Effect of Sub-Acute Intravenous Injection of Endothelin Receptor Agonist IRL-1620 in Mice
The present study was conducted to evaluate effect of sub-acute intravenous injection of ETB receptor agonist IRL-1620 in mice. Swiss albino mice (n = 24, weighing 20-25 g) were divided into two groups (12 animal each), control group (0 μg kg-1) and IRL-1620 treated group (1 μg/kg/min, i.v., for 30 days). Each group was further subdivided into two groups depending on sex of mice having 6 animals each. No significant change in animal body weight, hemogram, liver function test and renal function tests was observed on treatment with IRL-1620. Histological examination of liver was normal and Kidneys showed mild congestion. In conclusion, sub-acute dose (1 μg/kg/min, i.v.,) of endothelin receptor agonist ETB IRL-1620 did not cause toxic effect on liver and kidney.
Since, the first description of endothelins in 1988, it has become evident
that endothelins play important role in many physiologic functions. Endothelin
is synthesized in and produced by variety of normal cells, including endothelial
cells, vascular smooth muscle cells and variety of endothelial tissues (Penna
et al., 2006; Wesson, 2006). In mammals, endothelin
family comprise of three endogenous isoforms ET1, ET2 and ET3. All the three
are 21 amino acid residues peptides containing disulfide bridges (Yanagisawa
et al., 1999). These peptides mediate their actions through two receptors
types ETA and ETB (Sakurai et al.,
The ET axis is known to participate in growth and progression of variety of
tumors. IRL-1620 (M. wt. 1820.95) is a linear ET analog specific for ETB
receptor (Watakabe et al., 1992). The IRL-1620
stimulates ETB receptor causing dilation of tumor blood vessels.
The selective enhancement of tumor blood flow resulted in greater percentage
of drug reaching tumor. Than normal tissue (Rajeshkumar
et al., 2005, 2007; Takai
et al., 1992). Rai and Gulati (2003) reported
that IRL-1620 caused significant increase in blood flow to breast tumor tissue
than normal tissue in normal rats. There was significant increase in blood perfusion
to tumor tissue. The increase in blood perfusion was attenuated by BQ788 a specific
antagonist of ETB receptor suggesting an ETB receptor stimulation leads to vasodilation
in tumor tissues. Gulati and Rai (2004) reported that
ETB receptor stimulation with endothelin 1 increased tumor blood flow by 153%,
decrease in vascular resistance (147%) and tumor blood perfusion by 176% in
breast tumor rats. Increased delivery of cytotoxic agents to tumor will have
advantage of enhancing the benefits of cancer chemotherapy, while minimizing
systemic toxicity. It has been reported that endothelins have a short half-life
of 7-8 min. As an analog of ET-1, IRL-1620 may have a similar half-life (Rubin
and Levin, 1994) and this is of advantage for therapeutic uses.
No change in pharmaco-kinetic profile of chemotherapeutic agents were observed
when given along with IRL-1620 (Rai et al., 2005).
Such an approach may also lower the required dose and decrease the adverse effects.
Keeping the therapeutic importance of IRL-1620 in view, the present study was
planned to evaluate safety aspect of sub-acute dosing of ETB receptor
agonist IRL-1620 in mice.
MATERIALS AND METHODS
Swiss albino mice (n = 24, weighing 20-25 g) procured from Haffkine Institute, Mumbai, India in 2006 and were divided into two groups (12 animal each), control group and IRL-1620 treated group (1 μg/kg/min, i.v., for 30 days). Each group was further subdivided into two groups depending on sex of mice having 6 animals each. Mice were housed in polypropylene cages under hygienic conditions and acclimatized to the laboratory conditions for a period of seven days prior to initiation of dosing. Animals were kept in 12 h light and dark cycle and received food (Amrit Feeds Limited, Calcutta, India) and water ad libitum. The cages were placed randomly on the racks and their positions changed daily to avoid any bias or any influence due to the specific location of the cage. The groups were, control male mice group (CMM), IRL-1620 treated male mice group (IRL-1620-MM)), Control Female Mice group (CFM) and IRL-1620 treated female mice group (IRL-1620-FM). Animals were given freshly prepared intravenous injection of IRL-1620 for 30 days. Control group was injected with distilled water only. All the animals were observed for clinical signs and were monitored for mortality. The body weight were monitored weekly throughout the study.
Overnight fasted animals were sacrificed on 31st day, blood and tissues samples were collected. The Institutional animal ethics committee of Ramnarain Ruia College Matunga, Mumbai (India) had approved the study protocol.
The general behavioral symptoms and any local reactions were observed during
the treatment period. Mortality, if any, during the course of the experiment
Hemoglobin, Erythrocyte Sedimentation Rate (ESR), Red Blood Cell count (RBC),
Total Leukocyte Count (TLC) and platelet counts were recorded on automated cell
counter from Coulter.
Serum Glutamate Oxaloacetate Transferase (GOT), Glutamate Pyruvate Trans
aminase (GPT), alkaline phosphatase (ALP) activities, Blood Urea Nitrogen (BUN),
serum creatinine, total proteins, triglycerides, cholesterol and plasma sugar
levels were performed using diagnostic kits (Merck India Ltd., Mumbai, India)
on semi-auto analyzer STATFAX3300-1207(Ark Diagnostics, Mumbai, India).
Sodium, potassium and chloride were estimated on electrolyte analyzer (Roche Diagnostics, Mumbai, India).
