biological activities of hamycin
TRANSCRIPT
BIOLOGICAL ACTIVITIES OF HAMYCIN
A. C. MANIAR Bacteriology Department, Winnipeg General Hospital, Winnipeg, Manitoba
AND
SARALA MAVDIKAR Research Laboratories, Hindustan Antibiotics, Limited, Pimpri (Poona), India
Received March 18, 1965
Abstract After administration of an oral d m to mice, hamycin, an antifungalantibiotic,
penetrated the M y tissues and eliminated Crypt~coccus neoforwans present in the lungs. Out of 1 1 polyenes (harnycin, aureofungin, trichomycin, etmscomycin, amphotericin B, pimaricin, mycostatin, candidin, candicidin, PA150, and filipin), hamycin and aureofungin showed the highest "in vitro'hctivity a ainst Candida n l b h n s . Thc antilungal activity of hamycin, PA150, and aureofuungin was in- creased by the addition of *rum, plasma, or crystalline albumins. This increase in activity can hc used as an additional characteristic to differentiate some of the polyenes. We now believe that serum was responsible for our previous find- ings that "high concentrations of hanlycin in the organs of mice were detected". It has been shown that the complete albumin molecule was required to increase the activity of hamycin. The possibility that serum globulins or other proteins may increase the hamycin activity is not n r l d our by our experiments.
Introduction Hamycin, a new antifungal heptaene antibiotic was obtained from broth
cultures of Streptomyces pimprina Thirum isolated from Pimpri soil by Thiru- malachar, Menon, and Bhatt (61, who noted its high antifungal activity against Candida albicans. Purification methods were described by Bhate et al. (1). We previously reported finding an appreciable quantity of hamycin in the serum of albino mice 120 hours after a single oral dose of 800 mg/kg (4) and noted that certain blood components increased hamycin activity (3). In the investigation reported here, we have compared some biological activi- ties of hamycin "in vitro" as well as "in vivo" with those of other polyene antifungal antibiotics.
Procedures and Results Organisms
Stock cultures of Curvularia lunata (Wakker) Boedijn used for the micro- biological assay method were maintained by weekly transfer on Mz agar (2) slants incubated a t 27 "C for 3 to 4 days until the culture developed black spores. The culture of Cryptococcus neoformans was maintained on blood agar incubated a t 37 "C for 18 hours. Cultures of C. albicans were maintained on Mz agar incubated a t 37 "C for 18 hours.
Mz broth consisted of the following ingredients (2% agar was added for Mz agar).
Glycerol 1 .0 ml MgS0~7Hz0 0.005 g Glucose 1 . O g KHzPOI 10.00 mg Yeast extract 0.5 g Distilled water 100 ml NaCl 1 . O g pH adjusted to 7.0
Canadian Journal of Microbiology. Volume 12 (1966)
Can
. J. M
icro
biol
. Dow
nloa
ded
from
ww
w.n
rcre
sear
chpr
ess.
com
by
Uni
vers
ity o
f P.
E.I
. on
11/2
3/14
For
pers
onal
use
onl
y.
378 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 12, 1966
Hamycin (non-crystalline) and other polyenes were dissolved in 60% aqueous ethanol to an initial concentration of 0.8 mg/ml and these solutions were further diluted to the required strengths in distilled water containing 1 : 5000 dilutions of Tween 80. For oral adminsitration, hamycin was suspended in a 1 : 5000 dilution of Tween 80 in distilled water.
Experiment 1 Mouse Protection Studies In a previous paper, hamycin was detected by the authors (4) in the organs
of mice and rats, after a single oral dose, for a prolonged period. In the case of mice, hamycin concentrations ranged from 1.5 to 2.0 pg /g in Iungs, liver, kidney, spleen, and serum at the end of 3 hours. The organ concentrations decreased gradually during the 6- to 24-hour period and appreciabIe serum concentrations were maintained for 120 hours. In rats, though negligible amounts of hamycin were detected in the tissues tested a t the end of 3 and 6 hours of the oral administration, a t the end of 17 and 24 hours, however, tissue concentrations rose between 0.8 and 1.45 pg/g. Repeated administration of the antibiotic in mice gave high tissue levels from 1 to 3 pg/g, showing an additive effect. These findings indicate that hamycin can be used for the treatment of systemic fungal infections. To substantiate the above statement, mouse protection studies of the nature described below were planned.
