lHERAPEUTIC AND PROPHYlACTIC EFFECTS OF ROMURTIDE AGAINST EXPERIMENTAL ANIMAL INFECTIONS Yoshihito Niki and Osamu Tatara * Department of Primary Health Care and Preventive Medicine and Department of Internal Medicine* Kawasaki Medical School Kurashiki City, Japan INTRODUCTION Infectious complications are common and often life-threatening problems in immunocompromised patients. Despite remarkable developments of new anti-microbial agents and life-supporting treatments in the past decade, certain infections remain difficult to control when the immune condition of a patient is severely depressed. In these patients without adequate cellular and/or humoral defenses, even potent antimicrobial agents may become less effective. And patients who survive the first episode of infection are at high risk for recurrence of subsequent periods of immunosuppression. Immunopotentiating substances and biological response modifiers, such as muramyl dipeptide (MDP) and various kinds of cytokines have been increasingly recognized to be of major importance for treating infections. MDP is a minimal structural requirement for adjuvant activities of the bacterial cell wall, and the compound and its analogs have been revealed to enhance the resistance to infection (1). In the present report, we evaluated the biological activity of Romurtide, a promising synthetic analog of MDP, in our animal models of Klebsiella and Aspergillus pulmonary infection. Effects of Romurtide on K pneumoniae Pneumonia in Mice We investigated the synergistic effects of Romurtide and Cefmenoxime, a standard third generation cephalosporin, in the treatment of experimental Klebsiella pneumonia in mice. Mice were infected with 104 cfu of K pneumonia by inhalation of an aerosolized bacterial suspension. Quantitative cultures of lungs of mice showed rapid proliferation of the organism 2 to 3 days after the infection. About 95% of the untreated animals died of a systemic Klebsiella infection within one week. When mice were treated with a subcutaneous injection of Cefmenoxime twice daily for 3 days, starting 1 day after the infection, a dose-dependent increase of survival rates was observed. Microbial Infections, Edited by H. Friedman et aI. Plenum Press, New York, 1992
185
To clarify the synergistic effect of Romurtide, mice were treated with Cefmenoxime at a daily dose of 40 mg/kg (lower than the EDso). Romurtide was injected subcutaneously at a dose of 100 Ilg/animal on 3 different schedules. When Romurtide was administered one day prior to the infection, a significantly higher survival rate of 80% was observed on day 7. Even with a single dose of 100 Ilg of Romurtide administered one day after the infection, the survival rate observed was higher than that of mice treated with Cefmenoxime alone; still, more than 50% of the mice in this group died within a week. One additional injection of the compound at 3 days after the infection, however, improved the survival rate to 60%. Quantitative cultures of K pneumoniae in lungs of animals treated with Cefmenoxime alone showed a retardation of the proliferation compared to those of control mice. Most animals, however, showed higher counts at days three and four. In the lungs of mice treated with the combination chemotherapy, the growth of the bacteria was apparently suppressed, and rarely exceeded the initial counts of 104 cfu (colony forming unit) even 4 days after the infection. A histopathological study of lungs of these animals also showed evidence of a protective activity of Romurtide. In a lung section of a mouse treated with a prophylactic dose of Romurtide one day prior to infection and sacrificed 6 hr after infection, we can see an early response of macrophages and neutrophils into alveolar spaces, while no inflammatory cell infiltration was observed in the sections of lungs from control animals at this point. An accumulation of inflammatory cells in alveolar spaces became apparent 18 - 24 hr after the infection. A prophylactic dose of 100 Ilg of Romurtide affected the functions of the alveolar macrophages. Mean total cell count in alveolar lavage fluid from animals with Romurtide prophylaxis was significantly higher than that from PBS treated control animals at 24 hr after the infection; however, no significant difference was observed at 2 and 3 days after the infection. The chemotactic and phagocytic activities of alveolar macrophages harvested from mice 1 to 4 days after a single dose of Romurtide were also examined. As shown in Table 1, the migration of macrophages through a filter was enhanced significantly at 2 to 4 days after Romurtide injection. The phagocytic index of these macrophages was also significantly higher than that of PBS treated control at 24 hr after the infection. It is suggested that the enhancement of host resistance to experimental Klebsiella pneumonia by Romurtide is attributed in part to an augmentation of the functions of macrophages and neutrophils. Effects of Romurtide on A fumigatus Lung Infection in Rats Recently, not only bacterial but fungal infection has become another problem in immunocompromised patients. Pulmonary aspergillosis, in particular, is a potentially Table 1.
