IgG pharmacokinetics in a rat model of type 2 diabetes mellitus and diabetic nephropathy
Chadha, Gurkishan Singh
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Diabetes mellitus (DM) is one of the leading causes of physical impairment and death worldwide. Its global prevalence was about 8% in 2011 and is predicted to rise to around 15% by 2050. Type 2 diabetes mellitus (T2DM) accounts for 90-95% of all the cases. Furthermore, DM has a high association with co-morbidities such as heart disease, kidney disease, retinopathy, neuropathy and peripheral vascular diseases. Various studies has observed significant changes in the pharmacokinetics of small molecules with diabetes. On the contrary, only a few studies have evaluated the effect of the disease on the pharmacokinetics of large molecular weight molecules such as IgG and monoclonal antibodies. Given the reported changes in the disposition of small molecules with diabetes progression, we hypothesize that diabetes will also lead to alterations in the renal and total clearances of human IgG (hIgG) in Zucker Diabetic Fatty (ZDF) rats. We also hypothesize that diabetic nephropathy (DN) may also contribute to the alterations in the renal and total clearances of hIgG in diabetes. Furthermore, we hypothesize that the increased non-renal clearance in T2DM is due to increased renal (and potentially other tissue) catabolic clearance, due to altered expression and/or function of endocytosis proteins FcRn and/or megalin (LRP2)/cubilin. The main objectives for this dissertation were: a) to examine the effects of T2DM and DM/DN on the renal and total clearances of hIgG, in ZDF rats after IV and SC administration of hIgG (1mg/kg). The specific role of T2DM in the altered disposition of hIgG was evaluated by treating diabetic rats with pioglitazone, while the role of chronic kidney disease (CKD) was assessed using 5/6 nephrectomized Sprague Dawley rats; b) to develop a minimal physiologically based pharmacokinetic (mPBPK) model for evaluating the changes in renal and total clearance with T2DM; c) to evaluate the mechanisms underlying the changes in the renal and non-renal clearances of hIgG in T2DM. In these studies, we have observed and evaluated the changes in pharmacokinetics and bioavailability of hIgG, with T2DM and chronic kidney disease in ZDF rats and 5/6 nephrectomized rats, respectively. Significant increases in the biomarkers of the disease (blood urea nitrogen, urine albumin amounts and urine albumin to creatinine ratios) has been observed in ZDF rats. Also, renal clearance and total clearance of hIgG increased significantly (~100-fold; ~2.5-fold, respectively) in the diabetic animals, and these differences were more pronounced when the disease progressed with DN. Although increases in renal clearance were highly significant, much greater increases were observed in the total clearance of hIgG. Pioglitazone (a peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist) treatment reduces insulin resistance in the liver and peripheral tissues, and makes the diabetic animal's euglycemic. Both the total and renal clearances decreased significantly in diabetic animals after pioglitazone treatment, but the reversal in the changes were incomplete. After SC administration, bioavailability of hIgG was similar (>80%) amongst all the three group of animals; and the rate constant for absorption was similar among the three groups in early disease, but differed significantly with progression to DN. However, in the chronic kidney group with 5/6 nephrectomized rats, no differences in hIgG disposition between the control and the renal-compromised group were observed. Although significant differences were observed in the renal clearance in the two groups, no difference was observed in total clearance. A two-compartment model (2CM) has been used to characterize the concentration-time profiles of hIgG. A 2CM has been commonly used to describe and fit hIgG and monoclonal antibodies PK data in animals. To overcome the limitations in 2CM, an extended mPBPK for hIgG disposition with the plasma and urine data was developed. The model has been developed to incorporate the effect of DM and DN on total and renal clearance changes, along with the effect of pioglitazone treatment. The model incorporated a kidney compartment, and classified organs as leaky or tight based on capillary fenestrations. The model was able to successfully fit plasma and urine data from the 3 groups and was able to simulate tissue concentrations. The model was also able to quantify the changes in the renal function (glomerular filtration rate, sieving coefficient and fraction reabsorbed in the kidney) with diabetes and reversal in these changes with pioglitazone treatment. The mPBPK model provided a simplified approach towards physiological based modeling, and yielded a better understanding of distributional and elimination parameters of hIgG with and without DM/DN. To mechanistically evaluate the changes in the clearance of hIgG in DM/DN, tissue (liver and kidney) homogenate studies were conducted and the tissue catabolism of 125 I labelled hIgG among the three groups of rats was compared. The present studies demonstrated significantly increased catabolism of hIgG in the kidney and liver homogenates of diabetic rats. With pioglitazone treatment, the increased catabolism of hIgG in the liver tissues was reversed, however the kidney homogenates still demonstrated significantly increased catabolism of hIgG as compared to the control rats. The FcRn, megalin and cubilin expression in liver and kidney samples was also evaluated. The protein expression of FcRn, megalin and cubilin was significantly reduced in kidney cortex with DM/DN. While pioglitazone treatment was able to significantly reverse the decrease in protein expression of FcRn and cubilin, the protein expression of megalin remained significantly decreased as compared to the control rats. In the case of liver, the FcRn protein expression was similar among the three groups, while there was no detectable expression of either megalin or cubilin protein. There was no correlation between the mRNA and protein expression of FcRn, megalin or cubilin in the studies. To evaluate the role of FcRn in the disposition of hIgG, the changes in PK of chicken IgY (1mg/kg IV) in ZDF rats were evaluated. Chicken IgY represents the avian counterpart to mammalian IgGs, but it does not interact or bind with FcRn and thus its catabolism is FcRn-independent. The chicken IgY plasma concentration-time profiles and total clearances were similar in control and diabetic rats. The renal clearance increased by ~60-fold in the diabetic rats, but it was still lower than that seen with hIgG. This suggest that FcRn-dependent changes may contribute to the altered renal and non-renal clearance of hIgG. In summary, this dissertation provides novel insight into the effect of DM/DN on the total and renal clearance of hIgG, an isotype of mAbs. The studies have demonstrated the significance of pioglitazone treatment towards reversing the changes in hIgG clearance in DM and DM/DN. Furthermore, a novel mPBPK model was developed to quantify the renal function changes, which is also able to simulate the tissue concentrations of hIgG in all the three groups of animals. The studies have also observed hypercatabolism of hIgG in the kidney and liver of diabetic rats, and have mechanistically evaluated the changes in mRNA and protein expressions of FcRn, megalin and cubilin in the kidney cortex and the liver, and correlated these changes with the alterations in the renal and total clearances of hIgG, in the presence and absence of DM/DN.