Four groups of albino mice (30 mice per group) weighing 23-26 g were given hamycin orally. The first group received a single dose of 800 mg/kg on the first day only. The second group was given a dose of 400 mg/kg per day far 7 days. The third and the fourth p p s received dosages of 200 mgfkg: and 100 mg/kg respectively, per day, for 10 days. A separate group of 30 mice was used as a control. All the groups of mice, including the control group, were challenged with a single dnse of C. tteofomtzs on the first day of the drug treatment. The challenge dose was administered by the intravenous route in 0.2-ml volumes containing 1.2 to 1.4 million organisms. On the loth, 20th, and 30th day after the challenge dose, 10 mice from each of the groups were sacrificed and their lungs were collected by aseptic techniques. These lung samples were smeared separately on Mz agar and incubated a t 37 "C for 5 days and growth of cryptococci was observed. The results of this experiment are recorded in Table I. The lungs from control groups were all positive for C. aeoformans for the entire test periods of 10 to 30 days. A single oral dose of 800 mg/kg of hamycin did not eliminate cryptococci from the lungs except in three mice at t h e end of 10 days and one mouse at the end of 20 days. Repeated daily dosages tested down ta 100 mg/kg protected all the mice from cryptococci.
Experiment 2 Activity with Serum, Plasma, and Albumin Since hamycin was detected in mouse serum 120 hours after oral feeding
(4), we thought i t was not inactivated by serum. The following experiment was carried out to detect any change in antifungal activity of hamycin in the presence of serum, plasma, or albumin.
Solutions of hamycin containing 0.2, 0.4, and 0.8 pglml were prepared and 1 ml of each was added respectively to two sets of three test tubes. Each of
Can
. J. M
icro
biol
. Dow
nloa
ded
from
ww
w.n
rcre
sear
chpr
ess.
com
by
Uni
vers
ity o
f P.
E.I
. on
11/2
3/14
For
pers
onal
use
onl
y.
MANIAR AND MAVDIKAR: HAMYCIN 379
TABLE I Protective action of hamycin against Cryptococcus neoformans in mice*
Day on which lungs collectedf
10th 20th 30th Daily dosage Duration of
mg/kg treatment, Number of lungs showing positive growth (oral) days of cryptococci
800 1 400 7 200 10 100 10 Control -
'Hamycin was given orally; challenge dose was 0.2 ml of 1.2 to 1.4 million cells of cryptococci given intraven- ously.
?Ten mice were sacrificed for collection of lungs in each of the cases.
the tubes from one set received 1 ml of distilled water, whereas those in the second set received 1 ml of horse serum; thus, the first contained 0.1, 0.2, and 0.4 pg of hamycin per milliliter in distilled water and the second contained the same concentrations of hamycin in 50% horse serum. The tubes were incubated a t 37 OC for 30 minutes when their contents were tested for anti- biotic activity by an assay method (2) using C. lunata as the test organism. This experiment was repeated with the polyenes listed in Table 11.
TABLE I1 Antifungal antibiotics used for serum activity
Range of Names and source of antibiotics concentrations,
lJg/ml
1 Candicidin (Prairie Regional Laboratories,N.R.C., Saskatoon, Saskatchewan, Canada) 15-25
2 Candidin (Prairie Regional Laboratories, N.R.C., Saskatoon, Saskatchewan, Canada) 15-25
3 Etruscomycin (Lepetit, Rome, Italy) 10-15 4 Filipin (Upjohn Company, Kalamazoo, Michigan, U.S.A.) 50-100 5 Mycostatin (The Squibb Institute for Medical Research,
New Brunswick, N. J., U.S.A.) 100-175 6 Pimaricin (Koninklijke Nederlandsche Gisten
Spiritusfabriek, N.V., Delft, Holland) 30-50 7 Trichomycin (Fujisawa Pharmaceutical Company,
Tokyo, Japan) 2.5-10
This experiment was also repeated substituting, for horse serum, the serum and plasma of mice, rats, guinea pigs, rabbits, calves, and a man. Serum and plasma of a rabbit were dialyzed in sterile distilled water for 7 days, with cellophane paper as a membrane, and the experiment was repeated.
The results of antifungal activity of eight polyenes in the presence of horse serum are shown in Fig. 1. The activities of etruscomycin, pimaricin, candidin, candicidin, filipin, trichomycin, and mycostatin decreased in the presence of the serum while that of hamycin increased; similar results were obtained with
Can
. J. M
icro
biol
. Dow
nloa
ded
from
ww
w.n
rcre
sear
chpr
ess.
com
by
Uni
vers
ity o
f P.
E.I
. on
11/2
3/14
For
pers
onal
use
onl
y.