Stimulation of Alveolar Macrophage Functions in Mice Treated with 100 Ilg!mouse Romurtide
Function Chemotaxis
Phagocytosis
Treatment
Days after single dose treatment 1
2
3
PBS
95.8:!:19.2
45.6:!:28.3
37.2:!:17.1
69.2:!:25.1
ROM
64.6:!:18.7
160.3:!:15.0
91. 0:!:39.6
135.2:!:48.6
PBS
25.9:!: 7.2
27.7:!: 4.0
25.3:!: 7.1
27.5:!: 5.8
ROM
* 45.5:!:
36.3:!: 5.5
36.5:!:11.3
32.3:!: 7.5
0.9
*
4
Chemotaxis: number of chemotactic cells/HPF Phagocytosis: % of macrophages with Latex particles *: P < 0.05 186
*
life-threatening complication among neutropenic patients with leukemia or lymphoma and bone marrow transplant recipients. In spite of efforts to improve diagnosis and treatments over the last decade, early and specific diagnosis of this infection is still difficult, and treatment may be toxic and often fails. We have three different models of pulmonary aspergillosis: an acute model, a model of chronic infection, and a model where recurrent infection is induced by immunosuppression. The prophylactic and therapeutic effects of Romurtide were evaluated in these models. In the acute model, male SD rats were treated for two weeks with three doses per week of 100 mg/kg of cortisone acetate while being maintained on a low protein (8%) diet and receiving tetracycline via their drinking water. Animals were infected by surgically exposing their trachea and injecting a suspension of 106 conidia of A. fumigatus. Animals develop a rapidly progressive aspergillus infection that remains confined to the lungs. Mortality in this model was 100% by 7 to 10 days post infection. In this acute model, we administered 3 to 7 doses of 500 or 1,000 ~g of Romurtide beginning one day prior to infection. Doses were administered by subcutaneous injection or by gastric lavage. In all experiments, the survival of untreated control was 0% by day 8. In some experiments, Romurtide appeared to delay mortality, however, as sole therapy, the drug did not have a statistically significant effect on mortality in the acute model of pulmonary aspergillosis. We have used this acute model to evaluate the effectiveness of various antifungal agents, including aerosol amphotericin B treatment and prophylaxis (2-4). The aerosol route of amphotericin B is more efficient than the intravenous route in delivering the drug to the lung; it also should be less toxic since it limits accumulation in other organs. We also investigated the activity of Romurtide in the acute model, when combined with aerosol amphotericin B. Three daily doses of Romurtide were administered by subcutaneous injection beginning one day prior to infection; a single dose of 1.6 mg/kg of aerosol amphotericin B prophylaxis was administered 2 days prior to infection. In this experiment, the survival rate was not significantly different for animals that received both aerosol amphotericin Band Romurtide and animals that received aerosol amphotericin B alone (Figure 1). We also compared the effects of the compound when
100 80 ~
010
II>
....III
60
iii > >
40
Qj
-------------
t
____ Aero-AmB Control (n =45) - -: 1. 6 mg / kg x 1 :_, 48 hrs prior to Infection
-~
Infection
,
a::
/'
Aero-AmB + Romurtide 500).lg x 3
'-
:J VI
32.4%
._- _.
28.9%
20 Control (n 0
7
=40)
14
21
Days after Infection Romurtide; 500).lg s.c.,
day -1, 0, +1
Fig. 1. Combination therapy with amphotericin B and Romurtide against acute Rat Pulmonary Aspergillosis. 187
combined with intraperitoneal doses of a!llphotericin B. The doses of amphotericin B were highly effective, however, and the combination with Romurtide did not improve survival over that of amphotericin B alone. We were concerned that our inability to demonstrate the therapeutic effect of Romurtide might result from the fact that the high inoculum, acute model produced a very severe, rapidly progressive infection. Because the infection is quickly fatal, subtle enhancements of non-specific immunity are unlikely to significantly alter the outcome. So next we investigated the effects of Romurtide in models of more indolent or chronic infection. To produce a model of chronic infection, we used a lower inoculum, lOS conidia, and discontinued steroids and low protein diet at one week post infection. In this model, Romurtide, when given alone at high doses, did not prevent mortality; however, it significantly delayed mortality compared to controls. By day 21, the survival rate among controls was 23.5% and was 43% among animals that had received 9 doses of Romurtide. Figure 2 shows the effects of aerosol amphotericin B alone or combined with Romurtide in the chronic model. Romurtide, given daily for three days beginning one day prior to infection did not enhance the activity of amphotericin B. However, we did observe a reduction in mortality when amphotericin B treated animals were treated with three doses per week of Romurtide beginning on the first day post infection. It is well recognized that immunosuppressed patients who develop aspergillosis may experience a recurrence of the infection during a subsequent period of immunosuppression. In our chronic model, by the end of week four post infection, animals had regained more than 60% of the weight lost during the steroid induction period and appeared to be healthy. However, at necropsy, they had more than l(f cfu of A. fumigatus in their lungs. Histopathological sections of lungs from these animals showed many well developed focal lesions which were encapsulated by macrophages, lymphocytes and multi-nucleated giant cells. In the center of the lesions we could see branching septate hyphae and necrosis in the silver stained section. We adapted this chronic model to investigate the question of recurrence. Animals received a prophylactic dose of aerosol amphotericin B, steroids were discontinued one week post
