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pharmaceuticals
Article
Subchronic Toxicity Study of Oral Anthrafuran on Rabbits
Michael I. Treshchalin 1, * , Helen M. Treshalina 1 , Vasilisa A. Golibrodo 1 , Andrey E. Shchekotikhin 1,2
and Eleonora R. Pereverzeva 1
1 Gause Institute of New Antibiotics, 11 B. Pirogovskaya Street, 119021 Moscow, Russia;
treshalina@yandex.ru (H.M.T.); vasilisa2006@gmail.com (V.A.G.); shchekotikhin@mail.ru (A.E.S.);
pereverzeva-ella@yandex.ru (E.R.P.)
2 Mendeleyev University of Chemical Technology, 9 Miusskaya Square, 125047 Moscow, Russia
* Correspondence: funky@beatween.ru
Abstract: A new antitumor multi-target drug anthrafuran, with cellular targets such as topoisomerase
I/II and some protein kinases, was obtained in Gause Institute of New Antibiotics and was demonstrated
to have a reliable specific effect on different murine and human tumor models by oral administration. In
this study, we focused on the evaluation of subchronic toxicity of oral anthrafuran drug formulation (AF)
on Chinchilla rabbits. The absence of any changes in the condition or behavior of animals was shown for
oral anthrafuran. Changes with reversible and dose-dependent hepato- and nephrotoxicity at low doses,
as well as hemato- and gastrointestinal toxicity at high doses, were confirmed pathomorphologically.
The identified toxic properties are extremely valuable, since oral anthrafuran does not have the limiting
cardio- and myelotoxicity. Anthrafuran with 2 mg/kg/day or 6 mg/kg/day doses was administrated
orally over 15 days. Investigations include assessment of the body weight, hematological and serum
biochemical parameters and urinalysis, electrocardiography and pathomorphological evaluation of the
internal organs. Quantitative data were processed statistically with Student’s t-Test, p < 0.05. Revealed
Citation: Treshchalin, M.I.;
during the subchronic study were the favorable toxicological properties of oral anthrafuran as opposed
Treshalina, H.M.; Golibrodo, V.A.;
to clinical anthracyclines, oral idarubicin, or parenteral doxorubicin, which allows it to be considered
Shchekotikhin, A.E.; Pereverzeva, E.R.
Subchronic Toxicity Study of Oral
promising for further research.
Anthrafuran on Rabbits.
Pharmaceuticals 2021, 14, 900. Keywords: anthrafuran; oral administration; subchronic toxicity; rabbits
https://doi.org/10.3390/
ph14090900
Academic Editor: Joël Schlatter 1. Introduction
Heteroarenanthracenediones containing cyclic diamines in the side chain are known
Received: 17 August 2021 to have high antiproliferative activity [1–5]. Previously described multi-targeting (S)-3-[(3-
Accepted: 2 September 2021
amino-1-pyrrolidinyl)carbonyl]-4,11-dihydroxy-2-methylantra[2,3-b]furan-5,10-dione (here-
Published: 4 September 2021
inafter anthrafuran) is capable of simultaneously inhibiting topoisomerase 1 and 2, as well
as protein kinases, and induces the death of tumor cells of various histogenesis [6,7]. An
Publisher’s Note: MDPI stays neutral
investigation of the anthrafuran anticancer properties in vivo showed a high efficacy with a
with regard to jurisdictional claims in
wide dosage diapason against transplantable tumors by intraperitoneal administration [3].
published maps and institutional affil-
A study of the kinetics of dissolution of anthrafuran substance in bio-relevant media (for
iations.
example, in artificial gastric juice at 37 ◦ C) showed that anthrafuran refers to prepara-
tions with a low biopharmaceutical solubility (look for Biopharmaceutics Classification
System, BCS) [8]. To increase biopharmaceutical solubility, an oral dosage form based
on anthrafuran and liquid pharmaceutically acceptable carriers with a high release rate
Copyright: © 2021 by the authors. of the active ingredient are obtained [9]. Most anticancer drugs are applied parenterally,
Licensee MDPI, Basel, Switzerland.
as this route of administration provides fast and maximal bioavailability of the agents
This article is an open access article
and therefore, accurate dosing during the whole course of chemotherapy. Novel orally
distributed under the terms and
administrated agents have a well-delivered formulation to achieve adequate bioavailability.
conditions of the Creative Commons
High bioavailability makes it possible to achieve complete or partial remission with oral
Attribution (CC BY) license (https://
administration of certain alkylating agents, antitumor antibiotics, and other classes of
creativecommons.org/licenses/by/
4.0/).
