Sugen Life Sciences Pvt Ltd
Advanced Research Products
Study Report
In vitro Antimicrobial activity of ARP100101-ARP100125 Compounds
Study No: XXXX
Date of Submission: April28, 2012
Sponsor
Advanced Research Products
NJ, USA
Testing Facility
Sugen Life Sciences Pvt Ltd
#4/86, S.V. Nagar
Perumalla palli (post)
Tirupati – 517 505
Andhra Pradesh
India
Sugenlife.com
Confidential
Page 1 of 25
Sugen Life Sciences Pvt Ltd
S. No
Advanced Research Products
Table of Contents
Page No
1.0
Contact Personnel
03
2.0
Study Personnel
04
3.0
Quality Assurance Statement
05
4.0
Abbreviations
06
5.0
Study details
07
5.1
Chronology of study
07
5.2
Test guidelines
07
5.3
Materials and Methods
07
5.4
Investigational drug substance
07
5.5
Materials
08
5.6
Bacterial cultures
08
5.7
Media preparation
08
5.8
Preparation of stock solutions of drugs/ compounds
09
5.9
Reference standards
09
6.0
Disc diffusion method
09
7.0
Inoculum preparation
09
8.0
Inoculation of test plates
09
9.0
Application of discs to inoculated agar plates
10
10.0
Incubation
10
11.0
Interpretation of Results
10
12.0
Results
11
13.0
25
Appendix -1
Conclusion
ARP100101
Appendix -2
ARP100102
13
Appendix -3
ARP100104
14
Appendix -4a & 4b
ARP100105
15
Appendix -5a & 5b
ARP100106
17
Appendix -6
ARP100108
19
Appendix -7
ARP100111
20
Appendix -8
ARP100119
21
Appendix -9
ARP100120
22
Appendix -10
ARP100121
23
Appendix -11
ARP100122
24
Confidential
12
Page 2 of 25
Sugen Life Sciences Pvt Ltd
Advanced Research Products
1.0 Contact personnel
Study Director:
Name : Y. Raja Ratna Reddy, M.Sc
Address: Department of Microbiology
Sugen Life Sciences Pvt Ltd
# 4/86, S.V. Nagar
Perumalla Palli (Post)
Tirupati 517 505
Tel.: 91-877-2276118
Confidential
Page 3 of 25
Sugen Life Sciences Pvt Ltd
Advanced Research Products
2.0 Study Personnel
The following personnel participated in conducting the study.
Name of the Person
Study Role
Y. Raja ratna Reddy, M.Sc
Study Director
C. Krishna kumari, M.Sc
Microbiologist
S. Mamatha, M.Sc
Research Assistant
KSVP Ratnam, M.Sc
Quality Assurance
C. Sreenivasulu Reddy, B.Com
Confidential
Test Facility Management
Page 4 of 25
Sugen Life Sciences Pvt Ltd
Advanced Research Products
3.0 Quality Assurance Statement
The study has been conducted at Sugen Life Sciences Pvt Ltd in accordance with CLSI
(Clinical Laboratory Standards Institute) and in-house methods. This study was performed /
audited and the findings were reported to the study director once in a week.
Ms. KSVP. Ratnam, M.Sc
Quality Assurance Unit
Confidential
Page 5 of 25
Sugen Life Sciences Pvt Ltd
Advanced Research Products
4.0
CLSI
DMSO
Clinical Laboratory Standards Institute
Dimethyl Sulfoxide
µg
Microgram
ml
Milliliter
mm
Millimeter
MTCC
E.coli
Confidential
Abbreviations
Microbial Type Culture Collection
Escherichia coli
Page 6 of 25
Sugen Life Sciences Pvt Ltd
Advanced Research Products
5.0. STUDY DETAILS
Study Title: In vitro Antimicrobial activity of Compounds (ARP100101 to ARP100125)
The purpose of the study was to determine in vitro antimicrobial activity of the test items
viz. ARP100101-ARP100125 in Gram positive/Gram negative bacteria and fungus by disc
diffusion method at the microbiology department of Sugen Life Sciences Pvt Ltd.