Aero-AmB + Romurtide (n = 8) 100 80 Ul
2
III
t
Infection 1 x 105
60
100%
Aero-Am~- ---- - --: __8_8_. 9%
control (n=36Y87.5% Aero-AmB + Romurtide A (n = 32)
0:::
iii
.:;>
40
'j
VI
'--_ _2....:;3.5%
20
Control (n = 34)
o
7 14 Days after Infection
21
Aero-AmB; 1.6 mg/kg xl 48 hrs prior to Infection Romurtide A; 500,.ugx3 day -1,0, +1 Romurtide B; 500,.ug x 3/week day +1'" +20
Fig. 2. Combination therapy with amphotericin B and Romurtide against chronic Rat Pulmonary Aspergillosis. 188
infection and then restarted four weeks post infection. During the second period of steroid treatments, animals also were treated with Ceftriaxone to prevent bacterial infections. When steroids were discontinued, there was a slow but steady clearance of the fungus from the lungs, and when steroids are restarted the organism began to proliferate. The infection had spread rapidly throughout the entire lung and the mortality rate was 90% by the seventh week of the second course of steroids. We studied the activity of Romurtide in this model. Figure 3 shows the results of quantitative cultures during weeks 4 through 7 post infection. As mentioned previously, when steroids were discontinued, as shown on the left, there was slow clearance of aspergillus from the lungs; the rate of clearance was similar for the control and Romurtide treated animals. When steroids were restarted, as shown on the right, there was a proliferation of aspergillus in the lungs of control rats. In contrast, despite the reinstitution of steroid immunosuppression, aspergillus was cleared from the lungs when animals were treated with Romurtide. Total mortality rate of animals at the seventh week of steroid treatment was significantly higher in the control group. In this model of recurrent aspergillosis, a second course of steroids caused reactivation and progression of the infection, while treatment with Romurtide appeared to prevent reactivation and to promote clearance of the fungus. DISCUSSION Romurtide was selected as a compound showing higher biological activity but less pyrogenicity than other compounds through studies on the structure-activity relationship. The profile and underlying mechanisms of the stimulating effects of Romurtide on resistance to infection in animals have been investigated and reported by many investigators. The drug has been confirmed to enhance several immune-functions as shown in Table 2. It could stimulate not only animal macrophages but also human macrophages to induce interleukins, CSFs and complement component and so on.
Romurtide 500,u9 x 3/week s.c. 6
I
• 0
Cl C
:l
Steroid I
•
•
Control
• Control
Romurtide
o Romurtide
5
~
I
•
I
..J
.,.a.
II
....:lu
3
Cl
•
•
•
.. -~ .. ' .. ' .. -
III
185
To clarify the synergistic effect of Romurtide, mice were treated with Cefmenoxime at a daily dose of 40 mg/kg (lower than the EDso). Romurtide was injected subcutaneously at a dose of 100 Ilg/animal on 3 different schedules. When Romurtide was administered one day prior to the infection, a significantly higher survival rate of 80% was observed on day 7. Even with a single dose of 100 Ilg of Romurtide administered one day after the infection, the survival rate observed was higher than that of mice treated with Cefmenoxime alone; still, more than 50% of the mice in this group died within a week. One additional injection of the compound at 3 days after the infection, however, improved the survival rate to 60%. Quantitative cultures of K pneumoniae in lungs of animals treated with Cefmenoxime alone showed a retardation of the proliferation compared to those of control mice. Most animals, however, showed higher counts at days three and four. In the lungs of mice treated with the combination chemotherapy, the growth of the bacteria was apparently suppressed, and rarely exceeded the initial counts of 104 cfu (colony forming unit) even 4 days after the infection. A histopathological study of lungs of these animals also showed evidence of a protective activity of Romurtide. In a lung section of a mouse treated with a prophylactic dose of Romurtide one day prior to infection and sacrificed 6 hr after infection, we can see an early response of macrophages and neutrophils into alveolar spaces, while no inflammatory cell infiltration was observed in the sections of lungs from control animals at this point. An accumulation of inflammatory cells in alveolar spaces became apparent 18 - 24 hr after the infection. A prophylactic dose of 100 Ilg of Romurtide affected the functions of the alveolar macrophages. Mean total cell count in alveolar lavage fluid from animals with Romurtide prophylaxis was significantly higher than that from PBS treated control animals at 24 hr after the infection; however, no significant difference was observed at 2 and 3 days after the infection. The chemotactic and phagocytic activities of alveolar macrophages harvested from mice 1 to 4 days after a single dose of Romurtide were also examined. As shown in Table 1, the migration of macrophages through a filter was enhanced significantly at 2 to 4 days after Romurtide injection. The phagocytic index of these macrophages was also significantly higher than that of PBS treated control at 24 hr after the infection. It is suggested that the enhancement of host resistance to experimental Klebsiella pneumonia by Romurtide is attributed in part to an augmentation of the functions of macrophages and neutrophils. Effects of Romurtide on A fumigatus Lung Infection in Rats Recently, not only bacterial but fungal infection has become another problem in immunocompromised patients. Pulmonary aspergillosis, in particular, is a potentially Table 1.