anticancer drugs. In this regard, more and more oncologists have begun to give preference
Pharmaceuticals 2021, 14, 900. https://doi.org/10.3390/ph14090900 https://www.mdpi.com/journal/pharmaceuticals
Pharmaceuticals 2021, 14, x FOR PEER REVIEW 2 of 10
Pharmaceuticals 2021, 14, 900 2 of 10
other classes of anticancer drugs. In this regard, more and more oncologists have begun
to give preference to oral chemotherapy drugs, since this route of administration im-
to oral chemotherapy drugs, since this route of administration improves the quality of
proves the quality of life of patients and reduces their time in the clinic [10]. Thereby, the
life of patients and reduces their time in the clinic [10]. Thereby, the searches for novel
searches for novel orally bioavailable agents are real directions in anticancer drug devel-
orally bioavailable agents are real directions in anticancer drug development. Earlier, it
opment.
was Earlier,
revealed thatitmoderately
was revealed that moderately
subchronic toxicity subchronic toxicity
on the rodents (rats)on
bythe
therodents (rats)
oral route of
by the oral route of
administration [11,12].administration [11,12].
The aim
The aim ofof the
the present
present study
study was
was to
to investigate
investigate the
the toxicological
toxicological properties
properties of
of oral
oral
anthrafuran in rabbits.
anthrafuran in rabbits.
2. Results
2.1. Functional Indicators and 0bservations
Administration of anthrafuran formulation did not cause mortality in any of of the
the
treated groups.
groups. NoNosigns
signsofof dyspepsia, abnormal behavioral reactions or neurological
dyspepsia, abnormal behavioral reactions or neurological dis-
disorders
orders werewere observed.
observed.
2.2. Dynamics of
2.2. Dynamics of Body
Body Weight
Weight
The
The dynamics
dynamicsof ofthe
thebody
bodyweight
weightchanges
changesareare
presented
presentedin Figure 1. By
in Figure 1. day 7 after
By day the
7 after
treatment (22th experiment day), all male rabbits who obtained anthrafuran were
the treatment (22th experiment day), all male rabbits who obtained anthrafuran were los- losing
weight. By day
ing weight. By 15 post-treatment
day (30th experiment
15 post-treatment day), the
(30th experiment body
day), theweight in both in
body weight treated
both
groups did not differ from the control.
treated groups did not differ from the control.
4
3.5
*
3
2.5
Body weight, kg
2
Days after
administration
1.5
7 15
1
0.5
0 5 10 15 22 30
Days
Control AF Σ MTD AF Σ LD50
Figure
Note: * 1. Dynamic
Values of rabbit’s body
are significantly weight
different during
to the and
control, p ≤after
0.05,oral administration of anthrafuran
n = 6.
formulation (AF). Note: * Values are significantly different to the control, p ≤ 0.05, n = 6.
Figure 1. Dynamic of rabbit’s body weight during and after oral administration of anthrafuran for-
2.3. Hematological
mulation (AF). Tests
Peripheral blood tests did not reveal any changes in the quantity of the total and
2.3. Hematological
differential Tests
leukocyte count, erythrocytes and thrombocytes count, the value of hemoglobin,
or hematocrit.
Peripheral blood tests did not reveal any changes in the quantity of the total and
Biochemical
differential serum
leukocyte testserythrocytes
count, showed an increase in the alanine
and thrombocytes aminotransferase
count, (ALT)
the value of hemoglo-
and aspartate aminotransferase
bin, or hematocrit. (AST) levels in all groups immediately after the end of
drug administration course (Figure 2). By the end of the experiment, this parameter was
similar to control.
Biochemical serum tests showed an increase in the alanine aminotransferase (ALT)
Pharmaceuticals 2021, 14, 900
and aspartate aminotransferase (AST) levels in all groups immediately after the end3 of
of 10
drug administration course (Figure 2). By the end of the experiment, this parameter was
similar to control.
180
160
* * *
140
120 *
100
u/L 80
60
40
20
0
1 15 1 15
Days
Daysafter
postadministration
treatment Daysafter
Days postadministration
treatment
ALT AST
Control AF Σ MTD AF Σ LD50
Figure
Note: 2. Serum
* Values alanine different
significantly aminotransferase (ALT)
from control, p ≤and aspartate
0.05, n = 6. aminotransferase (AST) levels in
rabbits after the end of oral anthrafuran formulation (AF) administration course. Note: * Values
significantly
Figure 2. Serumdifferent from control, p ≤ (ALT)
alanine aminotransferase 0.05, nand
= 6.aspartate aminotransferase (AST) levels in rab-
bits after the end of oral anthrafuran formulation (AF) administration course.
2.4. Heart Functions
OnFunctions
2.4. Heart day 1 as well as on day 15 post treatment, the electrocardiographic (ECG) parame-
tersOn
in day
both1 groups
as well were
as on similar
day 15 to control.
post treatment, the electrocardiographic (ECG) param-
eters
2.5.inInternal
both groups
Organswere similar to control.