5.1
Chronology of Study
5.1.1
Study Initiation Date
:
February, 2012
5.1.2
Experiment Start Date
:
March, 2012
5.1.3
Experiment Completion Date
:
April, 2012
:
April, 2012
5.1.4 Study Completion Date
5.2
Test Guidelines
The study was performed in compliance with CLSI (Clinical laboratory standards
Institute) and Manual on Antimicrobial Susceptibility Testing (under the auspices of
Indian association of medical microbiologists).
5.3
Materials and Methods
5.4
Investigational Drug Substance
Table -1.0: Details of test substances
Confidential
S.No
1
Name of the Test Item
ARP100101
In-House code
ABT01
Solubility
DMSO
2
ARP100102
ABT02
DMSO
3
ARP100103
ABT03
DMSO
4
ARP100104
ABT04
DMSO
5
ARP100105
ABT05
DMSO
6
ARP100106
ABT06
DMSO
7
ARP100107
ABT07
DMSO
8
ARP100108
ABT08
DMSO
9
ARP100109
ABT09
DMSO
10
ARP100110
ABT10
DMSO
Page 7 of 25
Sugen Life Sciences Pvt Ltd
Advanced Research Products
11
ARP100111
ABT11
DMSO
12
ARP100112
ABT12
DMSO
13
ARP100113
ABT13
DMSO
14
ARP100114
ABT14
DMSO
15
ARP100115
ABT15
DMSO
16
ARP100116
ABT16
DMSO
17
ARP100117
ABT17
DMSO
18
ARP100118
ABT18
DMSO
19
ARP100119
ABT19
DMSO
20
ARP100120
ABT20
DMSO
21
ARP100121
ABT21
DMSO
22
ARP100122
ABT22
DMSO
23
ARP100123
ABT23
DMSO
24
ARP100124
ABT24
DMSO
25
ARP100125
ABT25
DMSO
The tests were performed in duplicates.
5.5 Materials
Ciprofloxacin Cf5 (5µg/disc) standard susceptibility test disc (Batch No.
0000131030), Amikacin Ak30 (30 µg/disc) (Batch No. 0000131028), Clindamycin
Cd2
(Batch
No.0000130591)
and
Fluconazole
Fu25
(25µg/disc)
standard
susceptibility test disc (Batch No. 0000124939) were procured from Hi-Media. All
media were procured from Hi-Media and chemicals from Sigma. Sterile cotton
swabs (Batch No. 06-402) and sterile Petri plates (Batch No. 09-401) procured from
Hi-Media laboratories. Triclosan (Positive control) was procured from Kumar
Organics Private Ltd, Bangalore, India. Nicotine was supplied by the sponsor.
5.6 Bacterial Cultures
The bacterial cultures Gram positive bacteria (Staphylococcus aureus, Enterococcus
faecalis), Gram negative bacteria (E. Coli and, Pseudomonas aeruginosa) and fungal
cultures (Candida albicans & Cryptococcus neoformans) were procured from
MTCC, Chandigarh, India.
5.7 Media Preparation
All the required media were prepared as per manufacturer’s instructions.
Confidential
Page 8 of 25
Sugen Life Sciences Pvt Ltd
Advanced Research Products
5.8 Preparation of stock solutions of drugs / compounds
5000 µg /ml stock solution was prepared in DMSO for each of the drugs. From this
stock solution, other working stock solutions (100 µg/ml, 50 µg/ml, 25 µg/ml
5µg/ml) were prepared by serial dilution. All the working stock solutions were
stored in a refrigerator up to two months. All the stock solutions were properly
labeled.
5.9 Reference Standards
Ciprofloxacin, Clindamycin, Amikacin, Flucanozole and Triclosan were used as
standards. Nicotine was used for comparative efficacy.
6.0 Disc diffusion method
The Kirby-Bauer disc diffusion method which is recommended by the CLSI
(Clinical Laboratory Standards Institute) was used to determine the antimicrobial
activity of test items. The accuracy and reproducibility of the test procedure is
validated and this is the most commonly used method for testing the in vitro
antimicrobial sensitivity.
7.0 Inoculum Preparation
The standard inoculum which is critical for disc diffusion method was prepared
using broth culture suspension. At least three to five well-isolated colonies of the
same morphological type of test organism was selected from an agar plate culture.
