytotoxicity against EOM cells and the severity of the eye muscle dysfunction expressed as an eye muscle index, was observed in patients with TAO. There was a significant negative correlation between the duration of eye disease and % cytotoxicity against EOM cells, suggesting higher titers of cytotoxic antibodies in the early stages of TAO. There was no correlation between % cytotoxicity and serum level of anti-TSH receptor antibodies, measured in a radioreceptor assay. These findings suggest that autoimmunity against Müller's muscle may play a role in the pathogenesis of persistent lid lag and retraction. The nature of the EOM and Müller's muscle autoantigens recognized by cytotoxic antibodies in the serum of patients with TAO and related eye disorders is unknown.

Significance of tissue specific and tissue non specific autoimmune reactions of Graves' disease.

The relationship between the many immunologic abnormalities demonstrated in the peripheral blood of patients with Graves' disease (GD) and the broad spectrum of clinical features with which patients may present has not yet been addressed in detail. In this review we examine the evidence to support the notion that GD could be considered a multi-system autoimmune disorder in which tissue damage is restricted to the thyroid gland, connective tissue of the skin and orbit, extra-ocular and other skeletal muscles and, possibly, the lacrimal glands. Apart from the well recognized reactions of autoantibodies and sensitized T lymphocytes with epitopes on the thyroid specific TSH receptor, thyroid peroxidase and thyroglobulin, in patients with hyperthyroidism, there is also good evidence for autoantibody and, to a lesser extent, T lymphocyte reactivity with several eye muscle, other skeletal muscle and connective tissue, antigens in patients with ophthalmopathy, systemic myopathy, dermopathy and acropachy. There is also some evidence for immunoreactivity against lacrimal gland antigens in patients with ophthalmopathy associated with other features of GD. There are, in addition, a variety of organ non-specific reactions in GD; antinuclear antibodies are detected in serum from about one-third of patients with hyperthyroidism and ophthalmopathy, while from 5% to 10% have antibodies reactive with several other ubiquitous tissue proteins. Cloned proteins which are autoantigenic in some patients with hyperthyroidism or Hashimoto's thyroiditis and ophthalmopathy include collagen XIII, nebulin, the calcium binding protein calmitine, and the Mac-II antigen. All antibodies reactive with eye muscle antigens, except the 64 kDa protein which is also expressed in the thyroid, cross react with the same, or a related, protein in other skeletal muscle. Future research should focus on the underlying mechanisms for this broad loss of tolerance to self antigens and the effect of environmental factors such as stress, radioiodine and viral infection of the thyroid gland and other target tissues, in precipitating disease.

Failure to demonstrate cell-mediated immunity to orbital tissue antigens and epitopic fragments of a 64 kDa protein in the majority of patients with thyroid-associated ophthalmopathy.

We have studied a possible role of T cell sensitization to eye muscle antigens in patients with thyroid-associated ophthalmology (TAO). Peripheral blood mononuclear cell (PBMC) proliferation in response to crude porcine orbital tissue antigens, partially purified porcine eye muscle membrane proteins and predicted epitopic fragments of the recombinant 64 kDa protein 1D, was determined in patients with TAO and thyroid autoimmunity without eye disease. When membrane and cytosol fractions were used as antigen PBMC from 43% of patients with TAO but only 12.5% of normal subjects were responsive to a crude orbital connective tissue membrane fraction, although this difference was not significant. We were unable to demonstrate specific recognition of partially purified eye muscle membrane fractions; although most of the fractions tested were occasionally recognized by T cells from patients with ophthalmopathy, this was also the case for patients with autoimmune thyroid disease without ophthalmopathy and normal subjects. We did not clearly identify epitopic sequences within the 1D protein, most of the predicted peptides tested being recognized not only by T cells from a small proportion of patients with TAO, but also by those from some patients with autoimmune thyroid disease without ophthalmopathy and normal subjects. It is noteworthy however that approximately 22% of TAO patients, but no normal subjects, were positive to one or more of three peptides, suggesting that reactivity to the 1D protein may play a role in the pathogenesis of the eye disorder in some patients with TAO. The inconsistent and generally low T cell responses to crude and purified antigens noted in a few patients with TAO could be explained by low numbers of specifically sensitized lymphocytes in peripheral blood.

Should Graves' disease be considered a collagen disorder of the thyroid, skeletal muscle and connective tissue?

