U.S. patent application number 14/296073 was filed with the patent office on 2014-10-02 for medicament for the treatment of fungal infections particularly aspergillosis.
The applicant listed for this patent is SIGMA-TAU Industrie Farmaceutiche Riunite S.p.A.. Invention is credited to Paolo Carminati, Luigina Romani, Giovanni Salvatori.
Application Number | 20140296135 14/296073 |
Document ID | / |
Family ID | 34968212 |
Filed Date | 2014-10-02 |
United States Patent
Application |
20140296135 |
Kind Code |
A1 |
Salvatori; Giovanni ; et
al. |
October 2, 2014 |
MEDICAMENT FOR THE TREATMENT OF FUNGAL INFECTIONS PARTICULARLY
ASPERGILLOSIS
Abstract
The combination of pentraxin PTX3 with antifungals is described
for the treatment of fungal infections and particularly for
infections caused by Aspergillus fumigatus.
Inventors: |
Salvatori; Giovanni;
(Pomezia (RM), IT) ; Carminati; Paolo; (Pomezia
(RM), IT) ; Romani; Luigina; (Perugia (PG),
IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SIGMA-TAU Industrie Farmaceutiche Riunite S.p.A. |
Rome |
|
IT |
|
|
Family ID: |
34968212 |
Appl. No.: |
14/296073 |
Filed: |
June 4, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11579805 |
Nov 7, 2006 |
8778389 |
|
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PCT/IT2005/000247 |
Apr 28, 2005 |
|
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14296073 |
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Current U.S.
Class: |
514/3.3 ;
424/450; 514/21.2 |
Current CPC
Class: |
A61K 31/7048 20130101;
A61K 31/7048 20130101; A61P 11/00 20180101; A61P 31/10 20180101;
A61P 43/00 20180101; A61K 38/1709 20130101; A61P 31/00 20180101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 38/1709
20130101 |
Class at
Publication: |
514/3.3 ;
424/450; 514/21.2 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61K 31/7048 20060101 A61K031/7048 |
Foreign Application Data
Date |
Code |
Application Number |
May 7, 2004 |
IT |
RM2004A000223 |
Claims
1. Combination of pentraxin PTX3 and an antifungal agent.
2. Combination according to claim 1, wherein the antifungal agent
is amphotericin B.
3. Combination according to claim 2, wherein the amphotericin B is
in deoxycholate form or in a liposomal formulation.
4. A pharmaceutical composition comprising the combination
according to claim 1.
5. The composition of claim 4 comprising a suboptimal dose of the
antifungal agent.
6. A method for the therapeutic treatment of fungal infections,
comprising administering an effective dose of a medicament
comprising the pharmaceutical composition of claim 4 to a subject
in need thereof.
7. The method of claim 6, wherein said medicament comprises a
suboptimal dose of the antifungal agent.
8. The method according to claim 6, wherein the fungal infection is
pulmonary aspergillosis.
9. A method for the therapeutic treatment of pulmonary
aspergillosis, comprising administering an effective dose of
pentraxin PTX3 and an antifungal agent to a subject in need
thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. application Ser.
No. 11/579,805, filed on Nov. 7, 2006, which is a U.S. National
Stage of PCT/IT2005/000247 filed on Apr. 28, 2005, which claims
priority to and the benefit of Italian Application No.
RM2004A000223 filed on May 7, 2004, the contents of each of which
are incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to a medicament consisting of
a combination of pentraxin PTX3 and an antifungal for the treatment
of aspergillosis.
BACKGROUND TO THE INVENTION
[0003] Invasive aspergilosis (IA) is the main cause of nosocominal
pneumonia and the death in allogeneic bone marrow (BM) transplants
with an infection rate estimated as ranging from 8% to 15% and an
associated mortality rate of approximately 90% (3, 16, 30, 36, 47).