The top of each colony was touched with a sterile loop, and the growth is transferred
into a tube containing 4 to 5 ml of a suitable broth medium. The broth culture was
incubated at 37±1°C until it reaches uniform turbidity. The turbidity of the broth
culture was compared and adjusted to match the 0.5 McFarland standard with sterile
saline or broth. For each microorganism the standard inoculum was prepared as
described in section 8.6.
8.0 Inoculation of Test Plates
Optimally, within 15 minutes after adjusting the turbidity of the inoculum, a sterile
cotton swab was dipped into the inoculum, soaked and pressed firmly on the inside wall
of the tube above the fluid level to remove excess inoculum from the swab. The dried
surface of a sterile agar plate was inoculated by streaking the swab over the entire agar
surface, rotating the plate approximately 60 each time to ensure an even distribution of
inoculum. After the inoculum was absorbed on the agar surface, filter paper discs were
Confidential
Page 9 of 25
Sugen Life Sciences Pvt Ltd
Advanced Research Products
impregnated with different concentrations of the test item and applied within 15
minutes.
9.0
Application of Discs to Inoculated Agar Plates
The discs were dispensed onto the surface of the inoculated agar plate. Each disc must
be pressed down to ensure complete contact with the agar surface with adequate
distance between the discs.
10.0
Incubation
Plates were incubated in an incubator at 37±1C for 24 hrs for bacteria and at 24±1°C
for 48-72 hrs for fungi within 15 minutes after dispensing the discs.
11.0
Interpretation of Results
After incubation, each plate was examined for the zone of inhibition. The diameter
of the zone of complete inhibition (as judged by the unaided eye) was measured in
mm, including the diameter of the disc. The diameter of inhibition zone of different
concentrations of test item was compared with the inhibition zones of reference
standards.
Confidential
Page 10 of 25
Sugen Life Sciences Pvt Ltd
Advanced Research Products
12.0 Results
Twenty five compounds were tested for their antibacterial and antifungal activities. Compounds
which did not show any activity against the organisms tested in this study are not included in the
results. Compounds which showed activity on any one of the organisms or more are presented in
the results. Of the 25 compounds tested, compounds # 3, 7, 9, 10, 12-18, 23, 24 and 25 did not
show any antimicrobial or antifungal activity under the experimental conditions described in this
report. Therefore results of these compounds are not included below. Compounds 01, 02, 04, 05,
06, 08, 11 and 19-22 exhibited antibacterial and/or antifungal activities and the detailed results are
included below.
Confidential
Page 11 of 25
Sugen Life Sciences Pvt Ltd
Advanced Research Products
Appendix-1:ARP100101 (Fungi)
Table -2: Zone of inihibition by disc diffusion method for the ARP100101 in different
concentrations
Diameter of Zone Inhibition (mm)
Name of the Fungi
100 μg
50 μg
25 μg
5 μg
Control
Candiada albicans
10
8
-
-
Cryptococcus neoformans
-
-
-
-
-
Negative
control
-
Positive
control
30
-
-
25
Positive control: 25 µg Fluconazole (Hi-Media)
Negative control: Diluent (DMSO)
‘- ‘indicates no zone of inhibition
mm- millimeter
Zone of inhibition (mm)
Antifungal activity of ARP100101
Candiada albicans
Cryptococcus neoformans
35
30
25
20
15
10
5
0
100 μg
50 μg
25 μg
5 μg
Control Negative Positive
control control
Concentraion (ug)
Fig.1: Graphical representation of zone of inhibition of ARP100101 on fungi.
Confidential
Page 12 of 25
Sugen Life Sciences Pvt Ltd
Advanced Research Products
Appendix - 2: ARP100102 (Fungi)
Table -3: Zone of inihibition by disc diffusion method for the ARP100102 in different
concentrations.
Diameter of Zone Inhibition (mm)
Name of the Fungi
100 μg
50 μg
25 μg
5 μg
Control
Candiada albicans
13
12
10
10
Cryptococcus neoformans
-
-
-
-
-
Negative
control
-
Positive
control
30
-
-
25
Positive control: 25 µg Fluconazole (Hi-Media)
Negative control: Diluent (DMSO)
‘- ‘indicates no zone of inhibition
mm- millimeter
Antifungal activity of ARP100102
Candiada albicans
Zone of inhibition (mm)
35
Cryptococcus neoformans
30
25
20
15
10
5
0
100 μg
50 μg
25 μg
5 μg
Control
Concentration (µg)
Negative
control
Positive
control
Fig.2: Graphical representation of zone of inhibition of ARP100102 on fungi.
