Indian Journal of Biotechnology
Vol 9, January 2010, pp 18-23
Borrelidin: A prospective drug
D V R N Bhikshapathi, Y Shravan Kumar, Y Madhusudan Rao and V Kishan*
University College of Pharmaceutical Sciences, Kakatiya University, Warangal 506 009, India
Received 23 December 2008; revised 20 April 2009; accepted 25 June 2009
Borrelidin, an antibiotic isolated from Streptomyces species and firstly isolated from Streptomyces rochei, is a possible
drug candidate due to its recently reported antiangiogenic activity and other biological activities. Keeping in view the
potential scope of the antibiotic, we performed computational studies like application of Lipinski’s rule for borrelidin, and
found from the properties of the borrelidin that it possessed good absorption or permeability across the biological
membrane. Influence of borrelidin on CYP3A enzyme was tested by pretreatment of the male Wistar rats at a concentration
of 100 µg/mL/rat daily for 5 d by oral administration, buspirone was used as a substrate; the results indicated the role of
borrelidin acting as an inhibitor of CYP3A. The effect of borrelidin on pretreated rat liver by micromorphological studies
was also done and results showed the damage on liver with borrelidin pretreatment. Influence of borrelidin on platelets was
studied at three different concentrations along with known antiplatelet aggregating agent, aspirin. Platelet aggregation was
noticed for all the three tested concentrations in comparison to control. Influence of borrelidin on prothrombin time was also
performed and compared with known anticoagulant, warfarin sodium, have little effect of borrelidin on the prothrombin
time which indicated the anticoagulant activity.
Keywords: Borrelidin, Lipinski’s rule, CYP3A enzyme, platelets, prothrombin time
Introduction Borrelidin, a 18 membered macrolide antibiotic
(Fig. 1), was first isolated from Streptomyces rochei
in 1949 by Berger and co-workers1, and later from
other species of Streptomyces including S. griseus BS
13252, S. parvulus Tu 4055
3, and S. californicus
MTCC 4401, an Indian soil isolate4.
The antibacterial activity of borrelidin has been
found to be due to selective inhibition of threonyl
tRNA synthetase and the activation of caspase-3 and
caspase-85-6
. Many researchers noticed a paradigm
shift in biological activity of borrelidin. Recent
reports indicated that borrelidin had potent
antiangiogenesis activity and it also induced apoptosis
of the capillary tube forming cells in dose dependent
manner7. The antiangiogenic effect of borrelidin was
mediated by two mechanisms i.e., threonine-
dependent and threonine-independent, and further
apoptosis was also induced in capillary endothelial
cells via Caspase-8/3 pathway8.
Borrelidin was produced by fermenting the
organism in medium containing 2% glucose, 2%
soyabean flour, 0.5% NaCl, and 0.3% CaCO3 at a pH
of 6.5 for 72-96 h9. S. rochei
10 also produced
borrelidin when grown in a medium containing
glycerol, soyabean meal and yeast extract.
Borrelidin was demonstrated to have various
biological activities such as antibacterial,
antimitotic11
, antimicrobial, antiviral, herbicidal,
insecticidal and antitumor. Borrelidin exhibited
excellent antimalarial activity against both
chloroquine sensitive and resistant strains in mice6. In
our search for antibiotics active against drug resistant
bacteria, S. californicus was isolated from compost
yard soil of Gundla Singaram village, Andhra
Pradesh, India, which also produced borrelidin4. In
accompanying paper (Bhikshapathi et al., under
preparation) we describe the antimicrobial effect of
borrelidin on Mycobacterium tuberculosis H37Rv
(ATCC 27294) and multidrug resistant M. tuberculosis.
Further, effects of borrelidin were studied for anti-HIV
reverse transcriptase activity and Ames mutagenicity
(Bhikshapathi et al., under preparation).
As borrelidin is speculated to be a possible drug
candidate due to various reported biological activities
and keeping in view the potential scope for further
physiological activities associated with borrelidin, we
applied the Lipinski’s rule12
for borrelidin to know the
absorption or permeability across biological
membrane. Studies were also conducted on the role of
_______________
*Author for correspondence:
Tel: 91870-2446259, 2432699; Fax: 91870-2438844, 2453508
E-mail: [email protected]
BHIKSHAPATHI et al: BORRELIDIN: A PROSPECTIVE DRUG
19
metabolizing enzymes like CYP3A during absorption
and also the possible effect of borrelidin on rat liver
parenchyma. Further, tests were also conducted to
know the haematological status due to influence of
borrelidin on blood platelets and prothrombin time.
