Renal studies in safety pharmacology and toxicology: A survey conducted in the top 15 pharmaceutical companies

JPM-06247; No of Pages 10 Journal of Pharmacological and Toxicological Methods xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Journal of Pharmacological and Toxicological Methods journal homepage: www.elsevier.com/locate/jpharmtox

Original article

Renal studies in safety pharmacology and toxicology: A survey conducted in the top 15 pharmaceutical companies Amanda Benjamin a,⁎, David J. Gallacher b, Andrea Greiter-Wilke c, Jean-Michel Guillon d, Cheiko Kasai e, David Ledieu f, Paul Levesque g, Katja Prelle h, Sian Ratcliffe i, Frederick Sannajust j, Jean-Pierre Valentin a,1 a

Safety Pharmacology Centre of Excellence, Drug Safety and Metabolism, AstraZeneca R&D Alderley Park, Macclesfield, Cheshire SK10 4TG, United Kingdom Center of Excellence for Cardiovascular Safety Research & Mechanistic Pharmacology Janssen, Pharmaceutical Companies of Johnson & Johnson, Beerse, Belgium Safety Pharmacology, Hoffmann-La Roche Inc., Basel, Switzerland d Sanofi R&D, Preclinical Safety, Safety Pharmacology, 13 quai Jules Guesde, 94400 Vitry sur Seine, France e Drug Safety Research Labs, Astellas Pharma Inc., 2-1-6, Kashima, Yodogawa-ku, Osaka 532-8514, Japan f Novartis Pharma AG, Preclinical Safety, Basel, Switzerland g Bristol Myers Squibb, Princeton, NJ 08534, USA h Safety Pharmacology, Bayer HealthCare, Wuppertal, Germany i Drug Safety Research and Development, Pfizer, Eastern Point Road, Groton, CT 0634, USA j Safety & Exploratory Pharmacology, Merck Research Laboratories, SALAR Division, 770 Sumneytown Pike, P.O. Box 4, West-Point, PA 19486-0004, USA b c

a r t i c l e

i n f o

Available online xxxx Keywords: Safety pharmacology ICH S7A Kidney DIKI biomarkers Strategy

a b s t r a c t Introduction: With the recent development of more sensitive biomarkers to assess kidney injury preclinically, a survey was designed i) to investigate what strategies are used to investigate renal toxicity in both ICH S7A compliant Safety Pharmacology (SP) studies after a single dose of a compound and within repeat-dose toxicity studies by large pharmaceutical companies today; ii) to understand whether renal SP studies have impact or utility in drug development and/or if it may be more appropriate to assess renal effects after multiple doses of compounds; iii) to ascertain how much mechanistic work is performed by the top 15 largest pharmaceutical companies (as determined by R&D revenue size); iv) to gain an insight into the impact of the validation of DIKI biomarkers and their introduction in the safety evaluation paradigm; and v) to understand the impact of renal/urinary safety study data on progression of projects. Methods: Two short anonymous surveys were submitted to SP leaders of the top 15 pharmaceutical companies, as defined by 2012 R&D portfolio size. Fourteen multiple choice questions were designed to explore the strategies used to investigate renal effects in both ICH S7A compliant SP studies and within toxicology studies. Results: A 67% and 60% response rate was obtained in the first and second surveys, respectively. Nine out of ten respondent companies conduct renal excretory measurements (eg. urine analysis) in toxicology studies whereas only five out of ten conduct specific renal SP studies; and all of those 5 also conduct the renal excretory measurements in toxicology studies. These companies measure and/or calculate a variety of parameters as part of these studies, and also on a case by case basis include regulatory qualified and non-qualified DIKI biomarkers. Finally, only one company has used renal/urinary functional data alone to stop a project, whereas the majority of respondents combine renal data with other target organ assessments to form an integrated decision-making set. Conclusion: These short surveys highlighted areas of similarity: a) urinary measurements are most commonly taken on repeat-dose toxicity studies, and b) renal SP studies are less often utilised. The two major differences are a) lack of consistent use of DIKI biomarkers in urinary safety studies and b) the way large pharmaceutical companies assess renal function. Finally, suggestions were made to improve the safety assessment methods for determining the safety of compounds with potential renal liability. © 2015 Elsevier Inc. All rights reserved.

Abbreviations: α-GST, alpha-glutathione S-transferase; B2M, beta-2 microglobulin; BUN, blood urea nitrogen; CROs, contract research organisations; Cr, creatinine; CysC, cystatin C; DIKI, drug-induced kidney-injury; EFPIA, European Federation of Pharmaceutical Industries and Associations; EMA, European medicines agency; ERBF, effective renal blood flow; FDA, food and drug administration; GFR, glomerular filtration rate; GLP, goodlaboratorypractice;ICH, International Conferenceon Harmonisation;IND,Investigational New Drugapplication; KIM-1, kidney injury molecule-1; NAG, N-acetyl Glucosaminidase; NCE, novel chemical entity; NGAL, neutrophils gelatinase-associated lipocalin; NHP, non-human primate; PAH, para-aminohippurate; PMDA, Pharmaceuticals and Medical Devices Agency; PSTC, Predictive Safety Testing Consortium; R&D, Research & Development; RPA-1, renal papillary antigen-1; SAFE-T, IMI Safer and Faster Evidence Based Translation; SCr, serum creatinine; SP, safety pharmacology; TFF-3, trefoil factor-3; uTP, urinary total protein. ⁎ Corresponding author. Tel.: +44 1625 515355. E-mail address: [email protected] (A. Benjamin). 1 Current address: Non-Clinical Development, UCB, Chemin du Foriest, Braine l'Alleud B-1420, Belgium.

http://dx.doi.org/10.1016/j.vascn.2015.01.004 1056-8719/© 2015 Elsevier Inc. All rights reserved.

