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: vbkishan@yahoo.com

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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