In this paper, we describe certain physiological
effects of borrelidin on CYP3A enzyme, on rat liver,
drug likeliness for absorption or permeation across
biological membrane by Lipinski’s rule, and the
activity of borrelidin on prothrombin time and platelets.
Materials and Methods
Application of Lipinski’s Rule for Borrelidin to Predict
Absorption through Biological Membranes
Christopher Lipinski’s rule of five analysis helped
to raise awareness about properties and strategies that
make molecules more or less drug-like. The
guidelines were quickly adopted as it helped to apply
absorption, distribution, metabolism and excretion
considerations early in preclinical developments and
could help to avoid preclinical and clinical failures13
.
Properties were calculated using software Cerius-2,
Version 4.11, Accelrys Inc., San Diego, California,
USA, 2005 and by Descriptor calculator in the QSAR
module at GVK Bio Sciences, Hyderabad. Effect of Borrelidin on CYP3A Enzyme
Buspirone was obtained from Sun Pharmaceuticals,
Sodium chloride, Acetonitrile HPLC grade and glucose
were obtained from Merck (India) Limited, Mumbai,
and Dulbecco’s Phosphate buffer (pH 7.4) from Hi
Media (India) Limited, Mumbai. Animals used for the
study were male albino Wistar rats. Control animals
were without pretreatment and test animals were
pretreated with borrelidin in the concentration of 100
µg/mL/rat daily for 5 d by oral route. The solution was
prepared using DMSO and distilled water (1:9).
Male Wistar rats were fasted overnight with free
access to water before the experiments. The animals
were anesthetized with pentobarbital (30 mg/kg/ip).
The rats were exsanguinated, and abdomen was
opened and small intestine was separated. Then whole
small intestine was flushed with 50 mL of ice-cold
saline and divided into 3 segments of equal length.
Normal sacs with a length of 10 cm were prepared.
Probe drug, buspirone, a known substrate for CYP3A,
at a concentration of 10 mg/mL was prepared in
pH 7.4 isotonic Dulbecco’s PBS (D-PBS) containing
25 mM glucose. The probe drug solution (1 mL) was
introduced into the normal sac (mucosal side), and both
ends of the sac were ligated tightly. The sac containing
probe drug solution was immersed into 40 mL of
D-PBS containing 25 mM glucose. The medium was
pre-warmed to 37°C and aerated under bubbling
condition with 5: 95% of CO2 : O2 for 15 min, the
transport of the buspirone from mucosal to serosal
surfaces across the intestine was measured by sampling
the serosal medium periodically up to 120 min
(15, 30, 45, 60 & 120 min)14
. The samples were
analyzed by HPLC under the following conditions:
Mobile phase : Phosphate buffer (pH: 2.5) (50 mM):
Acetonitrile (50:50)
Flow Rate :1 mL/min
Column :C-18 RP analytical column
(Phenomenex)
λmax :240 nm
Range :0.0050 AUFS
pH was adjusted to 2.5 with ortho phosphoric acid.
The HPLC system consisted of a LC-10AT vp
solvent delivery system (Shimadzu, Japan), a SPD-
10A vp UV-visible variable wavelength detector
(Shimadzu, Japan), and a 250 x 4.6 mm Luna 5µ C-18
reverse phase analytical column (Phenomenex,
Netherlands).
Effect of Borrelidin on Rat Liver
To know the effect of borrelidin on rat liver after
pretreatment with 100 µg/d for 5 d, liver from treated
animals and control animals were sent for the biopsy.
The slides were prepared and observed under
microscope for micromorphological changes in liver
after Leishman’s staining at a magnification of 10 ×100.
Effect of Borrelidin on Platelets
Platelet rich plasma was collected from Kakatiya
Blood Bank, Warangal, aspirin from Merck (India)
Limited, Mumbai. To 100 µL of platelet rich plasma,
Fig. 1—Structure of borrelidin
INDIAN J BIOTECHNOL, JANUARY 2010
20
100 µL of different concentrations (10, 30 &
100 µg/mL) of borrelidin and aspirin solutions were
added and kept aside for about 30 min. Then, 10 µL
of the mixture was transferred on to the slide.
