U.S. patent application number 09/244792 was filed with the patent office on 2002-01-17 for use of aerosolized cyclosporine for prevention and treatment of pulmonary disease.
Invention is credited to IACONO, ALDO T..
Application Number | 20020006901 09/244792 |
Document ID | / |
Family ID | 22924129 |
Filed Date | 2002-01-17 |
United States Patent
Application |
20020006901 |
Kind Code |
A1 |
IACONO, ALDO T. |
January 17, 2002 |
USE OF AEROSOLIZED CYCLOSPORINE FOR PREVENTION AND TREATMENT OF
PULMONARY DISEASE
Abstract
The present invention relates to methods and compositions for
prevention of graft rejection in lung transplant recipients and for
treatment of subjects with pulmonary disorders. Specifically, the
methods and compositions of the invention provide a means for
inhibiting immune response mediated inflammatory processes in the
lungs. The method of the invention comprises the administration of
aerosolized cyclosporine for prevention of acute and/or chronic
refractory rejection in lung transplant patients. The invention
further provides for the use of aerosolized cyclosporine to treat
subjects having immunologically mediated inflammatory pulmonary
disorders including, but not limited to, asthma, cystic fibrosis,
idiopathic pulmonary fibrosis, chronic bronchitis and allergic
rhinitis. The present invention, by enabling a method for the use
of aerosolized cyclosporine for inhibiting pulmonary inflammation
leading to prevention of graft rejection and treatment of pulmonary
disorders, provides a safer and less toxic treatment than those
methods that utilize systemic administration of cyclosporine.
Inventors: |
IACONO, ALDO T.;
(PITTSBURGH, PA) |
Correspondence
Address: |
BAKER & BOTTS
30 ROCKEFELLER PLAZA
NEW YORK
NY
10112
|
Family ID: |
22924129 |
Appl. No.: |
09/244792 |
Filed: |
February 5, 1999 |
Current U.S.
Class: |
514/310 ; 424/45;
514/1.7; 514/1.8; 514/20.5 |
Current CPC
Class: |
A61K 9/0073 20130101;
A61P 37/06 20180101; A61K 38/13 20130101; A61K 2300/00 20130101;
A61P 11/00 20180101; A61P 29/00 20180101; A61K 38/13 20130101; Y10T
428/2982 20150115 |
Class at
Publication: |
514/11 ; 514/9;
424/45 |
International
Class: |
A61K 038/00; A61K
031/47 |
Claims
I claim:
1. A method for prevention of graft rejection in a lung transplant
recipient comprising administering to the recipient an effective
dose of aerosolized cyclosporine directly following transplantation
in an amount sufficient to prevent graft rejection.
2. The method of claim 1 wherein the dose of cyclosporine is
sufficient to achieve deposition levels ranging between 15 and 30
mg in the lung.
3. The method of claim 1 wherein the cyclosporine is
co-administered with a second immunosuppressive agent.
4. The method of claim 1 wherein the cyclosporine is
co-administered with a anti-inflammatory reagent.
5. A method for ameliorating pulmonary inflammation in a subject
comprising administering to the subject an amount of aerosolized
cyclosporine effective to inhibit or ameliorate pulmonary
inflammation.
6. The method of claim 5 wherein the pulmonary inflammation is
associated with asthma, sarcoidosis, emphysema, cystic fibrosis,
isiopathic pulmonary fibrosis, chronic bronchitis, or allergic
rhinititis.
7. The method of claim 5 wherein the dose of cyclosporine is
sufficient to achieve deposition levels ranging between 5 and 30 mg
in the lung.
8. A method for prevention of graft rejection in a non-lung
transplant recipient comprising administering to the non-lung
transplant recipient an effective dose of aerosolized cyclosporine
in an amount sufficient to prevent graft rejection.
9. The method of claim 8 wherein the dose of cyclosporine is
sufficient to achieve circulating levels ranging between 50-250
ng/ml.
10. The method of claim 8 wherein the cyclosporine is
co-administered with a second immunosuppressive agent.
11. A method for inhibiting the immune response associated with a
T-cell mediated immune disorder in a subject comprising the
administering to the subject an amount of cyclosporine effective to
inhibit the immune response associated with the immune
disorder.
12. A composition comprising a suitable carrier and aerosolized
cyclosporine in doses sufficient to reduce pulmonary inflammation
in subjects having pulmonary disorders.
13. The composition of claim 12 wherein the aerosolized
cyclosporine has a particle size of between 1 and 5 microns.