Liver and kidneys were removed from the sacrificed animals and were preserved
in 10% buffered formalin for histological examination.
The results are expressed as Mean±SD. Student's t-test was used for
the statistical evaluation of data and p<0.01 was accepted as significant.
No significant change in behavior was noticed in the IRL-1620 treated mice
of either sex as compared to respective control groups.
No mortality was recorded in any group throughout the dosing period.
Increase in body weight was comparable in treated and control group animals.
There were no significant variations in the hemoglobin levels in both the
IRL-1620 treated groups as compared to respective control groups. In treated
groups of either sex, there were no significant variations in the Red Blood
Cell (RBC), Total Leucocyte Count (TLC) and platelet count as compared to respective
control groups. No significant variations in the ESR was observed in all IRL-1620
treated groups as compared to respective control group animals (Fig.
Clinical Biochemistry Parameters
Liver Function Tests
No significant change in serum GOT, GPT, ALP activities (Fig.
2) and bilirubin and total protein levels were observed in IRL-1620 treated
groups of either sex with respect to their respective control groups (Table
Kidney Function Tests
In treated mice groups of either sex, no significant change in BUN, creatinine
levels, Na, K and Cl levels were observed as compared to respective control
|| Effect of sub-acute dosing of IRL-1620 on hemogram in mice
||Effect of sub-acute dosing of IRL-1620 on serum GOT, GPT and
ALP activities (IU L-1) in mice
|| Effect of sub-acute dosing of IRL-1620 on biochemical parameters
|Data is presented as Mean±SD (n = 6 in each group)
No significant change in glucose, cholesterol and triglycerides levels were
observed in IRL-1620 treated groups as compared to respective control groups
In the microscopic evaluation of the histological sections of the liver
of IRL-1620 treated animals were found to be of normal morphology. Kidneys showed
mild vascular congestion.
Blood flow rate is a major factor influencing delivery and distribution of
drugs to tumors and their therapeutic outcome. Attempts has been made to influence
tumor blood flow rate using vasoactive agents like IRL-1620 (Rajeshkumar
et al., 2007) and formulation developed from it is under Phase I clinical
trial. As per our information and literature review this is the first publication
regarding sub acute toxicity of IRL-1620 and very limited information is available
This study was conducted to evaluate the safety aspect of sub-acute dosing
of IRL-1620 in mice. On intravenous injection of IRL-1620, no significant change
in the body weights of mice were observed as compared to respective control
group animals. This could be because of normal food and water intake through
out the dosing period which were not altered during the treatment time. These
results correlated well with recent reports. Rats treated with normal saline
and IRL-1620 showed comparable increase in body weights (Rajeshkumar
et al., 2007). IRL-1620 safety was further established it was not pruritic
and actually inhibited responses to histamine and ET-1, where as ET1 caused
prurtisis (Trentin et al., 2006).
Endothelin 1 and its receptor antagonists are reported to alter various hematological
parameters. Bosentan, endothelin receptor antagonist caused decrease in hemoglobin
levels in patients with severe heart failure (Packer
et al., 2005). Halim et al. (1993) reported
that on continuous infusion of endothelin 1 in rabbits caused decrease in platelet
counts. In contrast to published reports regarding ET1 and ET receptor antogonists,
in the present study IRL-1620 treatment did not cause significant change in
hematological parameters suggesting IRL-1620 being safer than ET1 and endothelin
Liver has intricate micro vascular system that allows homogeneous perfusion
throughout the organ. Endothelin induced intra hepatic disturbance may play
certain role in drug induced toxicity. Endothelin 1 was found to induce flow
redistribution to the deeper and hilar portions of liver (Masuda,
2006). There are conflicting reports of effect of endothelin on aminotransferases.
Cozzi et al. (2006) reported that endothelin
receptor antagonist caused increase in aminotransferases whereas, Halim
et al. (1993) reported that bolus injection of ET1 causes increase
in activity of aminotransferases in rabbits. Safety of IRL-1620 was further
established in our study where IRL-1620 (1 μg/kg/min, i.v.) did not cause
significant increase in aminotransferase activities in mice.
Endothelin also known to have pro-inflammatory and pro-fibrotic properties.
It is closely related with normal renal physiology and pathology (Neuhofer
and Pittrow, 2006).The ET-1 exerts a wide variety of biological effects,
including constriction of cortical and medullary vessels, mesangial cell contraction,
stimulation of extracellular matrix production and inhibition of sodium and
water reabsorption along the collecting duct. In addition, ET-1 is causally
linked to renal disorders characterized by increased renal vascular resistance,
including acute ischaemic renal failure, calcineurin inhibitor toxicity, hepatorenal
syndrome and others (Neuhofer and Pittrow, 2006; Kohan,
1997). In contrast, no significant alteration in BUN, serum creatinine,
sodium, potassium and chloride levels were observed on treatment with IRL-1620
(1 μg/kg/min, i.v.) suggesting normal renal functions. Our biochemical
observation correlated well with histological examination of liver and kidney
which showed normal morphology after the treatment with endothelin in mice of
In conclusion, sub-acute dose (1 μg/kg/min, i.v.) of endothelin receptor agonist ETB IRL-1620 did not cause toxic effect on liver and kidney.
Authors acknowledge S.P. Mandali's Therapeutic drug monitoring laboratory, Mumbai, India and The Animal Testing Unit, Ramnarain Ruia College, Mumbai, India for provide the necessary facilities for conducting the study. Authors are thankful to financial department of R and D Centre, Venus Remedies limited for the financial support.
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