Despite progress in early diagnosis and therapy with the new
anti-fungal agents (6, 31), the majority of cases of AI remain
undiagnosed and untreated (16). The most important risk factor for
AI historically used to be neutropaenia. However, the advances made
in the preparation of patients undergoing chemotherapy and
transplantation have led to a significant reduction in the
neutropaenia period. Numerous studies have documented that
aspergillosis manifests itself following bone marrow transplants
concomitantly with the onset of graft-versus-host (GvH) disease
(30). The occurrence of IA also in non-neutropaenic patients (17)
confirms the importance of specific defects in both the innate and
adaptive immune effector mechanisms in the pathogenesis of the
disease (13, 20, 22, 32, 39, 43, 44). In particular, the role of Th
lymphocytes in providing an essential secondary defence against the
fungus has recently been assessed (8-10, 12, 14, 23, 26). Since IA
is extremely rare in immunocompetent individuals, therapy aimed at
enhancing the host's immune response offers a new and promising
approach in the treatment of this infection.
[0004] The innate immune system has evolved in a complex,
multi-faceted manner to protect lung tissue against infections. The
protection of lung tissue is thought to comprise not only a
preventive control of microbial proliferation, but also the
execution of a balanced inflammatory response sufficient to contain
the infection without inducing dangerous degrees of alveolar
exudation and infiltration. The molecular components of the
alveolar lining have recently been the focus of considerable
attention as primary immunomodulators in infections (28, 29).
[0005] The pentraxins (PTX) are a superfamily of proteins conserved
during the evolution from Limulus polyphemus to man, generally
characterised by a pentameric structure (21). PTX3 is a prototype
of the long pentraxin that consists in an N-terminal portion
coupled to the pentraxin C-terminal domain, the latter being a
homologue of the short PTXs (7). PTX3 is secreted by various types
of cells, particularly by mononuclear phagocytes, endothelial cells
and dendritic cells (DC), in response to the primary inflammatory
cytokines in vitro and in vivo (11, 38, 18). Increases in
circulating levels of this protein have been detected in various
different infectious and inflammatory conditions (37, 19, 34, 41).
PTX3 binds a number of selected microbial agents (e.g. A. fumigatus
conidia and P. aeruginosa) and activates various effector pathways
of the immune system to combat the infectivity of the pathogen
(20). Analysis of PTX3-deficient mice has demonstrated that PTX3 is
a pattern recognition receptor (PRR) that plays a non-redundant
role in the resistance to selected pathogens (20). The
susceptibility of PTX3-deficient mice to A. fumigatus has been
associated with unsuccessful organisation of the type I adaptive
immune response, but is restored by the exogenous administration of
recombinant PTX3 (20).
[0006] There is a need for therapeutic advances in combating
aspergillosis, despite the recent expansion of the antifungal
armamentarium (45).
[0007] Researchers are beginning to explore new strategies with a
singular combination of antifungals and cytokines (46). Treatment
with amphotericin B, a first-choice drug, is limited by
dose-related nephrotoxity which precludes full-dose therapy in
patients who have been submitted to BM transplants (24). Various
amphotericin B lipid-based formulations have been developed to
reduce the toxicity associated with conventional D-AmB (25), as
well as with L-AmB (2). The pharmacokinetic profile of L-AmB
toxicity is more favourable than that of D-AmB, thus making
full-dose therapy possible. Nevertheless, the failure rate is still
a matter for concern (1). Published murine models of IA, in which
the efficacy of antifungal agents has been assessed, have relied on
neutropaenia induced by chemotherapy with or without
corticosteroids to make the mice less susceptible to infection.
Recently there has been an increase in the development of new
antifungal agents for the treatment of IA, with drug classes
characterised by new therapeutic targets (45), which are giving
rise to new expectations for the treatment of IA and which increase
the number of new combined therapies possible (45). On the basis of
the treatment of other infectious diseases (4), combination therapy
would appear to be an important therapeutic option.
SUMMARY OF THE INVENTION
[0008] It has now been found that pentraxin PTX3 exerts a
surprising synergistic effect when combined with other antifungal
agents, thus permitting the preparation of a drug characterised by
suboptimal doses of antifungal. This characteristic proves to be
advantageous in terms of the greater manageability of the
medicament, in that it is capable of substantially limiting the
side effects characteristic of its individual active
ingredients.
[0009] Therefore, one object of the present invention is a
combination of pentraxin PTX3 and an antifungal agent, as well as a
pharmaceutical composition containing it and the use of said
combination for the preparation of a medicament for the
prophylactic or therapeutic treatment of fungal infections,
particularly aspergillosis.
DETAILED DESCRIPTION OF THE INVENTION
[0010] Pentraxin PTX3 and its various therapeutic uses are
described in various patent applications filed in the name of the
present applicant.