14. The composition of claim 12 wherein the dose is sufficient to
achieve concentration levels of between 5-15 mg of cyclosporine in
the lung.
15. The composition of claim 12 wherein the carrier is propylene
glycol.
16. A composition comprising aerosolized cyclosporine as a dry
powder, having a particle size in the range between 1 and 5
microns.
17. A composition comprising a suitable carrier and aerosolized
cyclosporine in doses sufficient to prevent development of an
immune response that would lead to graft rejection in a transplant
recipient.
18. The composition of claim 17 wherein the cyclosporine has a
particle size of between 0.1 and 2 microns.
Description
INTRODUCTION
[0001] The present invention relates to methods and compositions
for prevention of graft rejection in lung transplant recipients and
for treatment of subjects with pulmonary disorders. Specifically,
the methods and compositions of the invention provide a means for
inhibiting immune response mediated inflammatory processes in the
lungs. The method of the invention comprises the administration of
aerosolized cyclosporine for prevention of acute and/or chronic
refractory rejection in lung transplant patients. The invention is
based on the observation that when aerosolized cyclosporine is
administered shortly after lung transplantation, the preparation is
well tolerated and the rate of acute rejection is substantially
reduced, compared to controls that receive conventional oral or
intravenous immunosuppression only. The invention further provides
for the use of aerosolized cyclosporine to treat subjects having
immunologically mediated inflammatory pulmonary disorders
including, but not limited to, asthma, cystic fibrosis, idiopathic
pulmonary fibrosis, chronic bronchitis and allergic rhinitis. The
present invention, by enabling a method for the use of aerosolized
cyclosporine for inhibiting pulmonary inflammation leading to
prevention of graft rejection and treatment of pulmonary disorders,
provides a safer and less toxic treatment than those methods that
utilize systemic administration of cyclosporine.
BACKGROUND OF THE INVENTION
[0002] The long-term success of lung transplantation is currently
limited by the high incidence of transplant-related lung disease
(Glanville, A. R., et al., 1987, Ann Intern Med 107:300-306;
Trulock, E. P., 1993, Chest 103:1566-1576; Kesten, S., 1995, 152:
1321-1324; Paradis, I. et al., 1993, 14:751-763). This complication
is related to the transplant recipients' ongoing immune response
against donor major histocompatability antigens. Such an immune
response generally leads to persistent acute rejection of the lung
allograft which is a predominant risk factor for the subsequent
development of chronic rejection and permanent allograft
dysfunction and failure resulting in excessive morbidity and
mortality. This is a tragic consequence of lung transplantation and
for this reason, is a leading area of research in this field.
Although the rates of short-term survival after lung
transplantation have improved compared to most other solid organ
transplants, the therapeutic benefit of lung transplantation is
still limited by poor longer-term outcomes principally due to
chronic rejection of the transplanted lung.
[0003] Patients, whose lung allografts are in acute and/or chronic
rejection, are currently treated by a variety of potent
immunosuppressive agents, such as azathioprine, tacrolimus,
mycophenolate mofetil and cyclosporine, generally given by the
intravenous or oral route, that profoundly inhibit the T cell
response to donor antigen within the transplanted allograft.
Unfortunately, these immunosuppressive agents diminish the
patient's ability to mount an effective response to viral, fungal
and bacterial pathogens thereby predisposing the patient to life
threatening opportunistic infections and other toxic events such as
kidney toxicity. Despite usage of conventional systemic (oral or
intravenous) immunosuppressive drugs, about 50% of the treated
patients develop refractory chronic rejection, characterized
histologically by bronchiolitis obliterans, followed by a
progressive decline in pulmonary function and eventually
respiratory failure and death.
[0004] Cyclosporine, an 11-amino acid cyclic polypeptide antibiotic
is frequently used to prevent rejection after solid organ
transplantation (Kahan, B. D., 1989, N Engl J Med., 321:1725-1738;
Kumar, M. S. A., et al., Transplant Proc., 20:407-413; Keenan R.
J., et al., Transplantation 53:20-25). Cyclosporine acts as an
immunosuppressive agent by selectively inhibiting immune responses
mediated by T lymphocytes (Iacono, A. T., et al., 1997,
Transplantation 64:263-269; Keenan, R. J., 1995, Surgery
118:385-391). Unfortunately, systemic cyclosporine has a narrow
therapeutic index, e.g., ratio between toxic and therapeutic doses,
and effective immunosuppressive doses often cannot be achieved due
to the risk of toxicity to the liver and kidney. In addition,
administration of systemic cyclosporine results in a high incidence
of infections with viral, bacterial and fungal pathogens.