Leishman smearing was made and examined under
magnification of 10 × 100 for the platelet aggregations.
Effect of Borrelidin on Prothrombin Time
Warfarin sodium a free gift from Aventis Sanofi,
Goa, sodium citrate, Merck (India) Limited, Mumbai,
Thromborel-S, Erba, Germany and Erba Coag Uno
Instruments, Germany were used. Citrated plasma was
prepared by taking 1.8 mL of blood from healthy
volunteers and adding 200 µL of sodium citrate (3.8%),
centrifuging for 15 min at 3000 rpm. To 25 µL of
citrated plasma, 100 µL of drug solutions were added
and mixed properly and to that 50 µL of PT reagent
(Thromborel-S) was added. Then prothrombin time
was checked15
. The experiments were conducted for
different concentrations of borrelidin and warfarin
sodium (25, 50, 100, 250 & 500 µM).
Results and Discussion Application of Lipinski’s Rule for Prediction of Absorption
through Biological Membranes
Lipinski’s rule is a computational approach
developed to predict the solubility and permeability
across biological membranes. According to Lipinski’s
rule poor absorption or permeation is more likely
when there are more than 5H-bond donors, more than
10H-bond acceptors, molecular weight greater than
500, C log P greater than 512
. To evaluate drug
likeness better, the rules have spawned many
extensions, for example: Partition coefficient, log
P in -0.4 to + 5.6 range, Molar refractivity from
40 to 13016
.
Properties of borrelidin with their calculated values
and desirable values as per Lipinski’s rule and its
extensions are shown in Table 1. As per Lipinski’s
rule of five, the drug borrelidin was expected to have
good permeability. Even the extensions of Lipinski’s
rule about molar refractivity supported this view since
its molar refractivity was less than 130. All these
desirable characters of borrelidin indicated good
absorption or permeability across biological
membrane.
Effect of Borrelidin on CYP3A Enzyme during Absorption
Buspirone, already known as a substrate for
CYP3A metabolism17
, was used as a marker
compound to study the influence of borrelidin on
CYP3A mediated metabolism. Results of control
(without pretreatment) and pretreatment
(with borrelidin, 100 µg/d for 5 d) of buspirone
transport across gastro-intestinal membrane at three
different regions, duodenum (Fig. 2a), jejunum
(Fig. 2b) and ileum (Fig. 2c) are shown. The transport
of buspirone from normal sac was increased by many
Table 1—Properties of borrelidin with their calculated and
desirable values as per Lipinski’s rule and its extensions
Properties Calculated values Desirable values
Molecular weight 489. 5 <500
H-bond acceptor 7 < 10
H-bond donor 3 <5
Log P
(molecule based)
5.65 - 0.4 to + 5.6
Molar refractivity 120.58 40 - 130
Fig. 2a-c—Transport of buspirone across rat intestine in non
everted sac method for pretreated (100 µg/rat for 5 d with
borrelidin) and control (without pretreatment): Influence of
borrelidin on buspirone transport; a. in duodenum; b. in jejunum;
& c. in ileum.
BHIKSHAPATHI et al: BORRELIDIN: A PROSPECTIVE DRUG
21
folds at different regions of small intestine due to the
pretreatment of borrelidin for 5 d. In the first 45 min,
1.5 to 4-fold increase was noticed in duodenum as
compared to control rats. But from 60-120 min, no
traces of buspirone were found transported in control
animals. This indicated that the CYP3A metabolism
played a big role in duodenum. The transport of
buspirone was increased and depended on the time.
Further, maximum transport of buspirone from
intestinal sac was observed from jejunum and ileum
when compared to that of duodenum.
Buspirone is a known substrate17
to study the effect
of borrelidin on CYP3A metabolism from normal
sacs. It is already known that rat enterocytes contain
CYP3A, and this enzyme in general would reduce the
intestinal absorption of drugs into systemic circulation
because of metabolism. As CYP3A enzyme levels
decreased from duodenum to ileum it was expected
that the buspirone transport would increase.