[0011] International patent application WO 99/32516 describes the
sequence of the protein and its use in infectious, inflammatory or
tumoral diseases. Other uses of the long pentraxin PTX3 are
described in WO02/38169, WO 02/36151, WO 03/011326, and WO
03/084561.
[0012] In a preferred embodiment of the invention, the antifungal
agent is amphotericin B, more preferably in the deoxycholate form
known on the market under the trade mark Fungizone (Bristol-Myers
Squibb) or in the liposomal formulation known on the market under
the trade mark AmBisome (GILEAD).
[0013] As regards the aspects relating to the industrial
applicability of the present invention, the long pentraxin PTX3 and
the antifungal will be in the form of a pharmaceutical composition
in which the active ingredients are solubilised and/or vehicled by
pharmaceutically acceptable excipients and/or diluents.
[0014] Examples of pharmaceutical compositions that can be used for
long pentraxin PTX3 are also those described in WO 99/32516.
[0015] The combination according to the present invention can be
administered by the enteral or parenteral routes.
[0016] The daily dose will depend, according to the judgement of
the primary care physician, on the weight, age and general
condition of the patient.
[0017] It is pointed out that the preparation of said
pharmaceutical compositions, including the slow-release ones, can
be accomplished using common techniques and instrumentation well
known to pharmacists and to experts in pharmaceutical
technology.
[0018] In a particular embodiment of the invention, the fungal
infection is invasive aspergillosis (IA).
[0019] The combination according to the invention has been assessed
in the murine bone marrow transplant model which replicates the
immunodeficiency observed in the same conditions in man. The mice
are submitted to different treatment regimens and assessed for
resistance to IA and innate and adaptive immunity parameters. The
results have shown that PTX3 induced total resistance to infection
and reinfection, that it activated type I protective responses and
that it considerably increased the therapeutic efficacy of the
antifungal agents when administered in combination.
[0020] The present invention will now be illustrated in detail,
also by means of examples and figures, in which:
[0021] FIG. 1 illustrates the effect of the administration of PTX3,
AmBisome (L-AmB) or Fungizone (D-AmB) in mice with invasive
aspergillosis. Lethally irradiated C3H/HeJ mice were infused with
T-cell-depleted BM cells of BALB/c mice (2.times.10.sup.6) one week
before intra-nasal infection with 2.times.10.sup.7 Aspergillus
conidia. The mice were treated with PTX3 or with the polyenes, at
the doses and via the routes indicated, 5 days before (Pre-) or
after (Post-) or concomitantly with infection (Con.), in each case
repeating the administration for the next 5 days. Resistance to the
infection was assessed as percentage survival. The fungal load in
the lungs, brains and kidneys of the infected mice was quantified
by serial plating on the Saboaraud dextrose medium of the lavages
of the various organs and the results were expressed as colony
forming units (CFU) (mean.+-.SE) in samples taken from the organs
indicated and performed at the time of death for the mice that died
prior to infection or otherwise at 6 days after infection. The
groups consisted of 6 animals.
[0022] * Indicates P<0.05 (treated vs. untreated mice).
[0023] FIG. 2 illustrates the effect of the administration of PTX3
on resistance to Aspergillus reinfection. BM-transplanted mice,
generated as described in detail in the legend to FIG. 1, were
infected intranasally with 2.times.10.sup.7 Aspergillus conidia one
week after the BM transplant and treated concomitantly with PTX3 at
the doses and via the administration routes indicated, starting on
the day of infection and continuing for the next 5 days. Two weeks
after the infection the surviving mice were reinfected
intravenously with 5.times.10.sup.5 Aspergillus conidia. The fungal
growth (CFU) in the kidneys was evaluated 3 days after reinfection.
* Indicates P<0.05 (treated, vs. untreated mice).
[0024] FIG. 3 shows that PTX3 reduces the lung disease in mice with
invasive aspergillosis. Periodic Schiff base sections were prepared
from the lungs of the BM-transplanted mice infected with
Aspergillus conidia either untreated (A) or treated (B) with 1
mg/kg PTX3 intraperitoneally from the day of infection and
continuing for another 5 days. Numerous Aspergillus hyphae (arrows)
infiltrate the pulmonary parenchyma, with extensive parenchymal
destruction and strong signs of damage to the bronchial wall, and
necrosis and poor cell recruitment are observed in the lungs of the
untreated mice (3 days after infection). PTX3-treated mice present
pulmonary tissue characterised by curative infiltrates of
inflammatory cells with no evidence of parenchymal destruction or
fungal growth (6 days after infection). Enlargement, .times.100 in
panels A and B; .times.400 in insets.