[0005] To date, oral cyclosporine, when combined with azathioprine
(AZA) and prednisone, has proven incapable of persistently
suppressing the alloresponse to the lung to an extent necessary to
provide an optimistic long-term outcome (Griffith, B. P., 1992, Ann
Thorac Surg 54:846-51). Other therapies for prevention of
transplant rejection include anti-CD3 antibody (OKT3),
methotrexate, lymphoid irradiation and mycophenolate mofetil.
Unfortunately, even with these treatments clinical efficacy has
been disappointing and associated with toxicity (Cahill, B. C.,
1996, J Heart Lung Transplant 15:1130-1137; Valentine, V. G., et
al., 1996, 109:1184-1189; Copeland, K. R. and Yatscoff, R. W.,
13:281-288) Thus, cyclosporine either alone or as part of a multi
drug immunosuppressive regimen has been imperfect in preventing
both acute and chronic rejection.
[0006] Recent data has indicated that immunosuppression by local
administration of cyclosporine may be beneficial. For example,
using a collagen matrix impregnated with cyclosporine, it was
demonstrated that controlled release of low dose cyclosporine,
significantly prolonged non-heterologous heart allograft survival
with negligible blood and kidney tissue cyclosporine concentrations
(Bolling, et al., 1990, J Heart Transplant 9:74-78; Stepkowski, et
al, 1989, Transplantation 47:17-23).
[0007] While most solid organ transplants are inaccessible to such
localized immunosuppress therapy, lung allografts are the
exception. Aerosolized pharmacologic agents have direct access to
the lung, and there is extensive experience in the use of inhaled
.beta.-agonists and nebulized antibiotics. In animal models,
aerosolized cyclosporine has been demonstrated to be safe and more
effective than systemic cyclosporine in preventing graft rejection
(Dowling R. D.,1990, Surgery, 108:198; Zenati, M., 1991, Eur. J.
Cardiothor. Surg., 5:266; Keenan, R. J. et al., 1992,
Transplantation 53:20-25; Rabinowich H., 1988, Transplant Proc.,
20:836). Local delivery of aerosolized cyclosporine has been
effectively used to deliver cyclosporine to the lungs of patients
with severe chronic graft rejection that was refractory to all
previous attempts at control (Burckart, G. J., 1989, J Clin.
Pharmaco. 29: 860; Iacono, A. T., et al., 1996, Am. J. Resp. Crit.
Care Med., 153:1451-1455). In addition aerosolized cyclosporine was
effective as therapy for refractory acute rejection in
lung-transplant subjects unresponsive to conventional therapy
(O'Riordan, T. G., et al., 1995, Am. J. Respir. Crit. Care Med.
151:516; Iacono, A. T., et al., 1997, Am. J. Resp. Crit. Care Med..
155:1690-1698; Keenen, R. J., et al., 1997, J. Thorac. Cardiovasc.
Surg., 1134:335-341).
SUMMARY OF THE INVENTION
[0008] The present invention provides compositions and methods for
using aerosolized cyclosporine for prevention of graft rejection in
lung transplant recipients. The invention further provides for the
use of aerosolized cyclosporine for amelioration of inflammatory
pulmonary disorders including, by way of example and not
limitation, asthma, sarcoidosis, emphysema, cystic fibrosis,
idiopathic pulmonary fibrosis, chronic bronchitis, allergic
rhinitis and allergic diseases of the lung such as hypersensitivity
pneumonitis, eosinophilic pneumonia, bronchiolitis obliterans due
to bone marrow transplantation or other causes, as well as
pulmonary fibrosis resulting from collagen, vascular, and
autoimmune diseases such as rheumatoid arthritis and lupus
erythematosis.
[0009] Delivery of cyclosporine to the transplanted lung by aerosol
inhalation achieves higher concentrations in the lung than delivery
of the drug by systemic (oral or intravenous) administration,
resulting in improved control of rejection, with reduced toxicity
due to limited absorption from the lung into the bloodstream.
[0010] Accordingly, the methods of the present invention comprise
administering aerosolized cyclosporine to a subject having received
a lung transplant. To prevent rejection of the lung transplant the
cyclosporine is administered directly following the transplant
procedure prior to the development of symptoms associated with
organ rejection. The administration of aerosolized cyclosporine
results in a substantially lower prevalence of acute rejection and
development of obliterative bronchiolitis (OB). In addition,
cytokines, chemokines and effector molecules normally expressed
within the allograft are suppressed, such that the recipient
requires less systemic immuno-suppression. Moreover, systemic
immunocompetence is preserved by maintenance of T-helper cell
memory, resulting in a lower incidence of opportunistic and
bacterial infection.
