Evaluation of Cell Proliferation in Rat Mammary Glands Is Not Predictive of the Carcinogenic Potential of Insulin In Vivo

Abstract

For nonclinical safety-assessment of insulin analogues in vivo, mitogenic effects are compared to that of human insulin. Besides histopathologic evaluation, this usually includes assessment of cell proliferation (CP) in mammary glands. Insulin analogue X10 is recommended as positive control, due to its known carcinogenic effect in rat mammary glands. Here, we discuss the mitogenic effect of insulin in vivo and use of X10 as positive control. We present results from 4 nonclinical rat studies evaluating effects of repeated dosing with insulin detemir (≤26 weeks) or degludec (52 weeks) in mammary glands. Studies included human insulin-dosed groups as comparators, CP, and histopathologic evaluation. One study included an X10-dosed group (26 weeks), another ≤3 weeks of dosing with X10 or human insulin evaluating effects of these comparators. Neither human insulin, insulin detemir, degludec, nor X10 induced mammary tumors or increased CP in the studies. The CP marker proliferating cell nuclear antigen varied within/between studies and was not correlated with the remaining markers or CP fluctuations during estrous cycle, whereas the other CP markers, Ki-67 and 5-bromo-2′-deoxyuridine (BrdU), correlated with estrous cycle changes and each other. In conclusion, we propose that the mitogenic effect of insulin in rat mammary glands is weak in vivo. Cell proliferation evaluation in nonclinical safety assessment studies is not predictive of the carcinogenic potential of insulin, thus, the value of including this end point is debatable. Moreover, X10 is not recommended as positive control, due to lack of proliferative effects. Typical CP markers vary greatly in quality, BrdU seemingly most reliable.

New Modalities and Carcinogenicity Assessment

“…7 Consequently, EMA started to recommend assessment of CP, and assessment of CP in the mammary gland has often been included in nonclinical studies evaluating the carcinogenic effects of new insulin analogues, in some cases using X10 as a positive control. 1,4,5 The value of this CP assessment has been challenged in recent years, particularly because histopathologic examination of mammary gland tissue and identified macroscopic changes are now included in more recent assessments.…”

“…In agreement with published data in rats, CP fluctuated during the estrous cycle, and thus, the applied methods were able to identify changes, if present. 4,5 Based on the four studies evaluated, the authors found that the mitogenic effect of insulin in rat mammary glands is weak and that in vivo CP evaluation in nonclinical studies was not predictive of the carcinogenic potential of insulin; thus, the value of including this endpoint is debatable, as is the inclusion of X10 as a positive control.…”

New Modalities and Carcinogenicity Assessment

“…7 Consequently, EMA started to recommend assessment of CP, and assessment of CP in the mammary gland has often been included in nonclinical studies evaluating the carcinogenic effects of new insulin analogues, in some cases using X10 as a positive control. 1,4,5 The value of this CP assessment has been challenged in recent years, particularly because histopathologic examination of mammary gland tissue and identified macroscopic changes are now included in more recent assessments.…”

“…In agreement with published data in rats, CP fluctuated during the estrous cycle, and thus, the applied methods were able to identify changes, if present. 4,5 Based on the four studies evaluated, the authors found that the mitogenic effect of insulin in rat mammary glands is weak and that in vivo CP evaluation in nonclinical studies was not predictive of the carcinogenic potential of insulin; thus, the value of including this endpoint is debatable, as is the inclusion of X10 as a positive control.…”

Quantitative Assessment of Epithelial Proliferation in Rat Mammary Gland Using Artificial Intelligence Independent of Choice of Proliferation Marker

“…2,3 This led to a recommendation from the European Medicines Agency (EMA) that quantification of mammary gland proliferation in vivo could be performed as part of the non-clinical safety assessment of novel insulin analogues, 4 and mammary gland proliferation has therefore been included as an endpoint in repeat-dose toxicity studies during non-clinical development of insulin analogues. A recent study described that proliferation of normal rat mammary gland epithelium is not a predictive marker of receptor-mediated carcinogenic effects of insulin analogues, 5 but assessing mammary gland cell proliferation is still often implemented in non-clinical development programs, based on current recommendations from regulatory authorities regarding non-clinical safety assessment of insulin analogues.…”

New Modalities and Carcinogenicity Assessment

“…7 Consequently, EMA started to recommend assessment of CP, and assessment of CP in the mammary gland has often been included in nonclinical studies evaluating the carcinogenic effects of new insulin analogues, in some cases using X10 as a positive control. 1,4,5 The value of this CP assessment has been challenged in recent years, particularly because histopathologic examination of mammary gland tissue and identified macroscopic changes are now included in more recent assessments.…”

“…In agreement with published data in rats, CP fluctuated during the estrous cycle, and thus, the applied methods were able to identify changes, if present. 4,5 Based on the four studies evaluated, the authors found that the mitogenic effect of insulin in rat mammary glands is weak and that in vivo CP evaluation in nonclinical studies was not predictive of the carcinogenic potential of insulin; thus, the value of including this endpoint is debatable, as is the inclusion of X10 as a positive control.…”

Quantitative Assessment of Epithelial Proliferation in Rat Mammary Gland Using Artificial Intelligence Independent of Choice of Proliferation Marker

“…2,3 This led to a recommendation from the European Medicines Agency (EMA) that quantification of mammary gland proliferation in vivo could be performed as part of the non-clinical safety assessment of novel insulin analogues, 4 and mammary gland proliferation has therefore been included as an endpoint in repeat-dose toxicity studies during non-clinical development of insulin analogues. A recent study described that proliferation of normal rat mammary gland epithelium is not a predictive marker of receptor-mediated carcinogenic effects of insulin analogues, 5 but assessing mammary gland cell proliferation is still often implemented in non-clinical development programs, based on current recommendations from regulatory authorities regarding non-clinical safety assessment of insulin analogues.…”

New Modalities and Carcinogenicity Assessment

“…7 Consequently, EMA started to recommend assessment of CP, and assessment of CP in the mammary gland has often been included in nonclinical studies evaluating the carcinogenic effects of new insulin analogues, in some cases using X10 as a positive control. 1,4,5 The value of this CP assessment has been challenged in recent years, particularly because histopathologic examination of mammary gland tissue and identified macroscopic changes are now included in more recent assessments.…”

“…In agreement with published data in rats, CP fluctuated during the estrous cycle, and thus, the applied methods were able to identify changes, if present. 4,5 Based on the four studies evaluated, the authors found that the mitogenic effect of insulin in rat mammary glands is weak and that in vivo CP evaluation in nonclinical studies was not predictive of the carcinogenic potential of insulin; thus, the value of including this endpoint is debatable, as is the inclusion of X10 as a positive control.…”

Quantitative Assessment of Epithelial Proliferation in Rat Mammary Gland Using Artificial Intelligence Independent of Choice of Proliferation Marker

“…2,3 This led to a recommendation from the European Medicines Agency (EMA) that quantification of mammary gland proliferation in vivo could be performed as part of the non-clinical safety assessment of novel insulin analogues, 4 and mammary gland proliferation has therefore been included as an endpoint in repeat-dose toxicity studies during non-clinical development of insulin analogues. A recent study described that proliferation of normal rat mammary gland epithelium is not a predictive marker of receptor-mediated carcinogenic effects of insulin analogues, 5 but assessing mammary gland cell proliferation is still often implemented in non-clinical development programs, based on current recommendations from regulatory authorities regarding non-clinical safety assessment of insulin analogues.…”