Please cite this article as: Benjamin, A., et al., Renal studies in safety pharmacology and toxicology: A survey conducted in the top 15 pharmaceutical companies, Journal of Pharmacological and Toxicological Methods (2015), http://dx.doi.org/10.1016/j.vascn.2015.01.004

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A. Benjamin et al. / Journal of Pharmacological and Toxicological Methods xxx (2015) xxx–xxx

1. Introduction The kidney is a complex and crucial excretory organ that plays an important role in numerous regulatory processes that include fluid and electrolyte balance (ultrafiltration, reabsorption and secretion), control of blood pressure and volume, acid-base balance, removal of waste products and endocrine function (Stockham & Scott, 2008). The kidneys receive 25% of cardiac output and filter large volumes of plasma and are key contributor to drug disposition, metabolism and excretion (Choudhury & Ahmed, 2006). It is not surprising therefore that druginduced kidney injury (DIKI) is associated with significant discontinuation in pre-clinical and clinical drug development (Garrett & Workman, 1999; Kola & Landis, 2004; Lesco & Atkinson, 2001; Liano & Pascual, 1996; Mehta et al., 2004; Redfern et al., 2010). The mechanisms by which drugs produce acute and/or chronic kidney injury are poorly understood and currently histopathology is considered by many to be the ‘gold standard’ by which DIKI is established. The nonclinical safety study recommendations for the marketing approval of a pharmaceutical usually include safety pharmacology (SP) studies and repeat dose toxicity studies, amongst other study types, and these are covered by the International Conference on Harmonisation (ICH) S7A (Anon, 2001) and ICH M3(R2) (Anon, 2009) guidelines, respectively. Under ICH S7A, the assessment of renal function is considered as supplementary and therefore might not be performed by all sponsors. The ICH S7A guidance states “urinary volume, specific gravity, osmolarity, pH, fluid/electrolyte balance, proteins, cytology, and blood chemistry determinations such as blood urea nitrogen, creatinine and plasma proteins” (Anon, 2001) can be used to assess drug effect on renal function; such parameters are typically measured in a study consisting in a single dose administration of a test compound to conscious animals with the compound at levels up to the maximum tolerated dose. Under the ICH M3(R2) guidance, there is reference to routine inclusion of serum creatinine (SCr) and blood urea nitrogen (BUN) in clinical pathology/biochemistry panels in non-clinical safety studies supporting clinical trials (Anon, 2012) in particular the 28 days (‘one month’) pivotal rodent and non-rodent toxicology studies. Such parameters can be helpful to assess functional consequences of histopathological changes (or vice versa), and can be informative for clinical safety monitoring. Over recent years, seven novel urinary biomarkers have emerged and qualified for use in rat studies (Dieterle, Perentes, et al., 2010; Ferguson, Vaidya, & Bonventre, 2008; Ozer et al., 2010; Rouse et al., 2011; Sasaki et al., 2011; Yu, Jin, Holder, Ozer, & Villarreal, 2010). Their appearance/excretion in urine offers the promise of greater sensitivity over functional kidney biomarkers, and greater utility to detect early stages of drug-induced kidney stress, before histopathologicallydefined DIKI has occurred. Therefore, with the recent development of the tools to assess kidney injury preclinically this survey was designed i) to investigate what strategies are used to investigate renal toxicity in both ICH S7A compliant SP studies after a single dose of a compound and within repeat-dose toxicity studies by large pharmaceutical companies today; ii) to understand whether renal SP studies have impact or utility in drug development and/or if it may be more appropriate to assess renal effects after multiple doses of compounds; iii) to ascertain how much mechanistic work is performed by the top 15 largest pharmaceutical companies (as determined by R&D revenue size); iv) to get an appreciation of the impact of the validation of DIKI biomarkers and their introduction in the safety evaluation paradigm; and v) to understand the impact of renal/urinary safety study data on progression of projects. 2. Method Safety pharmacology leaders (e.g., heads of SP departments) within the top 15 pharmaceutical companies, as defined by R&D revenue figures in 2012 (Table 1, Pharmaprojects®, 2012 Citeline), were invited

Table 1 List of the top 15 pharmaceutical companies as defined by R&D portfolio size in 2012. Pharmaprojects®, 2012 Citeline. Company

Ranking

No. of R&D products 2012

No. of originated products

GlaxoSmithKline Pfizer Merck & Co Novartis Hoffmann-La Roche Sanofi Takeda Bristol-Myers Squibb AstraZeneca Johnson & Johnson Eli Lilly & Co. Astellas Abbott Laboratories Amgen Bayer

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

257 225 223 218 198 178 149 146 144 142 125 104 96 91 91

147 152 150 151 147 90 80 113 85 85 102 66 67 79 62

to participate in two short anonymous surveys designed to investigate what strategies are used to study renal effects of candidate drugs in both ICH S7A compliant SP studies and within repeat-dose toxicology studies. The surveys were created using Survey Monkey™. There were a total of 14 questions to answer across the two surveys, each of which was set out in a multiple choice format with the option to select multiple answers for some questions (i.e., questions 1–6, 8 and 10–12) furthermore some questions had a separate field for free text (i.e., questions 4, 6–8 and 12; Table 2). This enabled individuals to add clarity to any answer provided. Participants received the questions via e-mail with a link to the website and were asked to complete the surveys within one month of receiving the invitations in October 2013 and June 2014. Once the deadline for survey completion had been reached, the responses were collated and reviewed. The results were shared during a teleconference, with participants contributing to the discussion and interpretation. The participants unanimously agreed to release the results of the survey in the public domain.

3. Results Ten out of the 15 invited pharmaceutical companies participated in the first survey and eight participated in the second survey (67 and 53%, respectively). The first question explored the types of renal studies that organisations conduct as part of the submitted regulatory package (Fig. 1). There were ten respondents to this question. Five companies (50%) conduct renal SP studies including excretory functions, but one of the respondents commented that they only run renal SP studies that include renal excretory function measurement and renal hemodynamic parameters when the compound being tested is thought to have a renal liability, and then studies are only performed in larger species such as dog or non-human primate (NHP). All of the five respondents that conduct renal SP studies also investigate renal excretory measurements after repeated doses of a compound (urinary collection on repeat dose toxicology studies). Nine companies (90%) conduct renal excretory measurements in toxicity studies. Two companies (20%) responded that they do not conduct any renal or urinary supplemental SP studies or renal endpoints in toxicology studies as part of their regulatory packages, however of these two, one respondent also stated that they can perform renal SP studies and renal excretory measurements in toxicology studies, so it has to be inferred that although they do have the capability to run these study-types they do not include renal SP studies as part of their standard regulatory package. The second survey had eight respondents, who all stated that they conduct renal/urinary studies, thus inferring that the two respondents from the original survey who stated that they did not perform any