[0011] In yet another embodiment of the invention, aerosolized
cyclosporine is administered to a subject having an inflammatory
pulmonary disorder. The method of the invention comprises
administering aerosolized cyclosporine to inhibit inflammation in a
subject having an inflammatory pulmonary disorder such that the
expression of cytokines is modulated and the symptoms of
inflammation are ameliorated.
[0012] The invention further provides for compositions comprising
cyclosporine in a suitable carrier which can be administered to a
subject, in aerosolized form, at an effective dose to prevent graft
rejection or ameliorate the inflammatory symptoms associated with
pulmonary disorders. For subjects with pulmonary disorders, the
compositions used in the practice of the invention comprise an
effective dose of cyclosporine that is generally lower than the
doses reportedly used for treating refractory acute lung rejection
or the doses described herein for prevention of lung rejection.
[0013] The invention is based on the observation that
administration of aerosolized cyclosporine given as a prophylaxis
after lung transplantation can prevent acute rejection. The present
invention, by providing methods for prevention of graft rejection
and amelioration of inflammatory pulmonary disorders using
aerosolized cyclosporine, reduces the toxicity and susceptibility
to life threatening opportunistic infections associated with
systemic use of cyclosporine.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1A, 1B AND 1C. Pharmacokinetics and bioavailability of
aerosolized cyclosporine. FIG. 1A. Five subjects studied on average
post-operative day number 20.8 underwent blood measurements of
cyclosporine after inhalation of a 300 mg dose. FIG. 1B.
Subsequently, a dose of intravenous cyclosporine (1 mg/kg over a 4
hour infusion) was administered and blood concentrations of
cyclosporine were determined by monoclonal immunoaassay over 24
hours following infusion. FIG. 1C compares blood measurements of
cyclosporine after inhalation versus intravenous
administration.
[0015] FIG. 2. Acute cellular rejection grade 2 or greater in the
first six months post-transplantation. The number of biopsy-proven
acute rejection events is decreased in subjects that received
aerosolized cyclosporine versus controls that received only
standard oral triple drug immunosuppression (2.278
episodes/rejection/subject.+-.0.113 versus 1.308.+-.0.398, p value
0.0196 (Mann-Whitney U test).
[0016] FIG. 3. A Kaplan Mayer survival curve in treated subjects
versus controls, demonstrating improvement in survival in those
subjects that received aerosolized cyclosporine (p=0.014) versus
controls receiving standard systemic immunosuppression.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The present invention relates to methods for preventing
graft rejection in lung transplant recipients wherein said methods
comprise the administration of aerosolized cyclosporine directly
following lung transplantation. The invention further relates to
methods for ameliorating inflammation in subjects having
inflammatory pulmonary disorders using aerosolized cyclosporine.
Subjects treated with aerosolized cyclosporine have reduced
pulmonary inflammation due to a cyclosporine mediated decrease in
inflammatory cytokines in the lung.
[0018] The methods of the invention provide a means for
ameliorating pulmonary disorders through direct delivery of the
immunosuppressive agent cyclosporine to the lung while avoiding the
toxicity associated with systemic use of cyclosporine, or other
systemic immunosuppressive drugs that frequently cause toxicity and
infection.
Use Of Aerolized Cyclosporine For Prevention Of Rejection In Lung
Transplant Recipients
[0019] The present invention relates to a method for prevention of
graft rejection in lung transplant recipients by administration of
aerosolized cyclosporine. The present invention is used as a
prophylactic means for inhibiting the onset of graft rejection in
lung transplant recipients. The method comprises the administration
of aerosolized cyclosporine to a transplant recipient directly
following transplantation by aerosol inhalation. In a preferred
embodiment of the invention, the initial maximum dose of
aerosolized cyclosporine is usually administered to the transplant
recipient within 10 days following transplantation or prior to the
development of any of the symptoms generally associated with lung
transplant rejection. The cyclosporine is delivered to the lung of
the recipient by inhalation of cyclosporine in aerosol spray form
using, for example, a pressurized delivery device or nebulizer. The
cyclosporine may be administered in either dry powder or wet
form.
[0020] Compositions suitable for use in the present invention
include compositions comprising cyclosporine in an effective amount
to achieve its intended purpose and one or more physiologically
acceptable carriers. More specifically, an effective amount means
an amount sufficient to prevent development of an immune response
that would lead to graft rejection in a lung transplant recipient.
