This document discusses methods for assessing drug effects on renal and gastrointestinal systems in safety pharmacology studies. For renal function, in vivo mammalian models using rats and dogs are commonly used to assess glomerular function through clearance tests, tubular function through urine analysis, and hemodynamic function through blood flow measurements. In vitro and in silico models are also discussed. For gastrointestinal function, methods described include assessing gastric emptying and intestinal motility using in vitro tissue/organ baths and in vivo animal models, measuring gastric secretion in cell preparations and ligated rats, modeling nausea and emesis in ferrets and dogs, and measuring absorption in Caco-2 cell cultures and perfused intestinal segments of rats.

1. Safety pharmacology
Tier 2
Ragini Anil Dani
2nd semester
PJLCP

2. Tier 2 Safety P’cological Studies
• Also called as Supplemental
pharmacological studies.
• Used to access potential adverse
pharmacodynamic effect organ
system function not addressed by
core battery studies.
• It includes
 Renal/Urinary system
 Gastrointestinal System
 Other Organ system
Skeletal system
Immune and endocrine functions

3. Renal studies in safety pharmacology
• The kidney is a complex excretory organ
• Playing role in
 Fluid and electrolyte balance
 Control of blood pressure
 Removal of waste products
 Drug disposition
 Endocrine function

4. • 30-40% drugs invented can cause
renal injury
• Still the safety P’cological studies
related to renal system in preclinical
system is small
Renal studies in safety pharmacology

5. Methods to assess drug effects on Renal Function
• It should cover
 Excretory function
 Hemodynamic function
 Endocrine aspects
• In-vivo models are frequently used.

6. Models Employed
• In-Vivo Mammalian models
• In-Vivo Non-Mammalian models
• In-Vitro Models
• In-Silico Models
Glomerular Function
Tubular Function
Hemodynamic Function

7. In-Vivo Mammalian Models
• Rats, Dogs, Pigs are commonly used
• Conscious, Freely moving animals
• Analysis of urine and plasma are done.
• Urinary bladder catherterization and metabolism cage
are used

8. Metabolism cage

9. a. Glomerular Function
• Clearance measurement of endogenous and exogenous small molecules
(urea, creatinine, 2-MPT, inulin, cystatin C, iohexol, or iodixanol)
• analysis of plasma and urine samples
• The noninvasive clearance (NIC)-kidney device that when mounted on the
back of laboratory animals enables the transcutaneous measurement of the
elimination kinetics of the fluorescent renal marker FITC-sinistrin
• This allows the measurement of the clearance of FITC-sinistrin from the
plasma in real time without the need for any blood Sampling

11. b. Tubular Function
• allows the identification of the
functional status of particular
nephron segments
• includes visual assessment of urine
(color, clarity), volume, specificgravity
or osmolality, pH, quantitative or
semiquantitative protein, and glucose
content.
• Dipstick test strips assess other
parameters, such as ketones,
bilirubin, urobilinogen, hemoglobin,
etc

13. c. Hemodynamic Function
1.Direct renal blood flow measurment
• requires the placement of a flow probe around the renal
artery
• conducted in large animal species such as dogs,(mini-)pigs, and
nonhuman primates
• usually coupled with systemic blood pressure monitoring, using a pressure
catheter placed into an artery
• Another hemodynamic endpoint is the renal vascular resistance (RVR),
calculatedas the ratio between RBF and mean arterial pressure (MAP)
• RVR can be increased in case of renal dysfunction or in case of systemic hypertension

15. 2. Indirect renal blood flow measurement
• para-aminohippuric acid(PAH) clearance test
• all PAH passing through the kidneys appears in the urine
• PAH clearance is directly proportional to the rate of plasma
flow through the kidneys
• If the hematocrit is known, the total renal blood flow can
be easily calculated from the eRPF value

16. In Vivo Non-mammalian Models
• The zebrafish (Danio rerio) larva has gained increasing interest
over the last decade as an alternative to mammalian in vivo
models.
• The zebrafish kidney is genetically and morphologically close
to that of mammals
• measurements of FITC-inulin intensity in the caudal artery and
excreted FITC-inulin
• validated using gentamicin and high salt loading
• high-throughput screening, visual transparency, low cost, and
genetic manipulation

17. • The kidney is highly complex, composed of a filter unit and a tubular segment, together
containing over 20 different cell types.
• Nephrotoxicants cause injury by selectively injuring specific cell types or by nonselectively injuring
multiple cell types within the kidney, depending on their mechanism of action
• Assessment of the potential nephrotoxic effects of pharmaceutical compounds on different
components of the kidney therefore requires the use of different model systems.
• in vitro model that recapitulates the in vivo response of renal cells to nephrotoxicants requires
appropriate expression of the transporters and receptors that interact with the drug of interest.
• In addition to detecting expression of transporters at the transcript and protein level by
quantitative PCR and immunohistochemistry, the function of transporters and endocytic receptors
can be investigated by assessing the effect of fluorescent substrates and transport inhibitors
In Vitro Models