The data
2.5. Internal on the drug influence on WI are presented in Table 1. In the group which
Organs
obtained a high dose of anthrafuran, by day 1 the thymus weight was decreased and liver
The data
weight on the drug
was increased as influence
comparedon to WI are presented
control. By day 15,inWITable 1. In
of the the group
internal which
organs in all
obtained a high dose of anthrafuran, by day 1 the thymus
groups which obtained anthrafuran were similar to control. weight was decreased and liver
weight was increased as compared to control. By day 15, WI of the internal organs in all
groups
Table which obtained
1. Weight anthrafuran
indices of were
internal organs of similar to control.
rabbits treated with anthrafuran orally.
Table 1. Weight indicesThymus
Groups of internal organs of rabbits treated
Heart with anthrafuran
Spleen orally.
Kidney Liver
Groups Thymus Day 1 post treatment
Heart Spleen Kidney Liver
AF, Σ MTD 0.16 ± 0.08 0.33 ±
Day 0.06 ± 0.02
0.09 treatment
1 post 0.34 ± 0.09 3.90 ± 0.77
AF,
AF,Σ ΣMTD
LD50 0.16
* 0.14± ±
0.08
0.03 0.33
0.24±±0.09
0.01 0.06
0.05± ±
0.02
0.01 0.34
0.26± ±
0.09
0.03 3.90 ± 0.77
* 7.53 ± 1.20
AF,Control
Σ LD50 * 0.14 ± 0.03
0.21 ± 0.02 0.24 ± 0.01
0.24 ± 0.01 0.05 ± 0.01
0.07 ± 0.02 0.26 ± 0.03
0.28 ± 0.02 * 7.53
3.67 ±1.20
± 1.34
Control 0.21 ± 0.02 0.24Day
± 0.01 0.07 ± 0.02
15 post treatment 0.28 ± 0.02 3.67 ± 1.34
Day 15 post treatment
AF, Σ MTD 0.16 ± 0.04 0.23 ± 0.03 0.05 ± 0.01 0.26 ± 0.02 2.47 ± 0.47
AF, Σ MTD 0.16 ± 0.04 0.23 ± 0.03 0.05 ± 0.01 0.26 ± 0.02 2.47 ± 0.47
AF, Σ LD 0.16 ± 0.09 0.24 ± 0.01 0.04 ± 0.02 0.25 ± 0.02 2.61 ± 0.17
AF, Σ LD5050 0.16 ± 0.09 0.24 ± 0.01 0.04 ± 0.02 0.25 ± 0.02 2.61 ± 0.17
Control 0.15 ± 0.05 0.26 ± 0.05 0.06 ± 0.01 0.24 ± 0.01 2.44 ± 0.22
Control 0.15 ± 0.05 0.26 ± 0.05 0.06 ± 0.01 0.24 ± 0.01 2.44 ± 0.22
Note: * Values significantly different from control, p ≤ 0.05. In the each group n = 6.
Note: * Values significantly different from control, p ≤ 0.05. In the each group n = 6.
2.6. Urine Analysis
2.6. Urine Analysis
The clinical urine analysis on day 1 post-treatment showed an increase of urobilinogen,
The clinical urine analysis on day 1 post-treatment showed an increase of urobilino-
ketones, and protein and a decrease of the specific gravity of urine in both tested groups
gen, ketones, and protein and a decrease of the specific gravity of urine in both tested
(Table 2). By the end of the observation, the results of urine tests of the experimental
animals did not differ from the control.
Pharmaceuticals 2021, 14, 900 4 of 10
Table 2. Clinical urine analysis of rabbits treated with anthrafuran orally.
Urobilinogen Ketones Protein Specific
Groups pH
(µmol/L) (mmol/L) (g/L) Weight
Day 1 post treatment
AF, Σ MTD * 17.0 * 0–0.5 6.6 *5 * 1.00
AF, Σ LD50 * 17.0 * 0–0.5 6.5 *5 * 1.00
Control negative negative 6.6 ≤0.33 1.02
Day 15 post treatment
AF, Σ MTD negative negative 6.3 ± 0.3 0.53 ± 0.4 1.020 ± 0.003
AF, Σ LD50 negative negative 6.5 ± 0.5 0.53 ± 0.4 1.010 ± 0.01
Control negative negative 6.5 ≤0.33 1.015 ± 0.005
Note: * Values significantly different from control, p ≤ 0.05, n = 6.
2.7. Histological Evaluation
The results of the pathomorphological study of the inner organs after drug administration
course with anthrafuran formulation are presented in Table 3 and in Figures 3–6.
Table 3. Pathomorphological findings after administration of anthrafuran formulation (AF) on days 1 and 15 post
treatment course.
Day 1st Day 15th
Organ Anthrafuran of Anthrafuran of Anthrafuran of Anthrafuran of
2 mg/kg/day 6 mg/kg/day 2 mg/kg/day 6 mg/kg/day
Vacuolar dystrophy of
hepatocytes around of
Vacuolar dystrophy of some central veins.
hepatocytes around of the Moderate edema around
Total pronounced vacuolar
central veins (Figure 3B). the triads.
dystrophy of hepatocytes
Liver Single small foci of micro Rare small micro necrotic
(Figure 3C). Micronecrotic
(Control: necrosis in the vicinity of Similar to control foci in the vicinity of the
foci of different sizes in the
Figure 3A) triads. Moderate triads. Hyperplasia of
vicinity of triads and
lympho-histiocytic hepatobiliary ducts and
central veins
infiltrates around the enlargement of connective
hepatobiliary ducts tissue around them,
sometimes fibrosis around
the triads (Figure 3D).