Stimulation of Alveolar Macrophage Functions in Mice Treated with 100 Ilg!mouse Romurtide
Function Chemotaxis
Phagocytosis
Treatment
Days after single dose treatment 1
2
3
PBS
95.8:!:19.2
45.6:!:28.3
37.2:!:17.1
69.2:!:25.1
ROM
64.6:!:18.7
160.3:!:15.0
91. 0:!:39.6
135.2:!:48.6
PBS
25.9:!: 7.2
27.7:!: 4.0
25.3:!: 7.1
27.5:!: 5.8
ROM
* 45.5:!:
36.3:!: 5.5
36.5:!:11.3
32.3:!: 7.5
0.9
*
4
Chemotaxis: number of chemotactic cells/HPF Phagocytosis: % of macrophages with Latex particles *: P < 0.05 186
*
life-threatening complication among neutropenic patients with leukemia or lymphoma and bone marrow transplant recipients. In spite of efforts to improve diagnosis and treatments over the last decade, early and specific diagnosis of this infection is still difficult, and treatment may be toxic and often fails. We have three different models of pulmonary aspergillosis: an acute model, a model of chronic infection, and a model where recurrent infection is induced by immunosuppression. The prophylactic and therapeutic effects of Romurtide were evaluated in these models. In the acute model, male SD rats were treated for two weeks with three doses per week of 100 mg/kg of cortisone acetate while being maintained on a low protein (8%) diet and receiving tetracycline via their drinking water. Animals were infected by surgically exposing their trachea and injecting a suspension of 106 conidia of A. fumigatus. Animals develop a rapidly progressive aspergillus infection that remains confined to the lungs. Mortality in this model was 100% by 7 to 10 days post infection. In this acute model, we administered 3 to 7 doses of 500 or 1,000 ~g of Romurtide beginning one day prior to infection. Doses were administered by subcutaneous injection or by gastric lavage. In all experiments, the survival of untreated control was 0% by day 8. In some experiments, Romurtide appeared to delay mortality, however, as sole therapy, the drug did not have a statistically significant effect on mortality in the acute model of pulmonary aspergillosis. We have used this acute model to evaluate the effectiveness of various antifungal agents, including aerosol amphotericin B treatment and prophylaxis (2-4). The aerosol route of amphotericin B is more efficient than the intravenous route in delivering the drug to the lung; it also should be less toxic since it limits accumulation in other organs. We also investigated the activity of Romurtide in the acute model, when combined with aerosol amphotericin B. Three daily doses of Romurtide were administered by subcutaneous injection beginning one day prior to infection; a single dose of 1.6 mg/kg of aerosol amphotericin B prophylaxis was administered 2 days prior to infection. In this experiment, the survival rate was not significantly different for animals that received both aerosol amphotericin Band Romurtide and animals that received aerosol amphotericin B alone (Figure 1). We also compared the effects of the compound when
100 80 ~
010
II>
....III
60
iii > >
40
Qj
-------------
t
____ Aero-AmB Control (n =45) - -: 1. 6 mg / kg x 1 :_, 48 hrs prior to Infection
-~
Infection
,
a::
/'
Aero-AmB + Romurtide 500).lg x 3
'-
:J VI
32.4%
._- _.