18. In Silico Models
• The SAPHIR project (a Systems Approach for Physiological
Integration of Renal, cardiac, and respiratory functions),
initiated in 2008
• targeting the short- and long-term regulation of blood
pressure, body fluids, and homeostasis of the major solutes
• for renal and urinary disorders, the predictivity was not very
high

19. A survey conducted in the top 15 pharmaceutical companies

20. Gastrointestinal Safety Pharmacology
• 2-3% discontinuation of drug project
• Related ADRs and AEs are not life threatening but
hampers quality of life for patients
• 18% of total ADRs
• Overuse of NSAIDs in US is a reason behind >1,00,000
hospitalizations and 17,000 deaths in a year (2003)
• Hence there is a need for improved and more extensive
GI Screening

22. Methods to Assess Drug Effects on Gastrointestinal
Function
1. Assessment of Gastric Emptying and Intestinal Motility
2. Assessment of Gastric Secretion
3. Models of Nausea and Emesis
4. Models of Gastrointestinal Absorption

23. Assessment of Gastric Emptying and Intestinal Motility
In Vitro Models
• subcellular, cellular, tissue, or whole organ to study the pharmacological effects
and mechanism of action of drugs on GIT
• Stomachs or isolated segments of GIT from small laboratory animals can be
suspended in organ baths to investigate the mechanisms underlying a novel drug
target and to validate physiological or pharmacological responses. Typically
muscular strips or whole segments from the GIT are suspended in organ baths
containing a suitable nutrient solution held at 37 C and gassed with carbogen
• study the enteric nerves is done by measuring changes in motility caused by
electrical field stimulation (EFS)

24. In Silico Organ Modeling
• Computational fluid dynamic (CFD) techniques (Ferrua and Singh 2010) predict the dynamic effects of
luminalcontents on GI motility, luminal content mixing, and propulsion
• This model showed a complex and highly three-dimensional flow profile during gastric contraction

25. In Vivo Models
• In rodents by administering meals containing markers such as phenol
red, barium sulfate (BaSO4), or charcoal, subsequent to the
administration of the test compound. The test meals can be used either
as an indicator of liquid (phenol red) or solid transport (charcoal,
BaSO4).
• For gastric emptying assessment, the stomach is weighed,after definite
time as its weight directly correlates with its content. Any difference in
the gastric weight between treatment groups indicates altered gastric
emptying.

26. • For intestinal motility assessment, the intestines are removed (usually
from duodenum to ileum) and the length of intestine filled with charcoal
or BaSO4 is measured and compared to the full gut length by visual
inspection.
• The percentage of intestinal length filled by the test meal is proportional
to the intestinal transit. Any difference between treatment groups
indicates an alteration in the intestinal motility (increase or decrease).
• With phenol red, the intestinal transit is evaluated by measuring the
spectral absorbance in specific subparts of the gut.

27. Assessment of Gastric Secretion
In Vitro Models
• performed on mucosal cell preparation from stomach mucosa obtained by enzyme
dispersion and separated by centrifugation to separate parietal cells (H+ secreting
cells)
• Measures the H+ concentration and pepsin secretion in the gastric effluent

28. In Vivo Models
• Rats fasted for 24 hrs prior to pyloric ligation.
• Randomly divided into 5 groups of 3 animals each.
Group I : Control vehicle
Group II : Standard drug (Omeprazole)
Group III : ‘x’ concentration of test drug
Group IV : ‘2x’ concentration of test drug
Group V : ‘ 4x’ concentration of test drug
• Drugs administered once for 2 days and 30 mins prior to ligation
• Rats anesthetized with ether.
• Pyloric ligation procedure done.
• Rats placed in separate cages and allowed to recover.
• 19 hrs after pyloric ligation, animals sacrificed by decapitation.
Pyloric ligation method

29. Models of Nausea and Emesis
In Vivo Models
• assessed in conscious animal models
• Ferrets and dogs are typically considered to be the best species for the evaluation of
emesis, dogs being usually more sensitive to emetic stimuli compared to humans
• Rats and mice are not able to vomit, but can display some behavioral reactions that
can represent nausea, such as gasping or pica
• Emesis evaluation in dogs, ferrets, or nonhuman primates usually consists of visual
recordings of retching and vomiting events, and possibly premonitory signs such as
licking, during a defined period following compound administration
• The number of events and the latency are the most frequent parameters reported.

30. Models of Gastrointestinal Absorption
In Vitro Models
• Cell culture-based permeability screening models are used for the rapid assessmentof
intestinal permeability of drug candidates
• The human monolayer of colon adenocarcinoma cells (Caco-2) model used to predict
the intestinal absorption of orally administered drugs or measurement of flux of a
marker molecule or active ion transport across epithelial cells.
• NSAID-induced mucosal damage has been demonstrated in gastric mucosa

31. In Vivo Models
• In situ absorption models have been used in the anesthetized rat, with intraluminal
administration of the test drug and collection of inflow and outflow of an isolated
segment of the gut
• In parallel, arterial blood is sampled and the disappearance rate of the substance is
evaluated

32. THANK YOU!