[0025] FIG. 4 shows that PTX3 accelerates cell recovery in mice
with invasive aspergillosis. BM-transplanted mice, generated as
described in detail in the legend to FIG. 1, were infected
intranasally with 2.times.10.sup.7 Aspergillus conidia and treated
(+) or untreated (-) with 1 mg/kg PTX3 intraperitoneally from the
day of infection and continuing for another 5 days. The numbers
refer to the percentage of positive cells, reported by the FACS
analysis in the lungs (A) and in the spleen (B), 3 or 6 days after
infection.
[0026] FIG. 5 shows that PTX3 induces functional recovery of the
Th1 cells. BM-transplanted mice, generated as described in detail
in the legend to FIG. 1, were infected intranasally with
2.times.10.sup.7 Aspergillus conidia and treated with 1 mg/kg PTX3
intraperitonealy both before infection (oblique line bars) and from
the day of infection and continuing for another 3 days (dotted
bars). At 3 days after infection, IL-12 p70 and IL-10 levels were
determined by specific ELISA analyses in bonchoalveolar lavage
fluids (A), the number of CD4.sup.+ T spleen, cells producing
cytokines were numbered by ELISPOT analysis (B) and the gene
expression of cytokine on CD4.sup.+ spleen cells was determined by
real-time PCR. Bars indicate standard errors, *P<0.05, infected
vs. non-infected mice; **P<0.05, PTX3-treated vs untreated mice.
Non-infected mice (white bars); infected mice (black bars).
[0027] FIG. 6 shows that PTX3 increases the therapeutic efficacy of
AmBisome (L-AmB) and Fungizone (D-AmB). BM-transplanted mice,
generated as described in detail in the legend to FIG. 1, were
infected intranasally with 2.times.10.sup.7 Aspergillus conidia and
treated intraperitoneally with each single agent alone or in
combination, before (Pre-) or after (Post-) infection. The doses
were: 0.04 and 0.2 mg/kg PTX3 before or after infection,
respectively; 1 mg/kg AmBisome and 2 mg/kg Fungizone. Resistance to
infection was evaluated as percentage survival and as CFU from the
lungs, performed at the time of death for these mice that died
before, or otherwise at 6 days after infection. *P<0.05, treated
vs. untreated mice; **P<0.001, combination treatment with
PTX3+L-AmB vs each single treatment alone; ***P<0.05,
combination treatment with PTX3+D-AmB vs D-AmB alone.
[0028] FIG. 7 shows that PTX3 reduces the production of TNF-.alpha.
and increases the Th1:Th2 cytokine ratio in mice treated with the
polyenes. BM-transplanted mice, generated as described in detail in
the legend to FIG. 1, were infected intranasally with
2.times.10.sup.7 Aspergillus conidia and treated intraperitoneally
with each individual agent alone or in combination after infection.
The dosages were as described in detail in the legend to FIG. 6.
TNF-.gamma. levels (pg/ml) were determined in bronchoaleveolar
lavage fluids 3 days after infection (A) and IFN-.gamma. and IL-4
levels (pg/ml) in the culture supernatants of the antigen-activated
spleen cells (B) performed at the time of death for those mice that
died before, or otherwise at 6 days after infection. Bars indicate
standard errors. (-, untreated mice). *P<0.05 treated vs.
untreated mice; **P<0.05, combination treatment with
PTX3+Fungisone (D-AmB) vs D-AmB alone: ***P<0.05, combination
treatment with PTX3+AmBisome (L-AmB) vs each single treatment
alone.
Materials and Methods
[0029] Animals. Female BALB/c and C3H/HeJ mice aged 8-10 weeks were
supplied by Charles River Breeding Laboratories (Calco, Italy). The
mice were bred under specific axenic conditions. BM-transplanted
mice were housed in small sterile cages (5 animals per cage) and
fed with sterile feed and water. All procedures regarding the
animals and their care were carried out in conformity with national
and international laws and standards. All in-vivo studies were
conducted in conformity with the national guidelines and with those
of the Committee for the Care and Use of Animals of the University
of Perugia.