Graves' disease comprises hyperthyroidism, ophthalmopathy, pretibial myxedema and acropachy, which occur separately or in various combinations. We have used the indirect immunofluorescence test to investigate reactivity of sera from patients with autoimmune thyroid disorders with and without ophthalmopathy, with porcine extra ocular muscle (EOM) and control tissue substrates. Sera from 75% of patients with Graves' hyperthyroidism (GH) and ophthalmopathy, which we call thyroid-associated opthalmopathy (TAO), contained one or more antibodies reactive with EOM compared to 32% of those with GH without the eye disorder, 41% of patients with Hashimoto's thyroiditis (HT), and 16% of normals. Antibodies reactive with an EOM connective tissue antigen(s), seen as fluorescence of the interstitium and endomysium, were found in sera from 10% of patients with TAO and 16% of those with GH, but not from any patient with HT or normal subject. Similar patterns of connective tissue reactivity were also found in lacrimal gland, skeletal muscle, kidney and salivary gland. Antinuclear antibodies were detected in sera from 31% of patients with TAO, but from only 8% with HT, in no patient with GH and in only 3% of normal subjects. The most common pattern was a fine speckled fluorescence, found in 45% of sera, consistent with reactivity against the Sm antigen or nuclear RNP. The finding of a high prevalence of ANA and, less often, anti-connective tissue antibodies in patients with thyroid autoimmunity and ophthalmopathy, is consistent with Graves' disease being a "collagen-like disorder". The reason why inflammation and resulting tissue damage is limited to the thyroid, connective tissue of the skin and orbit, skeletal muscle and, possibly, the lacrimal gland, is unclear. One possibility is cross reaction of ANA with tissue specific membrane proteins in these sites. The extent of immunologic abnormalities, and the resulting clinical features, in patients with Graves' disease may reflect the severity of a putative defect in immune regulation.

Native gel electrophoresis and isoelectric focusing of a 64-kilodalton eye muscle protein shows that it is an important target for serum autoantibodies in patients with thyroid-associated ophthalmopathy and not expressed in other skeletal muscle.

Although sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting are widely used to detect serum antibodies in patients with autoimmune disorders, this procedure unfolds and denatures proteins and may alter antibody-binding sites. We have used a gentle protocol for the preparation and purification of a 64-kilodalton (kDa) eye muscle (EM) membrane antigen associated with thyroid-associated ophthalmopathy (TAO) for use as antigen in immunoblotting. Pig EM membrane proteins were prepared from crude homogenates by high speed centrifugation and solubilized by hand homogenization. These native membrane proteins (NMprot) were then electrophoresed on an 8.5% polyacrylamide gel in the absence of SDS, reducing agents, or urea, and proteins from individual bands were eluted, applied to standard SDS-PAGE, and immunoblotted with selected TAO patient sera. A prominent 64-kDa protein, present in most of the bands, was recognized by autoantibodies in sera from 35% of the patients with TAO and 47% of those with Graves' hyperthyroidism without evident ophthalmopathy, but in only 4% of normal subjects. To further purify the 64-kDa protein and increase the sensitivity of immunoblotting, NMprot were separated by isoelectric focusing (IEF) in the absence of SDS, reducing agent, and urea. The 64-kDa protein appeared mainly in IEF fraction 7 and had an isoelectric point of 6.1-6.2. Similar results were found for a human EM protein of 64 kDa. Sera from groups of patients and normal subjects were tested in immunoblotting against a pig EM 64-kDa protein prepared from NMprot and purified in IEF. Tests were positive in 67% of patients with TAO, in 37.5% of those with Graves' hyperthyroidism without eye disease, in 11% of patients with Hashimoto's thyroiditis without eye disease, and in 9% of normal subjects. The 64-kDa protein was not found in other skeletal muscle. The demonstration that a native 64-kDa protein that is specifically targeted by autoantibodies in the serum of patients with TAO is expressed in EM, but not other skeletal muscle, greatly enhances its possible significance in the pathogenesis of this eye disorder.

The ocular muscle cell is a target of the immune system in endocrine ophthalmopathy.

The exact pathogenic mechanism of thyroid-associated ophthalmopathy (TAO) remains unclear, and extensive studies on this disorder have resulted in often conflicting data. Well-known technical difficulties including the limited access to orbital tissues from patients with active and early disease, lack of an animal model and poor reproducibility of some of the immunological techniques used are in part responsible for this confusing situation. Despite this there is considerable evidence for eye muscle (EM) tissue involvement in the autoimmune reactions of TAO. Although the primary EM antigen(s) recognized by immunocompetent cells and autoantibodies has not been definitely identified, some good candidates, among them a membrane antigen of 64 kD which is also expressed in the thyroid, have been partially characterized. While it is unclear which component of the autoimmune reaction against EM-humoral or cell mediated-plays the more important role, autoantibodies seem to be responsible at least in part for the clinical features of the eye disorder. On the other hand, the orbital connective tissue (OCT) cells, especially the fibroblasts surrounding the EM fibers, seem to be extremely sensitive to stimulation by cytokines and other soluble proteins and immunoglobulins released in the course of an immune reaction in the muscle cells. Fibroblasts secrete large amounts of glycosaminoglycans and also participate in maintaining the autoimmune reaction. It seems likely that the EM is the main and primary target of the orbital autoimmune process whereas inflammation of the OCT is probably secondary.