Fibrosis of some necrotic
nephrons passing through
cortical, juxtamedullary
Cortical zone: single foci of Cortical and medullary and medullary zones
vacuolar dystrophy and zones: severe perivascular (Figure 5B).
destruction of the edema. Cysts lined with squamous
convoluted tubules Cortical, juxtamedullary epithelium at the site of
Kidney
(Figure 4C). and medullary zones: destructed convoluted and
(Control: Similar to control
Medullary zone: cellular multiple small necrotic or medullary tubules
Figure 4A,B)
detritus and rare hyaline destructed foci in the (Figure 5C).
cylinders in the lumen of epithelial layer of the Thickening of the
some medullary tubules convoluted and medullary glomerular capsule and
(Figure 4D) tubules (Figure 5A) focal atrophy of the
glomerular capillary
network in the glomeruli
adjacent to the fibrosis area.
Pharmaceuticals 2021, 14, 900 5 of 10
Table 3. Cont.
Day 1st Day 15th
Organ Anthrafuran of Anthrafuran of Anthrafuran of Anthrafuran of
2 mg/kg/day 6 mg/kg/day 2 mg/kg/day 6 mg/kg/day
Pharmaceuticals 2021, 14, x FOR PEER REVIEW 5 of 10
Multiple extensive foci of
Myocardium Single small foci of toxic Two animals exhibited interstitial edema
(Control: cardiomyopathy multiple small foci of toxic Similar to control associated with toxic
Figure 6A) (Figure 6B) cardiomyopathy Moderate atrophy of lym- cardiomyopathy
Large germinal centers in indi-
(Figure 6C).
Spleen Similar to control Similar to control phoid tissue of some folli-
vidual follicles.
Moderate
cles atrophy Large germinal centers in
Spleen ModerateSimilar to control
atrophy of lym- Similar to control
Moderate atrophy of lym- of lymphoid tissue Moderate atrophy of lymphoid
individual follicles.
Thymus phoid tissue in the medullary phoid tissue in the medullary of some
Similar follicles
to control tissue in the medullary zone of
Moderate
zone of atrophy of
some lobules zone of lobules lobules.
Moderate atrophy of Moderate atrophy of
Lym- lymphoid tissue in the Moderate atrophy of lym-
Thymus lymphoid tissue in the Similar to control lymphoid tissue in the
phatic medullary
Similar zone of somephoid tissue of some lym-
to control medullary zone of lobules Similar to control Similar
medullary to control.
zone of lobules.
lobules
node phoid follicles
Moderate atrophy of
Lymphatic node Similar to control lymphoid tissue of some Similar to control Similar to control.
No pathological changes were observed in other organs.
lymphoid follicles
А В
С D
Figure
Figure 3. (A–D).
3. (A–D). Rabbit
Rabbit liver.
liver. (A) (A) Untreated
Untreated control.
control. (B) (B) AF,×15
AF, 15 × 2 mg/kg/day,
2 mg/kg/day, 1 day
1 day post
post treatment.
treatment. Vacuolar
Vacuolar dystrophy
dystrophy
of hepatocytes
of hepatocytes around
around of the
of the central
central vein. (C) (C)
vein. AF,×15
AF, 15 × 6 mg/kg/day,
6 mg/kg/day, 1 day
1 day post
post treatment.
treatment. Total
Total pronounced
pronounced vacuolar
vacuolar
dystrophy of hepatocytes. (D) AF, 15 × 6 mg/kg/day, 15 day post treatment. Edema around the triad. Small
dystrophy of hepatocytes. (D) AF, 15 × 6 mg/kg/day, 15 day post treatment. Edema around the triad. Small micronecrotic micronecrotic
focus in the vicinity of the triad (1). Hyperplasia of hepatobiliary ducts and enlargement of connective tissue around them.
focus in the vicinity of the triad (1). Hyperplasia of hepatobiliary ducts and enlargement of connective tissue around them.
(2) ×20.
(2) ×20.
Pharmaceuticals 2021, 14, 900 6 of 10
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Pharmaceuticals 2021, 14, x FOR PEER REVIEW 6 of 10
А В
А В
С D
С D
Figure 4. (A–D).
Figure Rabbit
4. (A–D). kidney.
Rabbit (A)(A)
kidney. Untreated
Untreatedcontrol. Cortical
control. zone.