Please cite this article as: Benjamin, A., et al., Renal studies in safety pharmacology and toxicology: A survey conducted in the top 15 pharmaceutical companies, Journal of Pharmacological and Toxicological Methods (2015), http://dx.doi.org/10.1016/j.vascn.2015.01.004

A. Benjamin et al. / Journal of Pharmacological and Toxicological Methods xxx (2015) xxx–xxx Table 2 List of the questions and multiple choice answers contained within the present survey. Questions marked with * had the option to include multiple answers. Question

Multiple choice options

1. What types of renal studies does your organisation conduct as part of your regulatory packages (please select all that apply)? *

Renal safety pharmacology study including renal excretory functions Renal safety pharmacology study including renal hemodynamic functions Renal excretory measurements in toxicology studies Renal hemodynamic function in toxicology studies We do not conduct renal or urinary supplementary safety pharmacology studies (ICH S7A) or renal endpoints in toxicology studies as part of our regulatory packages. Other (please specify) At candidate selection stage Prior to first in man studies Prior to submission for launch

2. If you conduct renal/urinary safety pharmacology studies (ICH S7A compliant), when do you perform them? Please select all that apply. * 3. What are the reasons for conducting/not conducting renal safety pharmacology studies (in accordance with ICH S7A)? Please select all that apply. *

It adds value to the overall safety pharmacology package. It is expected by some Regulatory Authorities. It is useful in conjunction with other general toxicity data (e.g. kidney histopathology). Renal safety pharmacology studies complement the renal assessment in toxicology studies. The urine and plasma analysis on a renal safety pharmacology study is more comprehensive/informative than that on a repeat-dose toxicology study. We do not conduct these studies unless there is a specific cause for concern. We never conduct renal safety pharmacology studies. We measure renal function in the repeat-dose toxicology (ICH M3(R2)) compliant studies so do not need a separate renal safety pharmacology study. In-house, GLP 4. Where, and to what standard, are In-house, non-GLP your renal/urinary studies Outsourced, GLP performed? Please select all that Outsourced, non-GLP apply. * We do not perform renal/urinary studies. Other (please specify) 5. If you outsource renal/urinary studies Strategic decision (either ICH S7A or ICH M3(R2) study Unavailability of technical and/or scientific expertise in house types), please give the reason why. Lower cost at CROs Please select all that apply. * Good quality at CROs Rapid turnaround time at CROs 6. If you perform renal studies, in which Not applicable Rat species do you do the experiments? Dog Please select all that apply. * Monkey Other (please specify) Not applicable 7. Which general experimental Anaesthetised condition do you routinely use to Conscious (metabolism cage) perform renal/urinary studies? Other (please specify) Do not measure any urinary parameters 8. Which parameters do you measure Urine volume and/or calculate in renal/urinary studies? Please select all that apply. * Urine pH Urine osmolarity Water consumption Urine electrolytes (Na+, K+, Cl−) Plasma electrolytes (Na+, K+, Cl−) Urinary glucose Plasma glucose Urinary creatinine Plasma creatinine

3

Table 2 (continued) Question

Multiple choice options

Creatinine clearance Blood urea Anion gap Haemoglobin Microscopic analysis (crystals etc.) Direct measurement of glomerular filtration rate (e.g. inulin clearance) Renal blood flow (PAH) Renal blood flow (imaging) Other (please specify) 9. With respect to the determination of Creatinine clearance is calculated from a single plasma sample and 24 h urinary creatinine clearance in renal/urinary creatinine excretion (snapshot). studies please select from the Creatinine clearance is calculated from following options to show what multiple, time-matched, plasma and applies to your studies. urine samples over 24 h. Creatinine clearance is calculated from multiple, time-matched, plasma and urine samples over multiple days of dosing. We do not calculate creatinine clearance. We only use Serum Creatinine as an index of GFR. 10. If you measure any of the regulatory Do not measure DIKI biomarkers approved drug-induced kidney-injury Cystatin c (DIKI) biomarkers, which ones do you Total protein measure? Please select all that apply. * Albumin KIM-1 β2-microglobulin Clusterin TFF-3 Do not measure DIKI biomarkers 11. If you measure any other DIKI α1-microglobulin biomarkers that have not been Calbindin approved by the regulatory bodies, Osteopontin which ones do you measure? Please Lipocalin-2 select all that apply. * TIMP-1 VEGF GST-α Tamm–Horsfall Urinary Glycoprotein 12. What are the factors that determine Not applicable which DIKI biomarkers you measure? Availability of qualified assays for individual biomarkers for different Please select all that apply. * species Choice of individual assays available at test facility Case by case, depending on science and species Other (please explain) Renal/urinary function is assessed after 13. When assessing renal/urinary function, do you do this after single or a single dose (ICH S7A safety pharmacology study). repeat dosing of compound? Renal/urinary function is assessed after If you answered that renal/urinary repeat doses (ICH M3(R2) study). function is measured in both single Renal/urinary function is assessed after dose and repeat dose studies, please state the reasons why in the text box. both single dose and repeat doses, but different parameters are measured in each study type. Renal/urinary function is assessed after both single dose and repeat doses with the same parameters being measured in each study type. We do not assess renal/urinary function. Yes. 14. Have you used renal/urinary functional study results alone to make No, but we would if the data supported such a decision. early ‘Stop’ decisions in projects? No, the data collected is just informative. No, the renal/urinary data is combined with other target organ assessments to form a larger decision-making data set. No, we do not measure renal function.

Please cite this article as: Benjamin, A., et al., Renal studies in safety pharmacology and toxicology: A survey conducted in the top 15 pharmaceutical companies, Journal of Pharmacological and Toxicological Methods (2015), http://dx.doi.org/10.1016/j.vascn.2015.01.004

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A. Benjamin et al. / Journal of Pharmacological and Toxicological Methods xxx (2015) xxx–xxx

Fig. 1. The types of renal studies conducted as part of regulatory packages. Multiple answers were possible.