[0030] BM-transplant model. Bone marrow (BM) cells from BALB/c
donor mice were prepared by differential agglutination with soy
agglutinin. T-lymphocyte-depleted cells (less than 1% of
contaminating T cells as measured by FACS analysis) were injected
intravenously (i.v.) at a concentration of
.gtoreq.4.times.10.sup.6/mL in recipient C3H/HeJ mice exposed to
the lethal dose of 9 Gy (33). Without the BM transplant, the mice
died within 14 days. According to previous studies (33), more than
95% of mice survived, which shows a haematopoietic chimerism of the
stable donor, as detected by the expression of MHC class I, of the
donor type, in cells from the spleen.
[0031] Micro-organism, culture conditions and infection. The A.
fumigatus strain was supplied by a fatal case of pulmonary
aspergillosis at the Institute for Infections Diseases of the
University of Perugia (13). For infection, the mice were lightly
anaesthetised by means of the inhalation of ethyl ether prior to
the instillation of a suspension of 2.times.10.sup.7 conidia/20
.mu.L saline solution, which was delivered slowly through the
nostrils using a micropipette with a sterile disposable tip. This
procedure was repeated for 3 consecutive days. For reinfection, the
mice surviving the primary intranasal (i.n.) infection were
inoculated with 5.times.10.sup.5 of Aspergillus conidia i.v. The
fungal load in the lungs, brain and kidneys of the infected mice
was quantified by serial plating on Sabourand dextrose medium and
the results (mean.+-.SE) were expressed as colony forming units
(CFU) in samples taken from the organs indicated. For selected
experiments, the fungal growth was also evaluated by chitin
analysis (10). For the histological analysis, the lung was removed
and immediately fixed in formalin. Sections (3 to 4 .mu.m) of
tissues embedded in paraffin were stained with the periodic acid
Schiff base procedure (13, 20).
[0032] Treatments: PTX3 (SIGMA-Tau, Pomezia, Rome, Italy) was
purified by immunoaffinity chromatography from the culture
supernatants of CHO cells CHO transfected with PTX3 and monitored
for the absence of endotoxins (20). PTX3, amphotericin B
deoxycholate (D-AmB, Fungizone, Bristo-Myers Squibb, Sermoneta,
Italy) and liposomal amphotericin B (L-AmB, AmBisome, GILFAD,
Milan, Italy) were diluted to the desired concentrations in sterile
saline solution (PTX3) or in a 5% glucose-water solution. The
treatments were carried out according to the following schedule:
different doses of PTX3, amphotericin B or AmBisome, alone or in
combination, were administered intraperitoneally (i.p.) or
intranasally (i.n.) (PTX3 only) for 5 days before infection with
Aspergillus (prophylactic treatment), concomitantly with infection
and for 5 days after infection, or for 5 days after the last
injection of conidia (therapeutic treatment). In the case of i.n.
administration, the PTX3 and the conidia were administered
separately. The control animals received only the diluent or
sterile saline solution.
[0033] Flow cytometry. The phenotype of the various cell types was
assessed using murine antibodies against the antigens indicated
with rat anti-mouse antibodies conjugated with FITC from PharMingen
(San Diego, Calif.). Prior to immunochemical identification, the
FcR was saturated by incubation of the cells with 5% normal serum.
Histotype antibodies were used as controls. The analysis was done
by means of FACScan (Becton Dickinson, Mountain View, Calif.). The
data obtained were evaluated as percentage of positive cells. The
histograms are representative of one of four independent
experiments.
[0034] Quantification of transcripts of cyctokines by real-time
RT-PCR. Total RNA (5 .mu.g, extracted from CD4.sup.+ T spleen cells
using the RNeasy Mini Kit (QIAGEN S.p.A., Milan, Italy)) was
retrotranscribed with Sensiscript reverse transcriptase (QIAGEN)
according to the manufacturer's indications. The primers for PCR
were obtained from Applied Biosystems (Foster City, Calif.). The
samples were subjected to 40 amplification cycles at 95.degree. C.
for 15 seconds, and then at 60.degree. C. for 1 min using the ABI
PRISM 7000 Sequence Detection System (Applied Biosystems). PCR
amplification of the enkaryotic 18S rRNA housekeeping gene was done
in order to allow normalisation of samples according to the
manufacturer's instructions (Applied Biosystems). The controls with
water were included in order to ensure specificity. All the data
were examined for integrity by analysis of the amplification graph.