Cortical zone.(B)(B)Untreated
Untreatedcontrol.
control. Medullary
Medullary zone. (C)AF,
zone. (C) AF,15 ×
Figure 4. (A–D). Rabbit kidney. (A) Untreated control. Cortical zone. (B) Untreated control. Medullary zone. (C) AF, 15 ×
15 ×2 mg/kg/day,
2 mg/kg/day, 1 day1 post
day treatment.
post treatment. Foci of destruction
Foci of destruction of the convoluted
of the convoluted tubules
tubules in renal in renal
cortex. cortex.
(D) AF, 15 × 2 (D) AF,
mg/kg/day,
2 mg/kg/day, 1 day post treatment. Foci of destruction of the convoluted tubules in renal cortex. (D) AF, 15 × 2 mg/kg/day,
1 day
15 ×12day post treatment.
mg/kg/day, Cellular
1 day Cellular detritus
post treatment. (1) and hyaline cylinders (2) in the lumen of some medullary tubules. ×20.
post treatment. detritus Cellular detrituscylinders
(1) and hyaline (1) and hyaline
(2) in thecylinders (2)some
lumen of in themedullary
lumen of tubules.
some medullary
×20.
tubules. ×20.
А
А
BB C
C
Figure
Figure 5. (A–C).
5. (A–C). Rabbit
Rabbit kidney.
kidney. (A)15
(A) AF, AF,× 15 × 6 mg/kg/day,
6 mg/kg/day, 1 day
1 day postpost treatment.
treatment. Cortical
Cortical zone.zone.
FociFoci of vacuolar
of vacuolar dystrophy
dystrophy
Figure 5. (A–C). Rabbit kidney. (A) AF, 15 × 6 mg/kg/day, 1 day post treatment. Cortical zone. Foci of vacuolar dystrophy
(1) and destruction in the epithelial layer (2) of the convoluted tubules. (B) AF, 15 × 6 mg/kg/day, 15 day post treatment.
(1) destruction
(1) and and destruction
in theinepithelial
the epithelial
layerlayer
(2) of(2)
theofconvoluted
the convoluted tubules.
tubules. (B)15AF,
(B) AF, × 615mg/kg/day,
× 6 mg/kg/day, 15 day
15 day postpost treatment.
treatment.
Fibrosis of some necrotic nephrons passing through cortical and juxtamedullary zones. (C) AF, 15 × 6 mg/kg/day, 15 days
Fibrosis of some necrotic nephrons passing through cortical and juxtamedullary zones. (C) AF,
Fibrosis of some necrotic nephrons passing through cortical and juxtamedullary zones. (C) AF, 15 × 6 mg/kg/day, 15 × 6 mg/kg/day, 15 days
post treatment. Juxtamedullary zone. Cysts lined with squamous epithelium at the site of destructed convoluted and me-
post
15 days treatment.
posttubules. Juxtamedullary
treatment. zone. Cysts lined with squamous epithelium at the site of destructed convoluted and me-
dullary ×20.Juxtamedullary zone. Cysts lined with squamous epithelium at the site of destructed convoluted
dullary tubules. ×20.
and medullary tubules. ×20.
Pharmaceuticals 2021, 14, 900 7 of 10
Pharmaceuticals 2021, 14, x FOR PEER REVIEW 7 of 10
А B C
6. (A–C).
FigureFigure RabbitRabbit
6. (A–C). myocardium. (A) Untreated
myocardium. control.control.
(A) Untreated (B) AF,(B) × 2 mg/kg/day,
15 AF, 1 day 1post
15 × 2 mg/kg/day, daytreatment. Foci ofFoci
post treatment. toxic
of toxic
cardiomyopathy. AF, 15 ×
cardiomyopathy. (C) AF, 15 × 6 mg/kg/day, 15 day post treatment. Extensive foci of interstitial edema associatedwith
(C) 6 mg/kg/day, 15 day post treatment. Extensive foci of interstitial edema associated with toxic
cardiomyopathy.××20.
toxic cardiomyopathy. 20.
No pathological changes were observed in other organs.
3. Discussion
Anthracyclines and structurally similar anthracenediones, which are highly effective
3. Discussion
Anthracyclinesagents
antineoplastic widely used
and structurally for the
similar treatment of malignant
anthracenediones, which are tumors.
highlyAnthrafuran,
effective a
heterocyclic derivative of anthracenedione, demonstrated
antineoplastic agents widely used for the treatment of malignant tumors. Anthrafuran, a a reliable antitumor effect on
different
heterocyclic murine tumor
derivative models for oraldemonstrated
of anthracenedione, administrationa [7]. The antitumor
reliable design of the subchronic
effect on
toxicity
different murinestudy on rabbits—15
tumor models fordaily oral oral administrations
administration [7]. Theat two doseoflevels
design of 2 mg/kg/day
the subchronic
(1/15
toxicity MTD)
study and 6 mg/kg/day
on rabbits—15 daily oral (1/15 LD50)—followed
administrations at twoby dose
a 15-day
levelsobservation
of 2 mg/kg/day period, al-
lowed us to identify all of the main toxic effects of anthrafuran.