An effective dose refers to that amount of cyclosporine sufficient
to inhibit an immune response in the lung of the transplant
recipient thereby preventing graft rejection. Determination of
effective amounts is well within the capability of those skilled in
the art.
[0021] The effective dose may be determined using a variety of
different assays. The progress of the transplant recipient can be
determined using assays that include serial transbronchial biopsies
to determine the presence and severity of rejection as measured by,
for example, a reduction in mononuclear cell inflammatory
infiltrate, characteristic of transplant rejection. In such
instances, the effective dose of aerosolized cyclosporine is that
amount required to sustain a local immunosuppressive effect in the
lungs, thereby preventing lung transplant rejection. In addition,
assays may be utilized to quantitate the deposition of aerosolized
cyclosporine in the lung of the recipient using radionucleotides.
Spirometry can be performed following inhalation of aerosolized
cyclosporine to assess how much air the lungs can hold as well as
how much and how quickly air can be exhaled. A reduction in forced
expiratory volume (FEV1) of greater than 15% in conjunction with
clinical symptoms of breathlessness indicates the need for reducing
the dose of aerosolized cyclosporine. In addition, symptoms of
pharyngeal soreness, cough and breathlessness may also indicate the
need for reducing the dose of aerosolized cyclosporine.
[0022] Serial pulmonary function tests, such as chest radiographs,
complete blood counts, assays for electrolytes and creatine levels,
and cytokine expression in bronchoalveolar lavage cells, as well as
histolgic analysis of the lung by transbronchial lung biopsy can be
performed to assess efficacy at 1-3 month intervals throughout the
course of aerosolized cyclosporine administration.
[0023] The amount of composition administered is also dependent on
the subject to whom the aerosolized cyclosporine is administered
and the judgement of the physician overseeing the subject. It
should be noted that the attending physician would know how and
when to terminate, interrupt or adjust the treatment to a lower
dose due to toxicity.
[0024] Conversely, the attending physician would also know to
adjust treatment to higher levels if the clinical response is not
adequate. This can be determined by measurement of the cyclosporine
in the lung using known radioisotopic techniques. In addition,
adjustments of concomitant administration of additional drugs may
be necessary.
[0025] In general, the total dose range of cyclosporine should be
sufficient to achieve allograft deposition levels ranging between
15 mg and 30 mg in the lung. For example, a dose of between 100-500
mgs of aerosolized cyclosporine may be inhaled, while most
preferably the usual dose of aerosolized cyclosporine to achieve
deposition in the lung between 15-30 mg is typically 300 mg.
[0026] The present invention relates to methods for prevention of
graft rejection in lung transplant recipients, therefore, the
initial aerosol treatment is administered prior to the development
of symptoms normally associated with transplant rejection. In
general, administration of aerosolized cyclosporine begins on Day
2-10 post-transplant, while most preferably the treatment begins on
Day 5-7 post-transplant. The treatment continues on a daily basis
for between 8-15 consecutive days, while most preferably the
treatment continues for 10-12 consecutive days. This initial dosing
is followed by administration of aerosolized cyclosporine three
times weekly for the duration of the life of the transplanted
lung.
[0027] It is further recommended that infants, children, and
subjects with impaired immune systems initially receive lower
doses, and they be titrated based on individual clinical response.
It may be necessary to use dosages outside the ranges disclosed
above in some cases as will be apparent to those of ordinary skill
in the art.
[0028] In general, the aerosolized cyclosporine is given as the
sole immunosuppressive agent if it is found to adequately control
rejection. However, aerosolized cyclosporine may be co-administered
to a transplant recipient in combination with other
immunosuppressive or anti-inflammatory reagents, including but not
limited to, oral cyclosporine (2.5-5.0 mg/kg); tacrolimus 0.01-0.04
mg/kg; prednisone 20 mg/kg or 0.3 mg/day. Aerosolized cyclosporine
may be give alone if the transplant recipient has a life
threatening infection cause by profound inactivation of the immune
system due to oral or intravenous immunosuppression, or experiences
toxicity, especially to the kidney, due to co-administration of
these drugs.