renal/urinary studies as part of their regulatory packages were the nonresponders to the questions in the second survey. The second question aimed to determine at which point ICH S7A compliant renal studies were performed by the surveyed companies and had 8 respondents. Three out of eight companies perform ICH S7A studies at candidate selection stage, six out of eight perform them prior to First in Man studies and two out of eight answered that they perform them prior to submission for launch. The reasoning behind the decision to conduct urinary SP studies was investigated in the third question (8 respondents; Fig. 2). Four companies stated that the renal SP studies complement the renal assessment in toxicology studies. Three answered that they measure renal function by urine collection only in repeat-dose toxicity studies and therefore did not perform standalone renal SP studies. Three companies only run renal SP studies if there is a specific cause for concern, but then the renal SP data is combined with data from repeat-dose toxicity studies to form an integrated risk assessment. Three companies commented that renal SP studies were useful in conjunction with other toxicology data such as kidney histopathology. One company responded that the urine and plasma analysis on a renal SP study is more comprehensive and informative than that on a repeat-dose toxicology study. One company answered that renal SP studies added value to the overall SP package. No respondents stated that renal SP studies were expected by the regulatory authorities or that they never conducted renal/urinary SP studies. In terms of where renal/urinary studies are performed and to what standard there was a similar split of outsourced and in-house studies performed, as determined by the answers to the fourth question, which had 10 respondents (Fig. 3). Six companies (60%) outsourced their renal studies to GLP compliant standards, five companies (50%) perform their renal studies to GLP compliant standards in-house, six companies (60%) did their studies in-house not to GLP compliant standards and two companies (20%) outsourced their studies but not to GLP standard. None of the companies questioned stated that they do not perform renal/urinary studies.

The reason for outsourcing renal/urinary studies was investigated in question 5 (8 respondents). Five respondents stated it was a strategic decision, four said it was due to unavailability of technical and/or scientific expertise in-house, two stated it was due to rapid turnaround time at contract research organisations (CROs), one respondent stated it was due to lower cost at the CRO and another respondent stated it was due to good quality at the CRO. The species that renal studies are performed in was determined by the responses to question six. The options were monkey (NHP), dog or rat. In most of the companies surveyed, multiple species were used for renal studies with seven (70%), nine (90%) and eight (80%) companies using NHP, dog and rat, respectively. One company performed renal function studies only in larger animals (monkey and dog). One company only investigated renal function in rats and the rest of the respondents used a combination of the three or all three. The following comments were also obtained: 1) In case of mechanistic studies, the rat is the preferred species and; 2) Renal endpoints in toxicity studies are taken in rat, dog and NHP. Question seven addressed the experimental methods used for renal experiments. All respondents (100%) used metabolic cages for the capture of urine samples and none of the companies investigated renal function in anaesthetised animals. One respondent stated that welltrained animals in slings or chair-restrained can be used for GFR measurements; as well as plasma clearance based methods such as iohexol for GFR and para-aminohippurate (PAH) for ERBF measurements. It was also commented by one respondent that in-house and CROs collected urine from dogs with the use of catheters rather than by metabolism cage. The wide variety of possible urinary and blood parameters that are measured and/or calculated in renal SP or toxicology studies was investigated in the eighth question which had ten respondents (Fig. 4). All respondents measure urine volume, urine pH and urinary Cr. Nine (90%) measure plasma electrolytes, SCr and urinary total protein (uTP), eight (80%) measure urine osmolarity, water consumption (it was assumed that this is measured over a set time), urine electrolytes and BUN.

Fig. 2. The reasons for organisations to conduct or not conduct renal/urinary safety pharmacology studies. Multiple answers were possible.

Please cite this article as: Benjamin, A., et al., Renal studies in safety pharmacology and toxicology: A survey conducted in the top 15 pharmaceutical companies, Journal of Pharmacological and Toxicological Methods (2015), http://dx.doi.org/10.1016/j.vascn.2015.01.004

A. Benjamin et al. / Journal of Pharmacological and Toxicological Methods xxx (2015) xxx–xxx

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Table 3 List of the regulatory approved DIKI-biomarkers that are measured in renal/urinary safety studies. #1 Do not measure reg. approved DIKI biomarkers Cystatin c Total protein Albumin KIM-1 β2-microglobulin Clusterin TFF3 Fig. 3. Where, and to what standard, renal/urinary studies are performed. Multiple answers were possible.

Seven (70%) measure urinary and plasma glucose, albumin and haemoglobin and calculate Cr clearance. Six (60%) perform microscopic analysis of the urine. Three (30%) calculated anion gap. Two directly measure GFR and ERBF (inulin and PAH clearance, respectively) and only one respondent investigated renal blood flow using imaging methodology. One respondent commented that the standard parameters measured in regulatory toxicity studies are: urine volume, urine pH, urine osmolarity, water consumption, plasma electrolytes, urinary and plasma glucose, urinary and SCr, uTP, albumin, blood urea, anion gap, haemoglobin and microscopic analysis. Another respondent commented that the inulin and PAH clearances could be done for specific questions during later stage of drug development. A respondent also commented that urine volume, urine pH, urine osmolarity, urine glucose, uTP, urine haemoglobin, urine albumin, microscopic analysis of urine, water consumption, plasma electrolytes, plasma glucose and SCr and BUN all are routine parameters measured in renal studies and that DIKI biomarkers (including urinary Cr) are only used when there is a renal liability, and the other parameters such as GFR, PAH/ERBF, and urinary electrolyte fractional excretion can also be utilised when required. Question nine delved into how Cr clearance is determined by the different companies and was answered by eight respondents. Four calculate Cr clearance from a single plasma sample and 24 h urinary Cr excretion (snapshot), two use SCr as an index of GFR, one company calculates Cr clearance from multiple, time-matched plasma samples over 24 h and the final respondent calculates Cr clearance also from multiple, time-matched plasma samples over multiple days of dosing. No respondents stated that they do not calculate Cr clearance.

