Biologics & the kidney – importance of transport & safety assessment

Biologics represent a new modality of drug molecules which is rapidly expanding in market share and in the number of biologics gaining regulatory approval each year.  Biologics are defined as a diverse range of synthetic molecules such as peptides, proteins, monoclonal antibodies, recombinant proteins, siRNA and Antisense Oligonucleotides (AONs).

Biologics are very different from ‘traditional’ small molecule drugs; they are large molecules with high molecular weights (up to 150KDa), are complex in structure, generally hydrophilic and have low stability.  They are however designed to be very specific in terms of target, have limited cell uptake and generally a low volume of distribution.^1,2  In contrast  to small molecules, biologics are not subject to hepatic metabolism as the primary route of breakdown but are metabolised by a diverse range of mechanisms involving proteases,  peptidases and nucleases  depending on structure, followed by cellular uptake and lysosomal degradation.³ For some classes of biologics renal clearance has been implicated. Several studies have demonstrated the biodistribution of peptide, siRNA and AONs is highly weighted to the kidney. This targeted uptake of siRNA and AONs by the proximal tubule has been exploited as a mechanism to deliver specific siRNA and AON molecules as a therapeutic tool to silence genes in the kidney. However the non-specific nature of the uptake process for the proximal tubule leading to accumulation of siRNA and AONs have also generated off target gene silencing in the kidney and nephrotoxicity. We are only now beginning to understand the mechanism of renal clearance and as to why some biologics are accumulated within the kidney resulting in nephrotoxicity.^4,5

To gain more understanding of the mechanism of renal proximal tubule accumulation of biologics we have investigated the potential utility of the aProximate™ model to investigate both the transport and nephrotoxicity of both siRNA and AON constructs in vitro.

The Newcells Biotech kidney proximal tubule epithelial (PTEC) cell assay (aProximate™) uses ethically sourced, primary human or animal cells, to generate proximal tubule cell monolayers on Transwell™  filter supports,  which express high levels of a diverse range of transporter proteins in the appropriate membrane orientation such as MATE1, MATE2-k, OAT-1, and OAT-3, OCT2.^6,7 The polarised monolayer format allows the measurement of the effects of both apical and basolateral dosing and has been used extensively to accurately predict renal drug handling and drug-drug interactions of a wide range of molecules prior to clinical trials.

Key to the renal uptake of biologics is the Megalin and Cubilin complex. Megalin, is a 600kDa membrane spanning glycoprotein and is member of the LDL receptor family. Megalin is polyspecific and is known to bind a very diverse range substrates, including plasma protein, antibiotics, vitamins and signalling molecules. To function, Megalin forms a duplex with Cubulin, a 460-kDa extracellular protein. Cubilin also has a diverse range of substrates including albumin. In physiology the endocytosis of small proteins bound to the Megalin-Cubilin–complex is the mechanism by which the glomerular filtrate is cleared of filtered protein.⁸ Importantly the Megalin-Cubulin complex has been implicated in the proximal tubule accumulation of biologics.^4,9 However, the renal pharmacokinetics and safety evaluation studies of large molecules are challenged due to the limited in vitro models, and availability of species with cross-reactivity for in vivo studies.² To address this, we have generated a set of validation data using aProximate™ cross species models. We have been able to show the megalin-cubulin mediated uptake of a range of siRNA, AONs and peptides. By using FDA approved biomarker of kidney injury we have screened a number of biologics for potential in vivo renal liability. This data showcases the utility of the aProximate™ kidney proximal tubule model to investigate both the transport and potential nephrotoxicity of both siRNA and AON constructs. We are conducting further studies including generating a validation panel of siRNA and AON constructs to produce ROC curves and assess the predictive power of in vitro proximal tubule cell monolayers.

 

References:

1. Prueksaritanont T, Tang C. ADME of biologics-what have we learned from small molecules? AAPS J. 2012 Sep;14(3):410–9.
2. Wan H. An overall comparison of small molecules and large biologics in ADME testing. Vol. 4, ADMET and DMPK. International Association of Physical Chemists; 2016. p. 1–22.
3. Datta-Mannan A Mechanisms Influencing the Pharmacokinetics and Disposition of Monoclonal Antibodies and Peptides Drug Metabol Disp 2019 47:1100 -1110
4. Moisan A, Gubler M, Zhang JD, Tessier Y, Erichsen KD , Sewing S, Gérard R,  Avignon B, Huber S, Benmansour F,  Chen X,  Villaseñor R, Braendli-Baiocco A, Festag M, Maunz A. Singer T,  Schuler F, Roth AB. Inhibition of EGF Uptake by Nephrotoxic Antisense Drugs In Vitro and Implications for Preclinical Safety Profiling.   Mol Ther Nucleic Acid 2017  6:89-105
5. Crooke ST, Baker BF,  Pham NC, Hughes SG,  Kwoh TJ, Cai D, Tsimikas S,  Geary, RS, Bhanot S. The Effects of 2′-O-Methoxyethyl Oligonucleotides on Renal Function in Humans.  Nucleic Acid Thera; 2018  28:10-16
6. Huang S-M. In Vitro Metabolism-and Transporter-Mediated Drug-Drug Interaction Studies Guidance for Industry DRAFT GUIDANCE [Internet]. 2009 [cited 2020 Feb 28]. Available from: http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm
7. Medicines Agency E. Guideline on the investigation of drug interactions [Internet]. 2012 [cited 2020 Feb 28]. Available from: www.ema.europa.eu/contact
8. Verroust PJ, Christensen EI Megalin and cubilin—the story of two multipurpose receptors unfolds  Nephrology Dialysis Transplantation, 2002 7:18671871
9. Weyer K, Nielsen R, Petersen SV, Christensen EI, Rehling M, Birn H. Renal Uptake of 99mTc-Dimercaptosuccinic Acid Is Dependent on Normal Proximal Tubule Receptor–Mediated Endocytosis 2013 J Nucl Med. 54:159-165