Following the same, a significant transport of
buspirone from normal sacs (with pretreatment of
borrelidin) was observed, which clearly indicated the
role of borrelidin as an inhibitor of CYP3A. From the
above results, it was concluded that borrelidin acted
on CYP3A mediated drug metabolism either by direct
inhibition or inhibiting the enzyme synthesis. It was
earlier reported that the macrolide antibiotics like
erythromycin, clarithromycin, etc., had the CYP3A
inhibition only18
, since this drug also shared some of
the structural features, it is expected that this also
inhibited the activity of enzyme rather than the
synthesis. Effect of Borrelidin on liver-micromorphological Studies
Rat liver biopsy slides with and without
pretreatment were examined under a microscope at a
magnification of 10 × 100. Results are depicted in
Fig 3a for control (without pretreatment), and
Figs 3b & c with pretreatment of borrelidin. Due to
pretreatment of rats with borrelidin, liver cells were
swollen, dilatation of central and hepatic veins were
noticed in comparison to normal cells and vein in
control rat liver. This observation clearly indicated the
damage to liver with the pretreatment of borrelidin; it
also indicated that borrelidin has a toxic effect on liver,
when treated with the concentration of 100 µg/d for 5 d.
Effect of Borrelidin on Platelets
Influence of borrelidin on platelet rich plasma was
studied at three different concentrations (10, 30 &
100 µg/mL) along with that of a known antiplatelet
Fig. 3a-c—Photomicrographs of rat liver stained with Leishman
stain (magnification 10 x100), showing: a. Control rat liver
(without pretreatment) with normal central vein (Arrow) and
normal liver cells without any damage.; b. Pretreated with
borrelidin for 5 d (100 µg/rat) showing cellular damage and
swollen cells; c. Pretreated with borrelidin for 5 d (100 µg/rat)
showing liver damage with dilated central vein.
INDIAN J BIOTECHNOL, JANUARY 2010
22
aggregating agent, aspirin. Results observed under
microscope for borrelidin and aspirin on platelets are
depicted in Figs 4a (control), 4b (borrelidin,
10 µg/mL), 4c (borrelidin, 100 µg/mL) and
4d (aspirin 100 µg/mL). Platelet aggregation was
noticed for all the three tested concentrations in
comparison to control. The magnitude of aggregates
varied with the concentrations of borrelidin. However,
at all the tested concentrations no effect of aspirin was
seen on platelets i.e., platelets remained without any
aggregation. The mechanism for platelet aggregation
is not yet known for borrelidin. Fig 4d depicts the
effect of highest concentration only.
Effect of Borrelidin on Prothrombin Time
Prothrombin time (in sec) was measured at different
time intervals on citrated blood plasma from 6 healthy
volunteers using Erba Coag Uno instrument. Normal
range for prothrombin time is 10 to 13 sec and a
calculated value INR (International normalized ratio) is
1.0 to 1.419.
The INR is calculated by the following
formula, INR= (Patient prothrombin time/mean normal
PT20
). The calculated mean normal PT is 13 sec in a
clinical setting (Jaya Hospital, Hanamkonda, AP). The
normal PT range is 11-14 sec15
. All the observed and
calculated values indicated that there was some effect
on the prothrombin time in comparison with standard
anti-coagulant drug i.e., warfarin (Fig. 5). This
indicated the tendency for bleeding or otherwise blood
clotting would be affected following the administration
of borrelidin. However the mechanism for the anti-
coagulant effect of borrelidin is not yet known.
Acknowledgement The authors would like to thank the AICTE, New
Delhi for providing research funds under MODROB
scheme vide File No. 8020/RID/MODROB-5/2001-
2002. DVRNB is thankful to AICTE for providing the
scholarship under Quality Improvement programme
for teachers and DAAD equipment grant, Germany.
Authors thank Prof. Axel Zeeck of Goettingen
University, Germany for gifting sample of borrelidin.
Further, the authors are also thankful to
Dr P V Beerbhadra Rao (VBR Diagnostics,
Hanamkonda), for helping in platelet activity and
micromorphological studies of rat liver and
Mr B Kiran Kumar, GVK Biosciences, Hyderabad for
calculating properties based on Lipinski’s rule for
borrelidin and Mr Ranjit Kumar, Biochemist, Jaya
Hospital, Warangal for helping in the activity of
borrelidin on prothrombin time.
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