#2

#3

#4

#5

#6

#7

#8

Total 0

√ √ √ √ √ √ √

√ √ √ √ √

√ √ √

√ √ √ √ √ √ √

√ √ √

√

√

√ √

√ √ √ √ √

4 7 7 7 4 3 2

Question ten determined which regulatory approved DIKI biomarkers, aside from the standard biomarkers such as BUN and Cr, are measured in renal SP and/or toxicity studies (Table 3) and question eleven determined which other, as yet non-regulatory approved, DIKI biomarkers are measured (Table 4). Both questions had eight respondents. Two respondents measured all 7 of the regulatory approved DIKI biomarkers, two measured CysC, uTP, albumin, KIM-1 and B2M, one measured uTP and albumin and the final respondent measured only KIM-1. In terms of the measurement of other DIKI biomarkers that are not yet approved by the regulatory bodies, as determined from the responses to question eleven, it was found that four respondents measure α1-microglobulin, osteopontin, alpha-glutathione-Stransferase (α-GST) and lipocalin-2, two of the respondents measure calbindin and one measures VEGF. None of the respondents measure Tamm–Horsfall Urinary Glycoprotein or TIMP-1. Two respondents do not measure any of the non-regulatory approved DIKI biomarkers. Several respondents commented on the measurement of urinary DIKI biomarkers; one respondent commented that various DIKI biomarkers were measured in dedicated mechanistic studies to investigate renal toxicity, another respondent commented that they also measure urinary NAG (N-acetyl Glucosaminidase) and a further respondent commented that DIKI biomarkers were not standardly measured and are only assessed if there was a renal liability. The factors determining which DIKI biomarkers are measured were addressed in question twelve. Six out of the 8 respondents answered it was on a case-by-case basis, depending on the science and species, four answered it depended on the availability of qualified assays for individual biomarkers for different species and one responded it was based on the choice of individual assays available at the test facility. No respondents answered that the DIKI biomarkers that were measured were chosen due to the limitation of the multiplex chip format. The number of doses of a test compound used prior to the assessment of renal/urinary function was addressed in the thirteenth question, which had eight responses (Fig. 5). Two respondents assess renal/urinary function after a single dose (ICH S7A SP study), two respondents assess renal function after repeat doses (ICH M3(R2) study), one respondent stated that renal/urinary function is assessed after both single and repeated

Table 4 List of the DIKI-biomarkers that are not approved by the regulatory authorities that are measured in renal/urinary safety studies. #1 #2 #3 #4 #5 #6 #7 #8 Total

Fig. 4. Parameters measured in renal/urinary studies. Multiple answers were possible.

Do not measure nonreg. DIKI biomarkers α1-microglobulin Calbindin Osteopontin Lipocalin-2 TIMP-1 VEGF GST-α Tamm–Horsfall urinary glycoprotein

√

√

√ √ √ √

√ √ √

√

√ √ √ √

√

√

√

√ √

√

2 √

4 2 4 4 0 1 4 0

Please cite this article as: Benjamin, A., et al., Renal studies in safety pharmacology and toxicology: A survey conducted in the top 15 pharmaceutical companies, Journal of Pharmacological and Toxicological Methods (2015), http://dx.doi.org/10.1016/j.vascn.2015.01.004

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Fig. 5. When renal/urinary function is assessed. Only a single answer was possible to this question.

doses of a compound, but different parameters are assessed in each study type and three respondents stated that renal/urinary function is assessed after both single and repeated doses of a compound, with the same parameters being assessed in each study type. The reasons why the renal/urinary function is assessed in both single and repeat-dose studies by the respondents were stated as follows: 1) Depending on the target, the mechanism of action and the types of compounds tested, generally, a 3–7 days repeat dosing is required to really assess a direct impact of the treatment on renal function, GFR etc.; 2) The choice of which study type to utilise was on a case-by-case basis for different projects and based on scientific question for the nature of potential findings (e.g., whether effect attenuates over time, or whether effect has a latent onset); 3) Both study types are run to evaluate time course of the biomarkers and distinguish between predictive/prodromal biomarkers vs. ‘monitoring’ biomarkers (plus eventually vs. ‘mechanistic’ biomarkers); and 4) Renal SP study types are only run when there is a renal liability, in line with ICH S7A recommendations and the urinary endpoints on repeat-dose toxicity studies are measured by dipstick. A further comment was made from a company who only perform urinary/renal functional measurements on repeat-dose toxicity studies stating that “Renal function is routinely assessed in repeat dose studies and that renal SP study types are only run in specific cases (with one renal SP study in the last 5 years)”. The final question (#14) aimed to determine the impact of renal/ urinary functional studies in terms of project progression and whether renal/urinary functional study results alone have been used to make early stop decisions in R&D projects (Fig. 6). Only one out of 10 respondents had used renal/urinary functional data alone to make an early “stop” decision in a project. Two respondents said they would use renal functional study data alone if the data supported such a decision. Six respondents said that the renal/urinary data is combined with other target organ assessments to form a larger decision-making set.

Fig. 6. Whether renal/urinary functional study results have been used alone to make early ‘Stop’ decisions in projects.

One respondent stated they do not measure renal function. No respondents collected the data just for information. 4. Discussion The aim of this survey was to evaluate the current strategies that are used to investigate renal toxicity in both ICH S7A compliant SP studies and repeat dose toxicity studies within the top 15 pharmaceutical companies. The survey was restricted to large pharmaceutical companies in order to determine and compare the strategies deployed by those companies of similar size since they account for a large proportion of all new medicines launched on a yearly basis and are thus significantly impacted by safety related drug attrition. Although out of scope of the current survey, it would be of value to expand the number of companies surveyed to include both medium and smaller size pharmaceutical companies, to determine if differences in approaches exist. Other similar surveys have been performed within the SP community (Ewart et al., 2012; Friedrichs, Patmore, & Bass, 2005; Lindgren et al., 2008), but this survey was, to the best of our knowledge, the first to specifically address renal/urinary studies within the context of SP and toxicology studies. The survey determined the details around the types of renal/ urinary studies performed by large pharmaceutical companies. It also assessed the current level of implementation of DIKI biomarkers within these study-types. Also, the impact of renal/urinary preclinical SP study data upon project progression was determined. In terms of the species and methods used to investigate renal function, rat, dog and NHP were used to similar degrees. Although one respondent investigated renal function solely in the rat and one other only used large animals, all the other respondents used a combination of rat, dog and NHP or all three species. All respondents investigated renal function in conscious animals using metabolism cages, with one respondent saying that due to lack of availability of metabolism cages at the CROs/in-house, that urethral catheters were sometimes used to look at renal function in dogs. Additionally it was commented that due to the more complex and labour intensive nature of GFR measurements and clearance experiments, well-trained animals in chair-restraints or slings can be used (Garner & Laks, 1976). It should be noted, however, that the use of catheters does not allow for timed urine collection for calculation of GFR or clearance measurements unless the bladder is first emptied and then there is a timed urine collection using the catheter. The percentage of outsourced studies highlighted in the current survey was slightly higher than that determined in a study by Lindgren et al. (2008), where it was found that companies outsourced approximately 50% of their respiratory, gastrointestinal and renal studies. In this survey 8/10 respondents answered that they outsourced renal studies with a 3:1 split of GLP compliant studies to those studies not designed to achieve GLP compliance. However, a high number of companies also perform their renal studies in-house with 5 performing studies to GLP compliant standards and 6 performing non-GLP compliant renal studies in-house. This may reflect a difference in outsourcing