Use Of Aeroslized Cyclosporine For Prevention Of Rejection In Organ
Transplant Recipients And For Treatment Of Immune Disorders
[0029] In yet another embodiment of the invention, aerosolized
cyclosporine can be administered to organ transplant recipients
other than lung transplant recipients using a delivery system that
utilizes a optimal cyclosporine particle size for systemic delivery
of cyclosporine via the lung. Such organ transplants include, but
are not limited to, transplants of the liver, kidney, heart and
bone marrow. The use of aerosolized cyclosporine provides an
effective system for maintaining a steady drug concentration in the
bloodstream thereby increasing the efficacy of the cyclosporine and
minimizing the toxic side effects associated with cyclosporine.
[0030] For pulmonary deposition, the cyclosporine particle size is
generally between 1 and 5 microns, a size that generally restricts
absorption into the bloodstream. In contrast, where the desired
goal is systemic delivery of the cyclosporine via absorption from
the lung into the bloodstream, the cyclosporine particle size is
reduced to an approximate size of between 0.1 and 2 microns.
Methods for producing acrosolized cyclosporine particles of
different sizes are routine and well known to those of skill in the
art.
[0031] Compositions suitable for use in treatment of organ
transplant recipients include compositions comprising cyclosporine,
in one or more physiologically acceptable carriers, in an effective
amount to achieve its intended purpose. More specifically, an
effective amount means an amount sufficient to prevent development
of an immune response that would lead to graft rejection in a
transplant recipient. An effective dose refers to that amount of
cyclosporine sufficient to inhibit an immune response in the
transplanted organ of the transplant recipient thereby preventing
graft rejection. In general, the total dose range of cyclosporine
should be sufficient to achieve circulating cyclosporine
concentrations of between 50-250 ng/ml, while most preferably the
usual dose of cyclosporine is sufficient to achieve circulation
levels of 200 ng/ml.
[0032] Determination of effective amounts is well within the
capability of those skilled in the art. The effective dose may be
determined using a variety of different assays. The progress of the
transplant recipient can be determined using assays that include
biopsies to determine the presence and severity of rejection as
measured by, for example, a reduction in mononuclear cell
inflammatory infiltrate, characteristic of transplant rejection. In
such instances, the effective dose of aerosolized cyclosporine is
that amount required to sustain a local immunosuppressive effect in
the transplanted organ, thereby preventing organ transplant
rejection. In addition, organ function may be monitored using a
variety of different assays, the use of which, will depend on
nature of the transplanted organ. For example, blood tests may be
performed to assay for normal liver or kidney function. In
instances where the transplant recipient has received a
transplanted heart, an electrocardiogram can be performed to test
for normal cardiac function.
[0033] In addition to the use of small particle size aerosolized
cyclosporine for treatment of non-lung transplant recipients, such
delivery systems may be used to treat subjects having T-cell
mediated immune disorders such as type IV cell mediated
(delayed-type) hypersensitivity, or autoimmune disorders.
Autoimmune disorders which may be treated using aerosolized
cyclosporine include, for example, systemic lupus erythematosus,
myasthenia gravis, Grave's disease, Hashimoto's thyroiditis,
rheumatoid arthritis, scleroderma, and pernicious anemia.
[0034] The dose of cyclosporine to be used in the method of the
invention is an amount sufficient to achieve its intended purpose.
More specifically, an effective amount means an amount sufficient
to inhibit the immune response associated with the immune
disorder.
[0035] The effective dose may be determined using a variety of
different assays including assays for detection of blood levels of
cyclosporine cytokines, and/or the presence of autoreactive
T-cells. In such instances, the effective dose of aerosolized
cyclosporine is that amount required to sustain an
immunosuppressive effect, thereby preventing the symptoms
associated with auto-immunity. Determination of effective amounts
is well within the capability of those skilled in the art and may
be readily ascertained.
Use Of Aerolized Cyclosporine For Amelioration Of Inflammatory
Pulmonary Disorders
[0036] There are a number of significant pulmonary diseases
resulting from abnormal accumulations of inflammatory cells in lung
tissue. Initially, the inflammatory cells and protein rich fluids
accumulate in the lung causing inflammation. If left untreated, the
inflammation commonly leads to replacement of normal lung tissue
with scarred tissue, which severely limits the ability of the lung
to function normally, leading to symptoms of progressive
breathlessness, exercise intolerance and eventually a very poor
quality of life. Some inflammatory lung diseases, asthma being a
common one, cause inflammation and respiratory disability without
causing lung scarring. Successful treatment of inflammatory
pulmonary disorders can be brought about by techniques which serve
to suppress the immune response.