Confidential
Page 13 of 25
Sugen Life Sciences Pvt Ltd
Advanced Research Products
Appendix -3:ARP100104 (Fungi)
Table - 4: Zone of inihibition by disc diffusion method for the ARP100104 in different
concentrations
Diameter of Zone Inhibition (mm)
Name of the Fungi
100 μg
50 μg
25 μg
5 μg
Candiada albicans
10
-
-
-
Cryptococcus neoformans
13
-
-
-
Control Negative Positive
control control
30
25
Positive control: 25 µg Fluconazole (Hi-Media)
Negative control: Diluent (DMSO)
‘- ‘indicates no zone of inhibition
mm- millimeter
Antifungal activity of ARP100104
Candiada albicans
35
Zone of inhibition (mm)
Cryptococcus neoformans
30
25
20
15
10
5
0
100 μg
50 μg
25 μg
5 μg
Control
Concentration (µg)
Negative
control
Positive
control
Fig.3: Graphical representation of zone of inhibition of ARP100104 on fungi.
Confidential
Page 14 of 25
Sugen Life Sciences Pvt Ltd
Advanced Research Products
Appendix -4a:ARP100105 (Bacteria)
Table – 5A: Zone of inihibition by disc diffusion method for the ARP100105 in different
concentrations.
Diameter of Zone Inhibition (mm)
Name of the Bacteria
100 μg
50 μg
25 μg
5 μg
Staphylococcus aureus
12
10
-
-
Enterococcus faecalis
-
-
-
-
-
Escherishia. coli
12
-
-
-
Pseudomonas aeruginosa
-
-
-
-
Control Negative
control
-
Positive
control 1
27
Positive
control 2
30
-
30
24
-
-
32
25
-
-
38
25
Positive Control 1: 5 µg Ciprofloxacin (Hi-Media)
Positive Control 2: 30 µg Amikacin (Hi-Media)
Negative control: Diluent (DMSO)
‘- ‘indicates no zone of inhibition
mm- millimeter
Antibacterial activity of ARP100105
40
Enterococcus faecalis
Escherishia. coli
35
Zone of inhibition (mm)
Staphylococcus aureus
Pseudomonas aeruginosa
30
25
20
15
10
5
0
100 μg
50 μg
25 μg
5 μg
Control Negative Positive Positive
control control 1 Control 2
Concentration (µg)
Fig.4a: Graphical representation of zone of inhibition of ARP100105 on different Gram
positive/Gram negative bacteria.
Confidential
Page 15 of 25
Sugen Life Sciences Pvt Ltd
Advanced Research Products
Appendix – 4b:ARP100105 (Fungi)
Table - 5B: Zone of inihibition by disc diffusion method for the ARP100105 in different
concentrations
Diameter of Zone Inhibition (mm)
Name of the Fungi
100 μg
50 μg
25 μg
5 μg
Candiada albicans
15
-
-
-
Cryptococcus neoformans
15
-
-
-
Control Negative Positive
control control
30
25
Positive control: 25 µg Fluconazole (Hi-Media)
Negative control: Diluent (DMSO)
‘- ‘indicates no zone of inhibition
mm- millimeter
Candiada albicans
Antifungal activity of ARP100105
Zone of inhibition (mm)
35
Cryptococcus neoformans
30
25
20
15
10
5
0
100 μg
50 μg
25 μg
5 μg
Control
Concentration (µg)
Negative
control
Positive
control
Fig.4b: Graphical representation of zone of inhibition of ARP100105 on fungi.
Confidential
Page 16 of 25
Sugen Life Sciences Pvt Ltd
Advanced Research Products
Appendix-5a:ARP100106 (Bacteria)
Table - 6A: Zone of inihibition by disc diffusion method for the ARP100106 in different
concentrations.