3 80 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 12, 1966
I I I I
FIG. 1. Antifungal activity of hamycin and seven other polyenes with and without horse serum. A = horse serum.
plasma. Figure 2 illustrates that hamycin in the presence of serum shows an increased zone of inhibition while amphotericin B and trichomycin show a decreased zone under similar conditions. (A part of the experiment is shown in Fig. 2 to demonstrate the differences in zones of inhibition by polyenes against C. lunata.) Dialyzed serum and dialyzed plasma of a.rabbit increased the activity of hamycin, which is shown in Fig. 3.
The above experiments were carried out by agar diffusion methods and i t was felt that serum or plasma might have increased the diffusibility of hamycin. T o exclude the possibility of this kind of physical phenomenon, an experiment to determine minimum inhibitory concentrations of 11 antifungal polyenes was carried out by the tube dilution method, with C. albicans as the test organism. The test was conducted in two sets using M2 broth (2). The in- oculum was prepared by suspending an 18-hour agar growth of C. albicans a t 37 "C in 0.85% saline and adjusting the suspension to 30% transmission a t 490 mp wave length in the Lumetron colorimeter. One drop of the inoculum (approximately 0.05 ml) was added to each tube. One set of tubes contained only MZ broth and the broth in the second set contained 10% horse serum. Two tubes were used for each of the concentrations in both the sets. All the broth tubes including the controls were incubated for 24 hours a t 37 O C and the results were read in the Klett-Summerson photoelectric colorimeter (blue filter No. 42).
Besides hamycin and the polyenes mentioned in Table 11, the following three antifungal antibiotics were included in the experiment:
PA150 (Pfizer Biochemical Laboratory, U.S.A.); amphotericin B (The Squibb Institute for Medical Research, New Bruns-
wick, N.J., U.S.A.); and
Can
. J. M
icro
biol
. Dow
nloa
ded
from
ww
w.n
rcre
sear
chpr
ess.
com
by
Uni
vers
ity o
f P.
E.I
. on
11/2
3/14
For
pers
onal
use
onl
y.
FIG. 2. Effect of serum on polyenes: (1) 0.25 pg/ml of hamycin in the presence of serum: (2) 0.25 pg/ml of hamycin (without serum); (3) 1.0 pg/ml of amphotericin B (without serum); (4) 1.0 pg/ml of amphotericin B with serum; (5) 0.5 pg/ml of trichomycin without serum: and (6) 0.5 pg/ml of trichomycin with serum. Note: Nos. 1, 3, and 5 show larger zones of inhibition than Nos. 2, 4, and 6 respectively.
Maniar and Mavdikar-Can. J. Microbial.
Can
. J. M
icro
biol
. Dow
nloa
ded
from
ww
w.n
rcre
sear
chpr
ess.
com
by
Uni
vers
ity o
f P.
E.I
. on
11/2
3/14
For
pers
onal
use
onl
y.
MANIAR AND MAVDIKAR: HAMYCIN
" D-si O I A L Y Z E D S E R U M
D-PI-. D I A L Y Z E D PLASMA
5 H = H C W V C I N
FIG. 3. Activity of hamycin with dialyzed rabbit serum and plasma.
aureofungin (7) (previously known as "Compound 496", Hindustan Anti- biotics Limited, Pimpri, India).
The above experiment was repeated with 5% crystalline egg albumin and 5yo bovine albumin.
I t can be seen from Table I11 that minimum inhibitory concentrations of hamycin and PA150 decreased in the presence of horse serum. In cases of other polyenes, the minimum inhibitory concentration increased. Similar results were obtained with bovine and crystalline egg albumin, showing that even albumins increase the activity of hamycin and PA150.
Experimenj 3 Activity of Proteolytic Enzymes The increase in activity of hamycin in the presence of albumin might be
due to (a) albumin itself (albumin combining with hamycin), or to (b) an
TABLE 111 Activity of 11 polyenes in MZ broth, with and without serum, expressed as minimum
inhibitory concentration
In MZ broth + serum In MZ broth only, Antibiotic b d m l
Hamycin Trichomycin Amphotericin B Candidin PA150 Candicidin Etruscomycin Filipin Pimaricine Mycostatin Aureofunein
NOTE: Results were read after 24 hours. * = 0.04 pg/ml after 48 hours.
Can
. J. M
icro
biol
. Dow
nloa
ded
from
ww
w.n
rcre
sear
chpr
ess.
com
by
Uni
vers
ity o
f P.
E.I
. on
11/2
3/14
For
pers
onal
use
onl
y.