Please cite this article as: Benjamin, A., et al., Renal studies in safety pharmacology and toxicology: A survey conducted in the top 15 pharmaceutical companies, Journal of Pharmacological and Toxicological Methods (2015), http://dx.doi.org/10.1016/j.vascn.2015.01.004

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strategies in large pharmaceutical vs. the entire bio-pharmaceutical sector and/or a change in the outsourcing strategies of the top 15 pharmaceutical companies in the last 5 years. Indeed, upon further questioning in the survey and during discussion with the respondents it became apparent that different companies have different strategies relating to where renal/urinary studies are conducted. Some outsource all Investigational New Drug applications (INDs)-enabling toxicity and SP studies, others keep their mechanistic investigatory studies in-house and they do not do these to GLPcompliance, whereas others assess kidney function in-house as part of their regulatory studies that are performed to GLP compliance. CROs are also utilised in times when the work is not feasible in-house due to tight timelines for candidate selection or if there is a scheduling conflict between projects in-house. Half of the companies outsourced studies due to unavailability of technical and/or scientific expertise in house. This is likely due to the fact that over the recent years there have been drivers within the pharmaceutical industry to reduce the operating costs in the drug discovery and development process. Consequently, one of the areas that has been affected as a result of this strategy is the ability of companies to run assays in-house as the skilled individuals who have the knowledge and experience of the relevant in vivo models are no longer available. There are multiple external providers available, but unfortunately CROs often have a much higher turnover of staff and thus the knowledge and experience base of key experts in operating these models inevitably becomes depleted, which is a concern for the future of renal safety assessment. In agreement with this, there were fewer respondents who stated that the decision to outsource renal studies is driven by CRO-specific drivers. The reasons given were the rapid turnaround time (two respondents), the lower cost (one respondent) or the good quality of the data provided by the CRO (one respondent). There was variability in the timing of when renal/urinary SP studies (ICH S7A compliant) are conducted by the companies surveyed. The majority perform these studies prior to first time in human studies, with a lower number (37.5%) investigating renal effects at the earlier candidate selection stage, when usually less is known about the safety liability of the NCEs in question, and a quarter of respondents performing renal SP studies much later in parallel to clinical development prior to submission for launch. It was apparent that not all companies include the same renal study types in their regulatory submissions. The majority perform renal excretory measurements in repeat-dose toxicology studies and renal SP single-dose studies are less often utilised. Only half of the respondents include renal SP studies as part of their regulatory package and then only do so if there is a specific cause for concern; stating that the data is useful in conjunction with other toxicology data such as histopathology, which remains to be considered by many to be the “gold standard” for assessing renal injury. Indeed, it was stated by several respondents that they do not have a standard approach for which studies to include as part of a regulatory package and that renal assessments are performed on a “case-by-case basis” and that “renal SP studies are only performed when a target or risk liability exists”. Thus, it is not a surprise to note that only one respondent thought that the renal stand-alone SP studies added value to the overall SP package. The most popular answer to the question determining the reasons for conducting or not conducting renal SP studies was that the renal SP studies complement the renal assessment in toxicology studies (50%), however, three out of eight respondents stated that they measure renal function in the repeat dose toxicology (ICH M3(R2) compliant) studies and therefore separate renal SP studies are not required. Indeed companies only perform renal SP studies when there is a specific cause for concern. The main reason for performing renal SP studies was that the data obtained is thought to be useful in conjunction with other general toxicity data such as histopathology. In agreement with this, only one respondent reported having discontinued a project using renal/urinary functional studies alone, but it was not established whether this was after a single dose of a compound or after chronic dosing.

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Twenty percent of respondents said that they would stop a project using solely renal/urinary functional data if the data supported such a decision, but the majority used the data from renal studies in combination with data from other target organs to form an integrated risk assessment. This could suggest that renal/urinary studies have little impact on project progression as standalone studies, or that evidence of renal dysfunction when not associated with histopathology findings or other correlates (e.g., genomics) is generally not a finding robust enough to take the decision of discontinuing a project. However, it could also be argued that renal findings are not usually the sole target organ findings. One respondent commented that it is unusual for the kidney to be the only target organ or for renal function to be the only organ function that changes, and that there are usually other drivers for potential decision making. For example, there could be hemodynamic changes or other organ toxicities in addition to nephrotoxicity. Therefore, these results suggest that most pharmaceutical companies embrace the ICH S7A guideline philosophy with respect to renal SP studies and only perform such studies when there is an identified or suspected cause for concern. However, it could also be postulated that the reason why the majority of the respondents measure renal function after repeat dosing of a compound relates to the fact that the kidneys have a high level of functional reserve; it is well-known that in chronic progressive renal failure as much as 80% of the functional renal mass can be lost before functional biomarker levels such as SCr and BUN rise (Sieber et al., 2009). Therefore, it is unlikely that changes in renal function would be seen after a single dose of a compound. Indeed Redfern et al. (2010) used published reviews and BioPrint® to perform an analysis of projects terminated in clinical development across all pharmaceutical companies between 1999–2009 and found that although 9% of projects were terminated due to renal toxicity, none of these toxicities were detected by the current, standard SP one-dose renal model, but rather they were detected by histopathology in the pre-clinical toxicity models or at later stages in development. Traditionally the primary purpose of general toxicology studies is to expose animals to sufficient dose levels of a compound over a sufficient length of time to observe pathological outcomes. Given the high functional renal reserve and the fact that histopathology is considered by many to be the gold standard for determination of DIKI, it makes sense to measure renal function after repeated dosing of a compound in assays that include histopathological endpoints. The majority of the respondents who perform both renal SP studies and urinary endpoints on repeat-dose toxicity studies stated that the same parameters are measured in both study types. However, one of the respondents stated that the measurements taken on a renal SP study are measured quantitatively and are much more comprehensive and the renal endpoints on repeat-dose toxicity studies are usually measured by dipstick analysis. Indeed, the actual endpoints measured in all renal/urinary functional tests varied across the different companies. SCr and BUN are small organic molecule by-products of muscle and nitrogen metabolism, respectively. Under steady-state conditions, increases in SCr and BUN, or decreases in their respective renal clearances, are indices of decreased GFR and therefore declining renal function. Thus, elevated SCr and/or BUN levels are the regulatory standard for pre-clinical and clinical diagnosis of nephrotoxicity. It is well-known that the use of SCr and BUN to determine nephrotoxicity is problematic since changes in these parameters happen after a substantial time delay or only after significant kidney injury (Ferguson et al., 2008). Additionally, it is not possible to determine the nature of the renal injury by the appearance of these markers as this could reflect pre- or post-renal injury. As a consequence of the high functional renal reserve SCr and BUN have been shown to be insensitive and inadequate markers to detect renal injury after acute nephrotoxic drug insult (Bonventre, Vaidya, Schmouder, Feig, & Dieterle, 2010). Regardless of this, the current regulatory guidance specifically requests routine inclusion of SCr and BUN in clinical pathology/biochemistry panels in non-clinical SP and toxicology studies supporting clinical trials (Anon, 2012). This is because they can be helpful to assess functional consequences of histopathological