(1/15 MTD) and 6 mg/kg/day (1/15 LD50 )—followed by a 15-day observation period, The study showed that AF
has a more favorable toxicological profile compared to doxorubicin
allowed us to identify all of the main toxic effects of anthrafuran. The study showed that and other anthracy-
AF hasclines.
a moreSigns of doxorubicin
favorable toxicologicaltoxicity observed
profile compared in theto chronic
doxorubicintoxicity
andstudy,
other such
anthra-as a re-
duction
cyclines. Signs of ofbody weight, cardiotoxicity,
doxorubicin toxicity observed depression of hematopoiesis
in the chronic toxicity study,and spermatogene-
such as a
sis, gastrointestinal
reduction of body weight, tract (GIT), and
cardiotoxicity, renal damage,
depression are well known
of hematopoiesis and have been de-
and spermatogenesis,
scribed by tract
gastrointestinal many(GIT),
authors
and[13–15].
renal damage, are well known and have been described by
many authorsAF, independent
[13–15]. of the dose, when administered daily, did not affect the consump-
tionindependent
AF, of food and of water and did
the dose, when notadministered
cause the death daily,ofdid
animals.
not affectAFtheinduced a short-term
consumption
of food and water
reduction andbody
of the did not causewhich
weight, the deathcouldof animals. AF induced
be interpreted a short-term
as indirect evidence reduc-
of gastro-
tion of the bodytoxicity.
intestinal weight, Nowhich could
other signs beof
interpreted as indirect
gastrointestinal evidence
toxicity were of gastrointestinal
detected either by clin-
toxicity. Nomorphological
ical or other signs ofmethods.
gastrointestinal toxicity were detected either by clinical or
morphological methods.
The major dose-limiting toxicities of doxorubicin, as well as daunorubicin, epirubi-
The
cin, major dose-limiting
and idarubicin, toxicities of doxorubicin,
are neutropenia and cardiomyopathyas well as[15].
daunorubicin, epirubicin,
Most researchers note that
and idarubicin, are neutropenia and cardiomyopathy [15]. Most
the hematological toxicity of anthracyclines is equivalent to their cardiotoxicity researchers note that theWhen
[16].
hematological toxicity ofhematological
using anthrafuran, anthracyclinesparameters
is equivalent to their within
remained cardiotoxicity [16]. Whennorm
the physiological
usingthroughout
anthrafuran, thehematological
study. A decrease parameters
in WI ofremained
thymus inwithin animals thetreated
physiological
with a high norm dose of
throughout the study. A decrease in WI of thymus in animals treated
anthrafuran may indicate a possible manifestation of hematological toxic properties dur- with a high dose
of anthrafuran
ing overdose. mayThe indicate a possible manifestation
pathomorphological of hematological
study confirmed toxic properties
that administration of AF at the
during overdose. The pathomorphological study confirmed
dosage level of 6 mg/kg/day causes moderate atrophy of lymphoid tissue of the that administration of AF
thymus,
at thespleen,
dosageand level lymph nodes. The structure of the spleen and the lymph nodes is the
of 6 mg/kg/day causes moderate atrophy of lymphoid tissue of fully re-
thymus, spleen,
stored. and lymphlobules
In individual nodes. of Thethestructure
thymusof it the spleen
persists forand
twothe lymph nodes is fully
weeks.
restored. In individual
Signs lobules ofwere
of cardiotoxicity the thymus
detectedit only
persists for two weeks.
by pathomorphological examination. After
Signs of cardiotoxicity were detected only by pathomorphological
administration of AF at the dosage level of total MTD, the morphological examination. After
features of toxic
administration of AF at the dosage level of total MTD, the
cardiomyopathy were moderately expressed, and by the end of the observation, themorphological features of my-
toxic ocardial
cardiomyopathy
structurewere was moderately
completely expressed,
restored. Aand highbydose
the end of the observation,
of anthrafuran caused the the same
myocardial structure was completely restored. A high dose of anthrafuran
changes as a low one on day 1 after the course, but after day 15 post treatment, they in- caused the
same changes as a low one on day 1 after the course, but after day 15 post treatment, they
tensified. It can be assumed that the drug, when the tolerated doses are exceeded, is able
intensified. It can be assumed that the drug, when the tolerated doses are exceeded, is able
to display cardiotoxic properties.
to display cardiotoxic properties.