382 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 12. 1966
TABLE IV Action of (partially) hydrolyzed albumin on hamycin
Zones of inhibition using Curvularia lunata (in mm)
0.25 pg/ml of hamycin in 0.25 pg/ml of albumin hydrolyzed by hamycin in al-
papaln at : bumin hydro- 0.25 pg(m! of lyzed by tryp- 0.25 pg/ml of hamyc~n m
37 "C 45 "C sin a t 37 "C hamycin 5% albumin
NOTE: Controls of 5 % albumin, 1.5 mg/ml of papain, and 0.002% trypsin did not give any zone of inhibition.
enzyme present in the albumin as an impurity. (A suitable enzyme might change the hamycin molecule to give higher activity.) To show that the al- bumin molecule is responsible for the higher activity of hamycin, the following experiments were carried out.
(A) Four milliliters of papain solution (3 mg/ml) were mixed with 4 ml of 0.5 pg/ml of hamycin. The mixture was incubated a t 37 OC for 30 minutes. The zone of inhibition by the above solution against C. lunata was compared with that of an equal quantity of hamycin (0.25 pg/ml) incubated in a similar manner. The experiment was repeated by incubating the papain hamycin mixture and hamycin alone a t 45 "C.
(B) Four milliliters of papain solution (3 mg/ml) were mixed with equal volumes of 15% bovine albumin. Half of the quantity was allowed to react a t 45 OC for 30 minutes and the remaining half of the mixture was reacted a t 37 OC for 30 minutes. These papain albumin mixtures were mixed with equal volumes of 0.5 pg/ml solution of hamycin and incubated a t 37 OC for 30 min- utes. Zones of inhibition produced by these mixtures were compared with a solution of 0.25 pg/ml of hamycin treated in a similar manner. Controls of papain alone, albumin alone, and a papain and albumin mixture were included. This experiment was repeated four times.
(C) The above experiments A and B were repeated with 0.004% trypsin instead of papain a t a reaction temperature of 37 OC.
At a reaction temperature of 37 "C, papain and trypsin did not appreciably alter the activityof hamycin. Similarly a t 4S°C, papain did not alter its activity.
Bovine albumin was hydrolyzed by papain and trypsin. The hydrolyzed fractions of bovine albumin were allowed to react with hamycin. I t can be seen from Table IV that hamycin did not show any increased activity with hydrolyzed albumin.
Discussion Based on the results presented, the following conclusions can be made. (1) Hamycin eliminated cryptococci present in the lungs of mice. (2) Serum, plasma, and albumins increased the antifungal activity of
hamycin.
Can
. J. M
icro
biol
. Dow
nloa
ded
from
ww
w.n
rcre
sear
chpr
ess.
com
by
Uni
vers
ity o
f P.
E.I
. on
11/2
3/14
For
pers
onal
use
onl
y.
MANIAR AND MAVDIKAR: HAMYCIN 383
(3) Hamycin activity was not affected by papain, trypsin, or partially hydrolyzed albumin. This indicated that the complete albumin molecule was necessary for increasing the activity of hamycin.
C. neoformans is a causative agent of meningitis but since these organisms can be detected in lungs 30 days after intravenous injection, the above experiment on mouse protection studies (Table I) was carried out. Since hamycin elimin- ated cryptococci from the lungs of mice, i t verifies our previous finding (4) that hamycin penetrates lung tissues. This experiment is not intended to demonstrate the protective action of hamycin in meningitis caused by C. neoformans, but merely to show that hamycin is active in tissues. The detailed work on the protection of mice was carried out by Padhye and Thirumalachar (5) and further work by them on this line will be published elsewhere.
Most of the polyenes are known to be inactivated in the presence of cho- lesterol or serum. Though hamycin is inactivated in high concentrations of cholesterol, its activity, surprisingly, is increased by the addition of serum. This increase in activity was also noted with plasma and sera of different animals and a man. Hence, t o rule out the fact that the presence of electrolytes in sera or plasma is responsible for the increase in activity of hamycin, rabbit serum and plasma were dialyzed for 7 days. The dialyzed serum and plasma also increased the activity of hamycin (Fig. 3) showing that electrolytes in serum or plasma have no effect on hamycin. The minimum inhibitory con- centration is an absolute value under given conditions of medium, tempera- ture. inoculum. and time of incubation. Hence. the results shown in Table I11 indicate that horse serum does increase the kctivity of hamycin. Similarly PA150 gave higher activity with serum. The activity of aureofungin was decreased after 24 hours incubation. At the end of 48 hours. however. its minimum inhibitory concentration was decreased in the presence of serum.