Please cite this article as: Benjamin, A., et al., Renal studies in safety pharmacology and toxicology: A survey conducted in the top 15 pharmaceutical companies, Journal of Pharmacological and Toxicological Methods (2015), http://dx.doi.org/10.1016/j.vascn.2015.01.004

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changes. Therefore, it is interesting to note that the three parameters that were measured by all respondents were urine volume, urine pH and urinary Cr, and the endpoints that are required by the regulatory bodies. SCr and BUN, were only measured by 90% and 80% of respondents, respectively. It is perhaps somewhat surprising that only 20% of respondents directly measure the hemodynamic parameter GFR (and sometimes ERPF), and that the majority of respondents (70%) use Cr clearance as a surrogate marker of GFR. This is despite the fact that Cr clearance measurements are thought to overestimate the GFR in rodents. Eisner et al. (2010) recently showed that the estimation of GFR by this measure led to an overestimation of the true GFR in healthy male mice due to a high renal proximal tubular secretion of Cr that contributed to nearly 50% of the overall Cr clearance. Also, it is important to note that drugs can induce increases in SCr and BUN and decreases in GFR, for example by inducing changes in renal perfusion pressure, or by affecting the proximal tubular transport of Cr or the reabsorption of urea, without detectable DIKI, and thus may give false positive signals in studies of renal function. When further detail was sought around how the Cr clearance is determined by the different companies it became apparent that different approaches are utilised. Four respondents (50%) calculate Cr clearance from a single plasma sample and 24 h urinary Cr excretion (snapshot), two companies use SCr as an index of GFR, one company calculates Cr clearance from multiple time-matched plasma and urine samples over 24 h and one calculates Cr clearance from multiple time-matched plasma and urine samples over multiple days of dosing. No respondents stated that they do not calculate Cr clearance. The reasoning behind the different approaches and the impact of the measurement of Cr clearance was not further investigated in this survey, however, it is clear that the accurate measurement of Cr clearance is only utilised by the minority of companies (25%) and the majority (75%) use either snapshot Cr clearance measurements or SCr as indexes of GFR. Indeed, the fact that GFR is not directly measured by the majority of respondents could be due to the more complex and labour intensive nature of the studies to measure GFR, and indeed ERBF, preventing these measurements from being added onto regulatory toxicology studies. Historically the determination of GFR in rodents has been measured using two approaches: by determination of a tracer's (infused to maintain a constant plasma concentration) urinary excretion rate using timed urine collections (Gabel, Ranaei, & Kivlighn, 1996; Qi et al., 2004), or, by determination of a tracer's elimination kinetics from the plasma following a single bolus injection (Katayama et al., 2010; Qi et al., 2004). Both methods are very time consuming and cumbersome, and involve anaesthesia and multiple blood sampling and thus a high burden on the animal and they therefore are not used routinely in the characterisation of animal models nor in clinical practice. Although there are now integrated platforms available that can simultaneously measure renal hemodynamics (GFR and ERBF), excretory function (quantitative urine analysis including DIKI measurements) and blood chemistry all in the same animal (Chen et al., 2013); these systems are yet to be utilised routinely within the pharmaceutical industry. However, it was encouraging to note that one company has implemented direct GFR evaluation by transcutaneous optical detection of sinistrinFITC as part of the investigative toxicity studies that are done prior to the regulatory repeat-dose toxicity studies (see Schock-Kusch et al., 2011; Schreiber et al., 2012). Finally, only one company stated that it includes renal hemodynamic functional measurements as part of its regulatory package. Due to the limitations of the measurement of SCr, BUN and Cr clearance and their utility in determination of kidney injury, it was hoped that the recent regulatory qualification of the DIKI biomarkers B2M, clusterin, CysC, KIM-1, albumin, uTP and TFF3 (Dieterle, Sistare, et al., 2010) would have impacted the assessment of nephrotoxicity in drug discovery. However, it would appear that the regulatory approved DIKI biomarkers are yet to be fully utilised and their regulatory approval has not impacted the design of preclinical safety studies within the