Diameter of Zone Inhibition (mm)
Name of the Bacteria
100 μg
50 μg
25 μg
5 μg
Staphylococcus aureus
-
-
-
-
Enterococcus faecalis
10
-
-
-
-
Escherishia. coli
-
-
-
-
Pseudomonas aeruginosa
-
-
-
-
Control Negative
control
-
Positive
control 1
27
Positive
control 2
30
-
30
24
-
-
32
25
-
-
38
25
Positive Control 1: 5 µg Ciprofloxacin (Hi-Media)
Positive Control 2: 30 µg Amikacin (Hi-Media)
Negative control: Diluent (DMSO)
‘- ‘indicates no zone of inhibition
mm- millimeter
Antibacterial activity of ARP100106
Enterococcus faecalis
40
Escherishia. coli
Pseudomonas aeruginosa
35
Zone of inhibition (mm)
Staphylococcus aureus
30
25
20
15
10
5
0
100 μg
50 μg
25 μg
5 μg
Control Negative Positive Positive
control control 1 Control 2
Concentration (µg)
Confidential
Page 17 of 25
Sugen Life Sciences Pvt Ltd
Advanced Research Products
Fig.5a: Graphical representation of zone of inhibition of ARP100106 on different gram
positive/gram negative bacteria.
Appendix – 5b: ARP100106 (Fungi)
Table - 6B: Zone of inihibition by disc diffusion method for the ARP100106 in different
concentrations.
Diameter of Zone Inhibition (mm)
Name of the Fungi
100 μg
50 μg
25 μg
5 μg
Candiada albicans
12
-
-
-
12
-
-
-
Cryptococcus
neoformans
Control Negative Positive
control control
30
-
-
25
Positive control: 25 µg Fluconazole (Hi-Media)
Negative control: Diluent (DMSO)
‘- ‘indicates no zone of inhibition
mm- millimeter
Antifungal activity of ARP100106
Zone of inhibition (mm)
35
Candiada albicans
Cryptococcus neoformans
30
25
20
15
10
5
0
100 μg
50 μg
25 μg
5 μg
Control Negative Positive Positive
control control 1 Control 2
Concentration (µg)
Fig.5b: Graphical representation of zone of inhibition of ARP100106 on fungi.
Confidential
Page 18 of 25
Sugen Life Sciences Pvt Ltd
Advanced Research Products
Appendix-6:ARP100108 (Fungi)
Table - 7: Zone of inihibition by disc diffusion method for the ARP100108 in different
concentrations.
Diameter of Zone Inhibition (mm)
Name of the Fungi
100 μg
50 μg
25 μg
5 μg
Candiada albicans
15
-
-
-
11
-
-
-
Cryptococcus
neoformans
Control Negative Positive
control control
30
-
-
25
Positive control: 25 µg Fluconazole (Hi-Media)
Negative control: Diluent (DMSO)
‘- ‘indicates no zone of inhibition
mm- millimeter
Antifungal activity of ARP100108
Cryptococcus neoformans
35
Zone of inhibition (mm)
Candiada albicans
30
25
20
15
10
5
0
100 μg
50 μg
25 μg
5 μg
Control Negative Positive
control control
Concentration (µg)
Fig.6: Graphical representation of zone of inhibition of ARP100108 on fungi.
Confidential
Page 19 of 25
Sugen Life Sciences Pvt Ltd
Advanced Research Products
Appendix-7:ARP100111 (Bacteria)
Table - 8: Zone of inihibition by disc diffusion method for the ARP100111 in different
concentrations.
Name of the
Bacteria
Staphylococcus
aureus
Enterococcus
faecalis
Escherishia. coli
Pseudomonas
aeruginosa
Diameter of Zone inhibition (mm)
Negative
Positive
5 μg Control
control
control 1
Positive
Control 2
Triclosan
24
30
15
-
30
24
20
-
-
38
25
25
-
-
38
25
-
100 μg
50 μg
25 μg
-
-
-
-
-
-
9
-
-
-
-
12
-
-
-
-
-
-
-
Positive Control 1: 5 µg Ciprofloxacin (Hi-Media)
Positive Control 2: 30 µg Amikacin (Hi-Media)
Triclosan 50 µg
Negative control: Diluent (DMSO)
‘- ‘indicates no zone of inhibition
mm- millimeter
Fig.7: Graphical representation of zone of inhibition of ARP100111 on different gram
positive/gram negative bacteria.
Confidential
Page 20 of 25
Sugen Life Sciences Pvt Ltd
Advanced Research Products
Appendix-8:ARP100119 (Fungi)
Table - 7: Zone of inihibition by disc diffusion method for the ARP100119 in different
concentrations.