Since plasma, serum, and "dialyzed serum" increased the activity of hamy- cin, it was thought that albumin might be responsible for this increase. I t was found, as expected, that 5% bovine albumin and 5% crystalline egg albumin increased the activity of hamycin and PA150.
The action of proteolytic enzymes was studied to determine whether any change in the activity of hamycin could be accomplished. I t was found that neither papain nor trypsin apparently affected the activity of hamycin. Since i t was shown that albumin is responsible for the increase in activity, the action of hydrolyzed bovine albumin (by papain a t 37 "C and 45 "C and by trypsin at 37 "C for 3 hour) on hamycin was studied. The partially hydrolyzed por- tions of albumin failed to increase the activity of hamycin (Table IV), which indicates that albumin increased the activity of hamycin either by chemical or physical combination. I t may be mentioned here that the degree of hydro- lysis by papain or trypsin was not measured and their specific action on albumin molecules was not determined. Hence, the word partial hydrolysis is used above. The activity of papain and trypsin on hamycin and albumin was resolved by the microbiological assay method. A chemical method might have given more accurate answers to such a problem. The possibility that the serum globulins or other proteins may increase the hamycin activity is not ruled out by the above experiments.
Can
. J. M
icro
biol
. Dow
nloa
ded
from
ww
w.n
rcre
sear
chpr
ess.
com
by
Uni
vers
ity o
f P.
E.I
. on
11/2
3/14
For
pers
onal
use
onl
y.
384 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 12. 1966
Action of papain and trypsin on hamycin was tried for the following reasons. (a) The structure of hamycin molecule is not, as yet, elucidated. Hamycin
is a polyene and it may contain some amino acids or a peptide group. I t is possible that hamycin may bind a protein molecule in the broth. If hamycin activity would have been changed by the proteolytic enzymes, the above possibility would have been indicated.
(b) Hydrolyzed albumin (Table IV) might contain some active papain or trypsin. The above experiment has served as a control showing that proteolytic enzymes have no action on hamycin.
One of the numerous enzymes present in serum and plasma might act on hamycin molecule giving higher activity. Since hydrolyzed albumin did not increase the antifungal activity and crystalline egg albumin increased it, it is reasonably safe to conclude that albumin molecule, in this case, was required to increase the activity of hamycin.
Acknowledgments We thank Dr. Peter Warner, Winnipeg General Hospital, for his interest,
valuable suggestions, and constructive criticisms in going through the manu- script. We are thankful to Dr. M. J. Thirumalachar, Hindustan Antibiotics Limited, for his help and appreciation. The technical help of Mr. M. N. Wamburker, Hindustan Antibiotics Limited, and Miss C. Kuz, Winnipeg General Hospital, is acknowledged with thanks.
References 1. BHATE, D. S., AMBEKAR, G. R., BHATNAGER, K. K., and HULYARKAR, R. K. 1961. Hamycin.
11. Isolation and chemical properties. Hindustan Antibiot. Bull. 3, 139. 2. MANIAR, A. C. and MAVDIKAR, S. 1962. Microbiological assay of hamycin and other anti-
fungal polyene antibiotics. Hindustan Antibiot. Bull. 4, 168-171. 3. MANIAR, A. C. and MAVDIKAR, S. 1963. Antifungal activity of hamycin and other polyenes
in the presence of serum. (Short note). Hindustan Antibiot. Bull. 6, No. 1, 19-21. 4. MANIAR, A. C. and MAVDIKAR, S. 1963. Tissue concentrations of hamycin by oral feeding
in laboratory animals. Hindustan Antibiot. Bull. 5, No. 4, 113-116. 5. PADHYE, A. A. and THIRUMALACHAR, M. J. 1963. Hamycin in the treatment of Cuyplococcus
neoformans infection in mice. (Preliminary communication). Hindustan Antibiot. Bull. 6, No. 2, 41-43.
6. THIRUMALACHAR, M. J., MENON, S. K., and BHATT, V. V. 1961. Hamycin. I. Discovery and biological studies. Hindustan Antibiot. Bull. 3, 136-138.
7. THIRUMALACHAR, M. J., RAHALKAR, P. W., SUKAPURE, R. S., and GOPALKRISHNAN, K. S. 1964. Aureofungin-a new heptaene antibiotic. Part I. Microbiological Studies. Hindustan Antibiot. Bull. 6, 108-111.
Can
. J. M
icro
biol
. Dow
nloa
ded
from
ww
w.n
rcre
sear
chpr
ess.
com
by
Uni
vers
ity o
f P.
E.I
. on
11/2
3/14
For
pers
onal
use
onl
y.