The data normalised with RNA 18S were expressed as relative niRNA
of the cytokines examined (.DELTA..DELTA.Ct) and compared with
those of naive mice (10).
[0035] Analysis of cytokines and "spot enzyme-linked
immuno-sorbent" (ELISPOT) analysis. The levels of cytokines in the
bronchoalveolar lavage fluids and in the supernatants of the
cultures of spleen cells stimulated with Aspergillus (9, 10),
thermally activated, were determined using the ELISA Kit (R&D
Systems, Inc. Space Import-Export srl, Milan Italy). The detection
limits of the analyses (pg/ml) were <16 for IL-12 p70, <32
for TNF-.alpha., <10 for IFN-.gamma. and <3 for IL-4 and
IL-10. For the numbering of the CD4.sup.+ T cells producing
cytokines, ELISPOT analysis was used on purified CD4.sup.+ T spleen
cells (9, 10). The results were expressed as the mean number of
cells producing cytokines (.+-.SE) per 10.sup.5 cells, calculated
using replicates of the serial cell dilutions.
[0036] Statistical analysis. The log-rank test was used for the
analysis of the paired data of the Kaplan-Meier survival carves.
Student's t-test or analysis of variance (ANOVA) and the Bonferroni
test were used to determine the statistical significance of the
differences in organ clearance and in the in-vitro analyses, as
indicated in the figure legends. Significance was defined as
P<0.05. The in-vivo groups consisted of 4-6 animals. The data
reported were pooled from the 3-5 experiments, unless otherwise
specified.
Results
[0037] As previously demonstrated, the exogenous administration of
PTX3 restored the antifungal resistance in PTX3-deficient mice with
IA (20). To assess whether PTX3 may have a beneficial effect in
otherwise susceptible mice, we used BM-transplanted mice whose
substantial susceptibility to IA is well documented (15). The mice
were subjected to treatments with different doses of PTX3
administered intranasally or intraperitoneally, before,
concomitantly with or after infection. The doses were selected on
the basis of preliminary experiments showing that PTX3 levels (at
the dose of 0.5 mg/kg/i.n.) were high in bronchoalveolar lavage
fluids for at least 24 h (from 70 to 25 ng/ml from 2 to 24 h) and
that they were higher than those observed in mice with IA one day
after infection (from 2 to 15 ng/ml) (20 and unpublished data). The
survival parameters and fungal load in the lung and brain were then
recorded and analysed in comparison with those obtained in mice
treated with different doses of AmBisome or Fungizone. The results
showed that PTX3 administered prophylactically (FIG. 1A) induced
total resistance to IA at any of the doses assayed, as revealed by
the increase in survival (.gtoreq.60 days) of the majority (from 85
to 95%) of the mice treated and by the signi-ficantly reduced
fungal load in the lungs and brain, particularly in the mice that
received the highest dose (1 mg/kg). Similar results were obtained
in mice that received PTX3 concomitantly with infection (FIG. 1B).
A dose-dependent effect was detected for this administration
period, in that the protective efficacy of PTX3 was lost at the
dose of 0.04 mg/kg. PTX3 administered after infection increased the
survival of the mice only at the higher dose, although at both
doses it significantly reduced the fungal load in the lung and at
the higher dose it significantly reduced the fungal load in the
brain (FIG. 1C). No difference was observed between the two
administration routes. Similar results were observed in mice
treated with 5 mg/kg of AmBisome, in that all the mice survived the
infection when treated before or after infection (FIG. ID).
Fungizone did not afford the same level of protection and an
increase in survival was observed as well as a decrease in fungal
load only at the highest tolerated dose (4 mg/kg) administered
after infection (FIG. 1E). In addition, we evaluated the
susceptibility of the cured mice, after treatment with PTX3, to
Aspergillus re-infection and found that the treatment with PTX3
also significantly increased the resistance to reinfection, as
revealed by the reduced fungal growth in the kidneys of the
reinfected mice (FIG. 2). PTX3 also improved the lung pathology.