pharmaceutical industry. Eight respondents measure regulatory approved DIKI biomarkers as part of their renal/urinary studies. When further detail around which DIKI biomarkers were measured was requested in the survey it was found that KIM-1, uTP and albumin were the only regulatory-approved DIKI biomarkers that were measured by all eight respondents The regulatory approved DIKI biomarkers TFF-3, CysC, B2M and clusterin were also measured by some respondents, but to a lesser degree. Other DIKI biomarkers that are, as yet, not approved by the regulatory bodies for use in pre-clinical settings, are also measured by some of the respondents. Osteopontin, α1 microglobulin, lipocalin-2 and α-GST are measured by four respondents, calbindin by two companies and VEGF by one. This suggests that these biomarkers add value to the determination of the renal liability of NCEs. The variability in which DIKI biomarkers were measured could be due to the availability of kits and assays for the measurement of these biomarkers. Indeed, the current availability of kits/assays for DIKI biomarkers in plasma and urine varies depending on the species in question (for a review see Fuchs & Hewitt, 2011). Rat DIKI biomarker kits are much more readily available with a variety of panels in different formats available from various providers which include KIM-1, albumin, osteopontin, lipocalin, RPA-1, α-GST, GSTYb1, neutrophil gelatinaseassociated lipocalin (NGAL), CysC, B2M, clusterin, glutathione S transferase μ, and calbindin. The availability of kits to measure canine DIKI biomarkers are limited, Myriad RBM® offer a Canine KidneyMAP® that measures KIM-1, albumin, clusterin and NGAL on a multi-analyte platform. The assessment of kidney injury in cynomolgus monkeys is limited by a lack of commercially available kits and thus assessment of DIKI in this species is usually performed using human multiplex immunoassay analysis (Guha et al., 2011). The measurement of DIKI biomarkers was the main area of discussion when the results were circulated and discussed by teleconference and it was confirmed that DIKI biomarkers are not used routinely within renal/urinary studies (after either single or repeated dosing of a compounds), but that they are used on a case-by-case basis when required and the selection of the biomarker(s) to measure was based on the location of injury along the nephron. The availability and cost of measuring DIKI biomarkers was a concern voiced by several participants and one participant was of the opinion that there is no additional benefit to measure DIKI biomarkers if histopathology was being used. This opinion perhaps reflects the current lack of understanding of the temporal relationship of the appearance of DIKI biomarkers and histopathological changes. Current experience with these urinary biomarkers is largely limited to well-controlled studies of drugs previously associated with DIKI (true positives) and untreated controls, and there is very little experience of true negatives, be those drugs that impact kidney function without DIKI and drugs that are known not to impact kidney function. Therefore, with the limited information that is currently available to assess the potential for false positive signals the policy to examine these biomarkers only after histologically-verified DIKI has been associated with an NCE, or DIKI is suspected based upon prior chemistry, pharmacology, or toxicology experience is understandable. However, the appearance of novel DIKI biomarkers, representing tissue injury response prior to overt cell death, has the potential for higher sensitivity and to prospectively identify DIKI (McIlroy, Wagener, & Lee, 2010). There was also a question from one attendee about the incorporation of DIKI biomarker measurement in the clinic; it was felt that the adoption of these endpoints is still growing, rather than being routine. Indeed, there are still significant challenges ahead to enable the qualification of translational safety biomarkers in the clinic. There are several groups that have been formed to address these challenges including the IMI Safer and Faster Evidence Based Translation (SAFE-T) consortium and the Predictive Safety Testing Consortium (PSTC, see Dieterle, Sistare, et al., 2010). The SAFE-T consortium is a unique partnership of 25 partners from the European Federation of Pharmaceutical Industries

Please cite this article as: Benjamin, A., et al., Renal studies in safety pharmacology and toxicology: A survey conducted in the top 15 pharmaceutical companies, Journal of Pharmacological and Toxicological Methods (2015), http://dx.doi.org/10.1016/j.vascn.2015.01.004

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and Associations (EFPIA), academics and small and medium enterprises, with a budget of 35.7 million Euros and a 6 year timescale. SAFE-T is working to address the current lack of sensitive and specific clinical tests to diagnose and monitor drug-induced injury to the kidney, liver and vascular systems in man (Matheis et al., 2011). Whilst providing data that can be used to help determine the strategies that are used within SP for the detection of kidney toxicity, the authors acknowledge several shortcomings of the survey. Firstly, it was not possible to differentiate which endpoints are measured in single dose SP type renal studies compared to urinary endpoints in repeat dose toxicity studies, or any of the specifics regarding urine collection; for example the time point the urine was collected or how long animals were in the metabolism cages. Secondly, the impact of the DIKI biomarker qualification on clinical study design within the pharmaceutical companies was not addressed and therefore we were unable to determine the current understanding of the translation of the DIKI biomarker from preclinical species to man. Based on the results of this survey some recommendations could be made to best assess the nephrotoxic potential of a NCE. First and foremost, the authors recommend that robust, quantitative renal functional measurements should be included in repeat-dose general toxicology studies, with additional biomarkers considered based on mechanistic risks, rather than in single-dose studies. In addition, if there is a nephrotoxicity hazard or risk identified, then further investigational studies should be performed. These studies could include a standalone SP renal study, as well as investigative, mechanistic or exploratory studies after single and repeat-doses of a compound. Either study type should include detailed assessments of renal function, such as assessment of renal reserve or hemodynamic function (Valentin & Hammond, 2006), snapshot calculations of Cr clearance should be avoided, and measurement of DIKI biomarkers. However, as general toxicology studies are run in a highly regulated environment, it can be difficult to add in exploratory or mechanistic biomarkers without creating exceptions to GLP compliance. The basic design of one-month rodent and nonrodent toxicology studies has been largely unchanged since the 1970s, although the pharmaceutical industry has been making progress with modernizing study designs and incorporating validated biomarkers and functional measures. As such, with greater experience in using renal markers, these endpoints could become more routinely incorporated in studies when needed. The second recommendation is based on the fact that DIKI biomarkers are not yet uniformly, comprehensively and consistently used by the companies surveyed mainly because of cost, uncertainty regarding their translatability and applicability to humans, and interpretation of current biomarker signals in a risk assessment context still requires concurrent kidney histopathology. The knowledge and understanding of DIKI biomarkers will only improve if companies build their experience and understanding of these potentially useful tools by measuring them in both their investigative renal studies and general toxicity studies. Therefore, if a NCE has a renal liability then DIKI biomarkers should be assessed in repeat dose studies, which also include histopathology endpoints (Redfern, Ewart, Lainee, Robinson, & Valentin, 2013). In conclusion, this survey has provided a snapshot in time of the current practices and approaches that are used to assess renal toxicity preclinically in large pharmaceutical companies. The majority of the respondents measure renal/urinary function in repeat dose toxicity studies and stated that renal SP studies (single dose) alone have little on project progression but rather are used in conjunction with the other SP studies as a larger decision making set when the project has a renal liability. Despite the regulatory qualification of seven DIKI biomarkers, these are yet to be used in earnest in preclinical drug discovery, with KIM-1, uTP and albumin being the only biomarkers that are used across the board. Further development and understanding of the translatability of DIKI biomarkers in the coming years will potentially impact on how the nephrotoxic potential of NCEs will be assessed by the pharmaceutical industry in the future.

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Please cite this article as: Benjamin, A., et al., Renal studies in safety pharmacology and toxicology: A survey conducted in the top 15 pharmaceutical companies, Journal of Pharmacological and Toxicological Methods (2015), http://dx.doi.org/10.1016/j.vascn.2015.01.004