[0037] The present invention provides methods for promoting local
immunosuppression in the lungs of subjects having pulmonary
disorders through inhalation of an aerosol of cyclosporine. The
method of the invention comprises the administration of aerosolized
cyclosporine to a subject having an inflammatory associated lung
disorder. The cyclosporine is delivered to the lung of the subject
by inhalation of cyclosporine in the form of an aerosol spray
using, for example, pressurized delivery devices or nebulizers. The
cyclosporine may be formulated in either a dry powder or liquid
form.
[0038] Among the pulmonary disorders whose symptoms can be
ameliorated by the use of aerosolized cyclosporine are inflammatory
pulmonary disorders wherein the symptoms of the disease result from
a local immune reaction in the lungs. Examples of such disorders
include, but are not limited to, asthma, sarcoidosis, emphysema,
cystic fibrosis, idiopathic pulmonary fibrosis, chronic bronchitis,
allergic rhinitis and allergic diseases of the lung such as
hypersensitivity pneumonitis and eosinophilic pneumonia.
[0039] In yet another embodiment of the invention, aerosolized
cyclosporine can be administered to patients receiving gene therapy
wherein said therapy involves the inhalation of nucleic acids, or
recombinantly engineered viruses, encoding a protein of interest.
The administration of such nucleic acids or recombinantly
engineered viruses can be associated with an inflammatory response
in the lungs resulting from the host immune response against the
nucleic acid or engineered virus. Thus, aerosolized cyclosporine
can be co-administered with nucleic acids or recombinant viruses to
reduce the inflammation associated with inhalation of such agents.
By reducing the level of inflammation, the therapeutic benefit
derived from the gene therapy may be prolonged.
[0040] Compositions suitable for use in the present invention
include compositions containing cyclosporine and a physiologically
acceptable carrier in an effective amount to achieve its intended
purpose. More specifically an effective dose refers to that amount
of cyclosporine sufficient to inhibit an immune response in the
lung of a subject suffering from a pulmonary disorder thereby
decreasing the inflammation associated with the disorder.
Determination of effective amounts is well within the capability of
those skilled in the art and may be readily ascertained.
[0041] The effective dose may be determined using a variety of
different assays. Transbronchial lung biopsies may be performed to
examine whether the lung tissue shows histological evidence of
inflammation; and/or assays can be performed to detect cyclosporine
mediated reduction in cytokine and chemokine gene expression from
bronchoalveolar lavage (BAL) cells and peripheral blood lymphocytes
(PBL) of the treated subject. Additionally, assays may be utilized
to determine the deposition of aerosol cyclosporine in the lungs
using, for example, radionucleotides. Serial spirometry can be used
to determine lung volume and flow rate, before and during
treatment.
[0042] Subject questionnaires with symptom scores will be completed
before and during treatment to assess a clinical response. A
cadiopulmunary exercise test can be performed at baseline and
during therapy to measure oxygen saturations and maximal oxygen
consumption during exercise. In such instances, the effective dose
of aerosolized cyclosporine is that amount required to sustain a
local immunosuppressive effect in the lungs thereby alleviating the
symptoms associated with pulmonary inflammation while maintaining
acceptable lung volumes and flow rates.
[0043] The amount of composition administered is also dependent on
the subject to whom the aerosolized cyclosporine is administered,
the pulmonary disorder the subject has, the severity of the
disorder's symptoms and the judgement of the overseeing physician.
In some instances it may be necessary to terminate, interrupt or
adjust the treatment to a lower dose due to toxicity as well as
adjusting the treatment to higher levels a suitable beneficial
response is not obtained.
[0044] In general, the total dose range of aerosolized cyclosporine
should be sufficient to achieve concentration levels ranging
between 5 mg and 30 mg in the lung, while most preferably a dose
range sufficient to achieve concentration levels ranging between 5
mg and 15 mg in the lung is desirable. For example, a dose of
between 20-400 mg of a aerosolized cyclosporine is administered,
while most preferably a dose of aerosolized cyclosporine of between
50-300 mg is administered. Overall, doses of aerosolized
cyclosporine may vary depending on the type and extent of lung
disease, however it is believed that doses needed to achieve a
beneficial response will be less then the doses of aerosolized
cyclosporine required to ameliorate transplant related
inflammation. It may be necessary to use dosages outside these
ranges in some cases, as will be apparent to those of ordinary
skill in the art.
[0045] Aerosolized cyclosporine may be administered several times
per day in small doses to ameliorate relatively mild airway
inflammation associated with disorders such as, for example,
asthma. Higher doses, given less frequently, may be required to
ameliorate more serious inflammation associated with pulmonary
disorders such as idiopathic pulmonary fibrosis.