Sections of lung from infected mice showed the presence of numerous
Aspergillus hyphae that infiltrated the pulmonary parenchyma, with
signs of severe damage to the bronchial wall and necrosis and poor
recruitment of inflammatory cells (FIG. 3A). These characteristics
were not observed in the mice treated with PTX3, the lungs of which
were characterised by healing infiltrates of poly- and mononuclear
inflammatory cells with no evidence of fungal growth or destruction
of the bronchial wall (FIG. 3B). These data provide evidence of the
therapeutic efficacy of PTX3 in the BM transplant condition where
antifungal agents normally display reduced activity (16, 31).
[0038] In mice with IA, resistance to infection correlates with
activation of Th1 cells that produce IFN-.gamma. (12, 13). To
assess whether PTX3 activated Th1 cell reactivity in
BM-transplanted mice with IA, we evaluated Cell recovery by FACS
analysis, local cytokine production and the antifungal activity of
the effector phagocytes. The quantitative evaluation of blood
leukocytes indicated that the absolute number of circulating
neutrophils increased significantly following treatment with PTX3
(data not shown). Nevertheless, since levels of neutrophils in the
blood do not enable us to predict susceptibility to aspergillosis
(5), the cytofluorometric analysis was performed on lung and spleen
cells. The numbers of CD4.sup.+ cells, CD8.sup.+ cells and
Gr-1.sup.+ neutrophils were significantly increased in the lungs of
mice treated with PTX3 (FIG. 4A). Recovery of neutrophils and,
partly, of CD4.sup.+ T cells was also observed in the spleens. No
difference was noted in the number of PTX3 cells of the lungs or
spleens with or without treatment with PTX3 (FIG. 4B). The cells
and lymphocytes that recovered proved functionally active, as
indicated by the production of pro- (IL-12) and antiinflammatory
(IL-10) cytokines in the pulmonary homogenates and by the frequency
of CD4.sup.+ Th1 (IFN-.gamma.) and Th2 (IL-4). FIG. 5A shows that
the treatment with PTX3 substantially increased IL-12 production
(approximately 4-fold); however, the production of IL-10 was only
halved (as compared to the untreated control), a result suggesting
that PTX3 exerts a subtle control over the inflammatory process at
the site of infection. Moreover, PTX3 treatment increased the
frequency of CD4.sup.+ Th1 cells in the spleen and reduced that of
the cells producing IL-4 (FIG. 5B), a finding confirmed by
evaluation of the mRNA expression levels of cytokines by means of
quantitative PCR. FIG. 5C shows that both prophylactic and
therapeutic treatment with PTX3 significantly increased the
expression of IFN-.gamma. and reduced that of IL-4. On assessing
the level of antifungal activity of the effector phagocytes, it was
found that the conidia-killing activity of the effector phagocytes
was higher in the PTX3-treated mice than in the untreated mice
(data not shown). Since in-vitro studies have ruled out any direct
killing activity of PTX3 on the fungus (data not shown), those data
qualify PTX3 as a new agent with substantial immunomodulatory
activity on both innate and adaptive antifungal immunity.
[0039] All the above-mentioned findings promoted us to assess
whether the immunomodulatory activity of PTX3 could be exploited to
increase the therapeutic efficacy of AmBisome or Fungizone, because
these agents are known to operate synergistically with antifungal
effector phagocytes (40). For this purpose the BM-transplanted mice
received PTX3 alone or together with the polenyes, at suboptimal
doses at which neither of the agents had achieved the maximum
therapeutic effect. The combined or single treatments were
administered either before or after infection. The mice were
monitored for survival, fungal growth and production of cytokines.
It was found that whereas each single agent administered alone
significantly reduced fungal growth in the lung, it did not
significantly modify the survival of the mice, with the exception
of PTX3 administered alone prior to infection. However, combined
therapy with PTX3 and AmBisome, whether before or after infection,
cured the mice of infection, as judged by the increased survival
(>60 days) and the reduced fungal growth. The combined
administration of PTX3 and Fungizone signi-ficantly increased the
resistance to fungal infection compared to that of Fungizone alone
when administered after infection (FIG. 6). Analysis of cytokines
in the pulmonary homogenates and in the supernatants of cultured
antigen-stimulated spleen cells demonstrated that PTX3 greatly
reduced the production of TNF-.alpha. in the lungs of mice that
received Fungizone, as compared to the levels observed in mice
treated with the drug alone; the level of production of TNF-.alpha.