Clinical, laboratory, and morphological investigations have demonstrated that AF
Clinical, laboratory, and morphological investigations have demonstrated that AF
applied in both doses studied exhibits hepato- and nephrotoxic properties. Hepatotoxicity
applied in both doses studied exhibits hepato- and nephrotoxic properties. Hepatotoxicity
was manifested in an increase of transaminases levels in the blood serum of rabbits and
was manifested in an increase of transaminases levels in the blood serum of rabbits and was
was enhanced in the mass coefficient of the liver. Lesions of the liver structure were ex-
enhanced in the mass coefficient of the liver. Lesions of the liver structure were expressed
pressed moderately after a low dose administration of anthrafuran formulation on day 1
post treatment and 2 weeks later, liver morphology was similar to control. When applying
Pharmaceuticals 2021, 14, 900 8 of 10
Pharmaceuticals 2021, 14, x FOR PEER REVIEW 8 of 10
moderately after a low dose administration of anthrafuran formulation on day 1 post
treatment and 2 weeks later, liver morphology was similar to control. When applying
anthrafuran in
anthrafuran in aa dose
dose totaling
totaling LD LD5050,, the
the structure
structure of of the
the liver
liver byby the
the end
end of
of the
the experiment
experiment
was restored
was restored only only partially.
partially.
Signs of nephrotoxicity were manifested in an increase in the level level of
of protein
protein andand
urobilinogen
urobilinogen in urine and a decrease in its specific gravity.
gravity. The degree of
degree failurefailure of kidney
morphology and the rate of recovery depended depended on the the value
value of of the
the applied
applied dose.
dose. A A high
high
dose of anthrafuran caused necrosis in the cortical and medullary medullary regions
regions of of the
the kidney.
kidney.
The damaged areas areas have
have undergone
undergone fibrosis.
fibrosis.
It is
is well
wellknown
knownthat thatdoxorubicin
doxorubicin induces
induces considerable
considerable testicular
testiculartoxicity in men
toxicity and
in men
animals
and animals[17,18]. Importantly,
[17,18]. Importantly, no pathological
no pathological changes
changesafterafter
AF AFadministration
administration were ob-
were
observed
served in in thethe rabbit
rabbit testis.
testis.
Toxic
Toxic properties
properties of oforal
oralanthrafuran
anthrafuranrevealedrevealedininthis thisstudy
studycorrespond
correspondtotothosethoseprevi-
pre-
ously
viouslydetected
detected in in
ratsrats
[11]. Some
[11]. Some differences
differences in the results
in the of the
results of investigations,
the investigations,apparently,
appar-
are associated
ently, with with
are associated the fact
the that chronic
fact that toxicity
chronic in rats
toxicity was
in rats wasexamined
examinedusing usingthethe drug
drug
substance, and in the study on rabbits, a drug formulation was used. In previous previous pharma-
cokinetic researches, it was found that after oral administration of the drug formulation,
AF was absorbed
absorbed faster,
faster, andand itsits maximum
maximum concentration
concentration in the the blood
blood waswas higher
higher than
than
after
after the administration of a substance [9]. AF substance is absorbed 2 times slower from
the administration of a substance [9]. AF substance is absorbed 2 times slower from
the
the gastrointestinal
gastrointestinal tract tract into
into the
the blood;
blood; therefore,
therefore, its its damaging
damaging effect effect on
on the
the gastric
gastric and
and
intestinal
intestinal mucosa
mucosa of of rats
rats is
is more
more prolonged
prolonged and and isis manifested
manifested to to aa greater
greater extent
extent than
than in
in
rabbits.
rabbits. A A longer
longer circulation
circulation of of the
the drug
drug substance
substance in in the
the blood
blood cancan also
also explain
explain the
the more
more
pronounced
pronounced hematological
hematological toxicitytoxicity of of AF
AF onon rats.
rats.
Altogether,
Altogether, these results provide evidence that
these results provide evidence that toxicity
toxicity of of oral
oral anthrafuran
anthrafuran in in both
both
cases
cases depended on dose, and multiple administration of therapeutic doses of the drug
depended on dose, and multiple administration of therapeutic doses of the drug
produced
produced transient
transient completely
completely reversible
reversible toxic
toxic effects.
effects.
4. Materials and Methods
4. Materials and Methods
A substance of anthrafuran (Figure 7), purity 99%, (S)-3-(3-aminopyrrolidine-1-carbonyl)-
A substance of anthrafuran (Figure 7), purity 99%, (S)-3-(3-aminopyrrolidine-1-car-
4,11-dyhydroxy-2-methylanthra[2,3-b]furan-5,10-dione methanesulfonate dyhydrate was syn-
bonyl)-4,11-dyhydroxy-2-methylanthra[2,3-b]furan-5,10-dione methanesulfonate dyhy-
thesized following the previously reported method [2,3,6].
drate was synthesized following the previously reported method [2,3,6].
Figure 7.
Figure 7. Anthrafuran chemical structure.
All experiments
experimentsin invivo
vivowere
wereperformed
performedinin accordance
accordance with thethe
with European
European Convention
Conven-
for
tionthe
forProtection of Vertebrate
the Protection Animals
of Vertebrate [19],[19],
Animals and and
the National standard
the National standardof the Russian
of the Rus-
Federation R 53434-2009
sian Federation «Good
R 53434-2009 Laboratory
«Good Practice»
Laboratory [20]. [20].