28.9%
20 Control (n 0
7
=40)
14
21
Days after Infection Romurtide; 500).lg s.c.,
day -1, 0, +1
Fig. 1. Combination therapy with amphotericin B and Romurtide against acute Rat Pulmonary Aspergillosis. 187
combined with intraperitoneal doses of a!llphotericin B. The doses of amphotericin B were highly effective, however, and the combination with Romurtide did not improve survival over that of amphotericin B alone. We were concerned that our inability to demonstrate the therapeutic effect of Romurtide might result from the fact that the high inoculum, acute model produced a very severe, rapidly progressive infection. Because the infection is quickly fatal, subtle enhancements of non-specific immunity are unlikely to significantly alter the outcome. So next we investigated the effects of Romurtide in models of more indolent or chronic infection. To produce a model of chronic infection, we used a lower inoculum, lOS conidia, and discontinued steroids and low protein diet at one week post infection. In this model, Romurtide, when given alone at high doses, did not prevent mortality; however, it significantly delayed mortality compared to controls. By day 21, the survival rate among controls was 23.5% and was 43% among animals that had received 9 doses of Romurtide. Figure 2 shows the effects of aerosol amphotericin B alone or combined with Romurtide in the chronic model. Romurtide, given daily for three days beginning one day prior to infection did not enhance the activity of amphotericin B. However, we did observe a reduction in mortality when amphotericin B treated animals were treated with three doses per week of Romurtide beginning on the first day post infection. It is well recognized that immunosuppressed patients who develop aspergillosis may experience a recurrence of the infection during a subsequent period of immunosuppression. In our chronic model, by the end of week four post infection, animals had regained more than 60% of the weight lost during the steroid induction period and appeared to be healthy. However, at necropsy, they had more than l(f cfu of A. fumigatus in their lungs. Histopathological sections of lungs from these animals showed many well developed focal lesions which were encapsulated by macrophages, lymphocytes and multi-nucleated giant cells. In the center of the lesions we could see branching septate hyphae and necrosis in the silver stained section. We adapted this chronic model to investigate the question of recurrence. Animals received a prophylactic dose of aerosol amphotericin B, steroids were discontinued one week post
Aero-AmB + Romurtide (n = 8) 100 80 Ul
2
III
t
Infection 1 x 105
60
100%
Aero-Am~- ---- - --: __8_8_. 9%
control (n=36Y87.5% Aero-AmB + Romurtide A (n = 32)
0:::
iii
.:;>
40
'j
VI
'--_ _2....:;3.5%
20
Control (n = 34)
o
7 14 Days after Infection
21
Aero-AmB; 1.6 mg/kg xl 48 hrs prior to Infection Romurtide A; 500,.ugx3 day -1,0, +1 Romurtide B; 500,.ug x 3/week day +1'" +20
Fig. 2. Combination therapy with amphotericin B and Romurtide against chronic Rat Pulmonary Aspergillosis. 188
infection and then restarted four weeks post infection. During the second period of steroid treatments, animals also were treated with Ceftriaxone to prevent bacterial infections. When steroids were discontinued, there was a slow but steady clearance of the fungus from the lungs, and when steroids are restarted the organism began to proliferate. The infection had spread rapidly throughout the entire lung and the mortality rate was 90% by the seventh week of the second course of steroids. We studied the activity of Romurtide in this model. Figure 3 shows the results of quantitative cultures during weeks 4 through 7 post infection. As mentioned previously, when steroids were discontinued, as shown on the left, there was slow clearance of aspergillus from the lungs; the rate of clearance was similar for the control and Romurtide treated animals. When steroids were restarted, as shown on the right, there was a proliferation of aspergillus in the lungs of control rats. In contrast, despite the reinstitution of steroid immunosuppression, aspergillus was cleared from the lungs when animals were treated with Romurtide. Total mortality rate of animals at the seventh week of steroid treatment was significantly higher in the control group. In this model of recurrent aspergillosis, a second course of steroids caused reactivation and progression of the infection, while treatment with Romurtide appeared to prevent reactivation and to promote clearance of the fungus. DISCUSSION Romurtide was selected as a compound showing higher biological activity but less pyrogenicity than other compounds through studies on the structure-activity relationship. The profile and underlying mechanisms of the stimulating effects of Romurtide on resistance to infection in animals have been investigated and reported by many investigators. The drug has been confirmed to enhance several immune-functions as shown in Table 2. It could stimulate not only animal macrophages but also human macrophages to induce interleukins, CSFs and complement component and so on.
Romurtide 500,u9 x 3/week s.c. 6
I
• 0
Cl C
:l
Steroid I
•
•
Control
• Control
Romurtide
o Romurtide
5
~
I
•
I
..J
.,.a.
II
....:lu
3
Cl
•
•
•
.. -~ .. ' .. ' .. -
III