as a reaction to AmBisome was lower compared to that induced by
treatment with Fungizone and was not modified by treatment in
combination with PTX3 (FIG. 7A). The production of IFN-.gamma. by
spleen cells was significantly increased as compared to untreated
mice after each single treatment, and was further increased in the
mice treated with PTX3 and AmBisome; in contrast, the production of
Il-4 was greatly reduced both by the treatment with PTX3 and/or
AmBisome, and by the combined treatment with PTX3 and Fungizone,
albeit to a lesser extent (FIG. 7B). Therefore. PTX3 would appear
to operate synergistically with AmBisome, more than with Fungizone,
in reducing the pulmonary inflammatory response and in promoting
Th1 antifungal reactivity.
[0040] PTX3 according to the present invention induced a curative
response in mice with IA with minimal disease. Bearing in mind that
PTX3 was effective when administered prophylactically and that it
shows no direct activity on fungal cells, it would appear that the
beneficial effect depends on its ability to activate a
Th1-dependent protective resistance.
[0041] PTX3 activates at least two effector pathways against the
pathogenic infectivity, namely, the classic complement activation
pathway, in the C1q binding (35), and the promotion of phagocytosis
through interaction with one or more as yet unidentified cell
receptors (20). It is likely that internalisation of the conidia by
the resident mononuclear cells may serve to limit the fungal
infectivity and permit recovery of the myeloid and lymphoid cells
in the lung. PTX3, however, also activates DCs through the
production of IL-12 and expression of co-stimulatory molecules in
response to Aspergillus conidia (20). Thus, the rapid onset of the
production of PTX3 in DCs through members of the Toll-like receptor
(TLR) family (18) implies a direct role of PTX3 in the
amplification of innate resistance and in orienting adaptive
immunity.
[0042] The production of IL-12 was increased and that of IL-10
reduced in the lungs of infected mice treated with PTX3, a result
that indicates an inflammatory response. Nevertheless, the
production of TNF-.alpha. was not increased by treatment with PTX3,
which suggests that PTX3, like a number of collectins, may act as a
fine regulator of the equilibrium between pro- and
anti-inflammatory stimuli (42, 48).
[0043] According to the present invention, the therapeutic efficacy
of AmBisome and Fungizone was evaluated in the BM transplant
infection model which parallels the profound immunopathology
witnessed in BM transplant recipients, where the susceptibility to
invasive fungal infections is causally related to the development
or otherwise of protective Th responses (15, 33). We found that
AmBisome displayed superior activity as compared to Fungizone in
mice with IA after BM transplant. Both daily prophylactic and
therapeutic treatments with 5 mg/kg AmBisome cured the mice of
infection and reduced the fungal load in the lung. With D-AmB, we
observed only a slightly increased resistance to infection at the
highest tolerated doses (e.g. 4 mg/kg) administered after
infection.
[0044] It has been postulated that the toxicity of D-AmB, including
fever and shivering, is the result of pro-inflammatory cytokine
production by the innate immune cells, through a TLR-dependent
mechanism (43). The murine macrophages and human cell lines that
express TLR2, CD14 and the MyD88 adaptor protein responded to D-AmB
with the release of pro-inflammatory cytokines, including
TNF-.alpha.. Here we have discovered that the production of
TNF-.alpha. was higher in mice treated with D-AmB than in those
treated with L-AmB. However, the combined treatment with PTX3
greatly reduced the production of TNF-.alpha. induced by the
treatment with Fungizone, whereas it con-comitantly increased the
therapeutic efficacy of the drug, as shown by the increased
survival and reduced fungal load in the mice treated with the
combination of PT3 and D-AmB Fallowing infection. Combined therapy
with PTX3 also increased the efficacy of the suboptimal dose of
AmBisome without affecting TNF-.alpha. production levels, as
compared to those observed with each single treatment. Therefore,
the activity of PTX3 in co-administration, with antifungal agents
may depend on an effect that goes beyond the lowering of
TNF-.alpha. production. In this connection, it is known that the
efficacy of anti-fungal chemotherapy depends on the immune
reactivity of the host (32) and that the different formulations of
amphotericin B exert an additional antifungal activity in
combination with the effector phagocytes against A. fumigatus (40).
It has also been reported that PTX3 increases phagocytosis and the
killing activity of the effector phagocytes against Aspergillus
conidia (20).
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