Practice»
The study was carried out on adult male Chinchilla rabbits (1.8–2.0 kg) obtained obtained from
from
the animal breeding unit of the FMBA (Scientific center
(Scientific center for biomedical
biomedical technologies of
technologies of the
the
Federal biomedical agency, Moscow,Moscow, Russia).
Russia). The animals
animals were
were randomly
randomly divided
divided into
into
groups
groups (n
(n = 6). The
= 6). The animal
animal study
study was
was approved
approved by by the
the Ethics
Ethics of
of Animal
Animal Experimentation
Experimentation
of
of Gause
Gause Institute
Institute of
of New
New Antibiotics
Antibiotics (protocol
(protocol No
No 11/2020).
11/2020).
The
The treatment regimen was
treatment regimen was 1515 ××22mg/kg
mg/kgand and1515× × 6 mg/kg,
6 mg/kg, withwith a 24-h
a 24-h interval
interval be-
between
tween administrations. Total doses were equivalent to the maximum tolerated dose
administrations. Total doses were equivalent to the maximum tolerated dose
(MTD)
(MTD) oror aa medium
medium lethal
lethal dose
dose (LD
(LD50 ) of anthrafuran, respectively. Doses were calculated
50) of anthrafuran, respectively. Doses were calculated
from
from appropriate doses for rats established in
appropriate doses for rats established in the
the study
study of
of acute
acute toxicity
toxicity of
of the
the drug,
drug, using
using
the dose conversion factors [11,21]. Untreated animals were used
the dose conversion factors [11,21]. Untreated animals were used as a control. as a control.
Anthrafuran formulation (AF) were in the form of gelatin capsules prepared individ-
ually for each animal, based on its body weight. Capsules were placed on the root of the
tongue and 10 mL of water was introduced into the oral cavity using a soft silicone probePharmaceuticals 2021, 14, 900 9 of 10
Anthrafuran formulation (AF) were in the form of gelatin capsules prepared indi-
vidually for each animal, based on its body weight. Capsules were placed on the root of
the tongue and 10 mL of water was introduced into the oral cavity using a soft silicone
probe plastic syringe. Dosage preparation for administration was carried out once a week
according to a change in the weight of the test animals. Doses before administration were
stored at room temperature, but not higher than 25 ◦ C.
In the course of the observation periods, the animals were followed up for any adverse
effects. Body weight and food consumption were evaluated weekly. The hematological
tests were performed using an automated hematology analyzer (Abacus Junior Vet, Diatron,
Budapest, Hungary). Blood was withdrawn from the marginal ear vein in healthy animals
before drug administration, then during the drug administration course (days 0, 7, 15), and
after the end of the drug administration course (days 3, 7, 15). The following parameters
were checked: leukocyte count with differentiation type, erythrocyte count, hemoglobin,
thrombocyte count, and hematocrit.
The following clinical biochemistry parameters were determined automatically using a
biochemical analyzer (ChemWell, Awareness Technology, Inc., Palm City, Florida, USA) on
days 1 and 15 post-treatment: alanine aminotransferase (ALT), aspartate aminotransferase
(AST), alkaline phosphatase, creatinine, glucose, serum urea, bilirubin total and direct, total
protein, and albumin.
The urinalysis tests were performed on days 1 and 15 post-treatment automatically
using a Laura Smart analyser (Lachema, Prague, Czech Republic).
Electrocardiographic examination (second standard lead) was performed on days 1
and 15 post-treatment (electrocardiograph AКSION EK1Е-07, Moscow, Russia).
The animals were euthanized on days 1 and 15 after the end of drug administration
course. Necropsy was performed for the all animals. Thoracic and abdominal cavities
and internal organs were inspected macroscopically. The organs were fixed in 10% neu-
tral formalin, embedded in paraffin, and cut. The sections 5 µ thick were stained with
hematoxylin-eosin.
Statistical analysis was performed using the Student’s t-Test. Mean values and stan-
dard deviations were calculated for body weights, hematological parameters, and relative
organ weights. The difference between the groups was considered significant at p ≤ 0.05.
5. Conclusions
Thus, the evaluation of subchronic toxicity of AF on the rabbits demonstrated less
toxicity for oral administration compared to doxorubicin and other anthracyclines and
anthracenediones. Most of its side effects are reversible within 15 days. Overall, the
results of the studies indicate the high potential for the further development of this novel
anticancer drug.
Author Contributions: Conceptualization, formal analysis, investigation, and writing, M.I.T., E.R.P.,
H.M.T., and A.E.S.; methodology and validation, M.I.T., H.M.T., and E.R.P.; data curation, visual-
ization and project administration, M.I.T., V.A.G., and E.R.P.; resources, supervision, and funding
acquisition, A.E.S. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: The animal study was approved by the Ethics of Animal
Experimentation of Gause Institute of New Antibiotics (protocol No 11/2020).
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are contained within the article.
Conflicts of Interest: The authors declare no conflict of interest.Pharmaceuticals 2021, 14, 900 10 of 10
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