Name of the Fungi
100 μg
50 μg
Candiada albicans
Cryptococcus
neoformans
10
9
12
11
Diameter of Zone inhibition (mm)
Negative Positive
25 μg
5 μg Control
control
control
30
-
-
-
-
Triclosan
25
Positive control: 25 µg Fluconazole (Hi-Media)
Triclosan 50 µg
Negative control: Diluent (DMSO)
‘- ‘indicates no zone of inhibition
mm- millimeter
Fig.7: Graphical representation of zone of inhibition of ARP100119 on fungi.
Confidential
Page 21 of 25
20
23
Sugen Life Sciences Pvt Ltd
Advanced Research Products
Appendix-9:ARP100120 (Fungi)
Table - 8: Zone of inihibition by disc diffusion method for the ARP100120 in different
concentrations.
Name of the Fungi
100 μg
50 μg
Candiada albicans
Cryptococcus
neoformans
9
0
9
0
Diameter of Zone inhibition (mm)
Negative Positive
25 μg 5 μg Control
control
control
0
0
0
0
30
0
0
0
0
25
Positive control: 25 µg Fluconazole (Hi-Media)
Triclosan 50 µg
Negative control: Diluent (DMSO)
‘- ‘indicates no zone of inhibition
mm- millimeter
Fig.8: Graphical representation of zone of inhibition of ARP100120 on fungi.
Confidential
Page 22 of 25
Triclosan
20
23
Sugen Life Sciences Pvt Ltd
Advanced Research Products
Appendix-10:ARP100121 (Fungi)
Table - 8: Zone of inihibition by disc diffusion method for the ARP100121 in different
concentrations.
Name of the Fungi
100 μg
50 μg
Candiada albicans
Cryptococcus
neoformans
12
10
-
-
Diameter of Zone inhibition (mm)
Negative Positive
25 μg 5 μg Control
control
control
30
-
-
-
-
25
Positive control: 25 µg Fluconazole (Hi-Media)
Triclosan 50 µg
Negative control: Diluent (DMSO)
‘- ‘indicates no zone of inhibition
mm- millimeter
Fig.9: Graphical representation of zone of inhibition of ARP100121 on fungi.
Confidential
Page 23 of 25
Triclosan
20
23
Sugen Life Sciences Pvt Ltd
Advanced Research Products
Appendix-11:ARP100122 (Fungi)
Table - 9: Zone of inihibition by disc diffusion method for the ARP100122 in different
concentrations.
Name of the Fungi
100 μg
50 μg
Candiada albicans
Cryptococcus
neoformans
13
-
-
-
Diameter of Zone inhibition (mm)
Negative Positive
25 μg 5 μg Control
control
control
30
-
-
-
-
25
Positive control: 25 µg Fluconazole (Hi-Media)
Triclosan 50 µg
Negative control: Diluent (DMSO)
‘- ‘indicates no zone of inhibition
mm- millimeter
Fig.10: Graphical representation of zone of inhibition of ARP100122 on fungi.
Confidential
Page 24 of 25
Triclosan
20
23
Sugen Life Sciences Pvt Ltd
Advanced Research Products
13.0 Conclusion
Based on the above results, it is concluded that of the 25 compounds tested for antimicrobial
activity, ARP100103, ARP100107, ARP100109,ARP100110, ARP100112 to ARP100118
and ARP100123 to ARP100125 compounds did not show any activity on both bacteria and
fungi tested in this report. Nicotine did not show any antibacterial activity even at 1000 μg /
ml.
ARP100105 and ARP100106 compounds having both antibacterial and antifungal activity.
ARP100105 showed activity at the concentrations of 100μg and 50μg and ARP100106
showed the activity at 100μg. The compounds ARP100101 (100μg and 50μg), ARP100102
(100μg, 50μg, 25μg, and 5μg) ARP100104 (100μg), ARP100108 (100μg), ARP100119 (100
μg and 50μg), ARP100120 (100μg), ARP100121 (100μg and 50μg) and ARP100122 (100
μg) exhibit antifungal activity. ARP100111 showed antibacterial activity at 100μg on both
gram positive and gram negative bacteria.
14. Signatures
Y. Raja Ratna Reddy, M.Sc
Ms. C.Krishnakumari, M.Sc
Confidential
Page 25 of 25