[0046] In certain instances, it may be desirable to co-administer
to a subject exhibiting pulmonary disorder symptoms, aerosolized
cyclosporine in conjunction with an additional agent. Such agents
include, for example, antibiotics, antivirals, immunosuppressives
or anti-inflammatory agents. Anti-inflammatory drugs include, for
example, inhaled steroids 4.times.220 mgs/puff/day, prednisone
20-60 mg day, methotrexate 5-15 mg/week, azathioprine 50-200
mg/day. Determination of effective amounts of these additional
compounds is well within the capability of those skilled in the
art.
Compositions For Aerolized Delivery Of Cyclosporine
[0047] The compositions for use in accordance with the present
invention may be formulated in a conventional manner using one or
more physiologically acceptable carriers or recipients.
Cyclosporine for use in the practice of the invention is
commercially available and may be obtained from manufacturers, such
as Novartis Pharmaceuticals (East Hanover, N.J.).
[0048] The cyclosporine can be formulated in pharmaceutically
acceptable compositions suitable for delivery to the lungs.
Particular formulations include dry powders, liquid solutions or
suspensions suitable for nebulization and propellant formulations
suitable for use in metered dose inhalers. The preparation of such
formulations is well know to those skilled in the art, and is
described in U.S. Pat. Nos. 5,814,607 and 5,654,007 the disclosures
of which are incorporated herein by reference.
[0049] Dry powder formulations will comprise cyclosporine in a dry,
lyophilized, form with a particle size within a preferred range for
deposition within the lung. Typically the particle size for
deposition in the lung will range between 1 and 5 microns. When
systemic delivery of the cyclosporine via absorption from the lung
into the bloodstream is desired the cyclosporine particle size is
generally between 0.1 and 2 microns in size. The preferred size
range of particles can be produced using methods such as
jet-milling, spray drying and solvent precipitation, for
example.
[0050] Dry powder devices typically require a powder mass in the
range from about 1 mg to 10 mg to produce an aerosolized dose.
Thus, the cyclosporine will typically be combined with a
pharmaceutically acceptable dry bulking powder. Preferred dry
bulking powders include sucrose, lactose, trehalose, human serum
albumin (HSA) and glycine. Dry powders can be administered to the
subject in conventional dry powder inhalers.
[0051] For liquid formulations the cyclosporine can be dissolved in
any recognized physiologically acceptable carrier for use in
delivery of aerosolized formulations. Such carriers include
ethanol, propylene glycol and ethanol-propylene combinations.
Although cyclosporine is relatively insoluble in water, it is
soluble in lipids and organic solvents, having a solidity of about
80 mg/ml in alcohol at 25.degree. C. In a preferred embodiment the
cyclosporine is dissolved in propylene glycol. The choice of
propylene glycol is based on its reported use as a solvent to
administer aerosolized formulations to individuals (Miller, W. C.
et al., 1991, J. Aerosol, Medical. 4:293-297). Such preparations
are stable at up to 60 days following preparation.
[0052] For administration by inhalation, the compositions for use
according to the present invention are conveniently delivered in
the form of an aerosol spray administered via pressurized packs or
a nebulizer, with the use of a propellant, e.g.,
dichlorordifluoromethane, dichloroterafluoroethane or other
suitable gas. Preferably, for incorporation into the aerosol
propellant, the cyclosporine of the present invention will be
processed into respirable particles as described above for the dry
powder formulations. The particles are then suspended in the
propellant, typically being coated with a surfactant to enhance
their disbursement. In the use of a pressurized aerosol the dosage
unit may be determined by providing a valve to deliver a metered
amount.
[0053] Commercially available jet nebulizers are available and may
be used to deliver aerosolized cyclosporine to a subject. Such jet
nebulizers include, but are not limited to, those supplied by
AeroTech II (CIS-US, Bedford, Mass.). In addition, for delivery of
aerosolized cyclosporine to the lungs of a subject an oxygen source
can be attached to the nebulizer providing a flow rate of, for
example, 10 L/min. In general, inhalation is performed over a 30-40
minute time interval through a mouthpiece during spontaneous
respiration.
[0054] The present invention provides for novel compositions
comprising a suitable carrier and aerosolized cyclosporine in doses
sufficient to reduce or ameliorate pulmonary inflammation in
subjects having pulmonary disorders. Such doses are lower than
those generally used to ameliorate rejection in transplant
recipients. In general, the compositions of the invention should be
sufficient to achieve concentration levels of between 5-30 mg,
while most preferably achieving 5-15 mg in the lung.
Example: Administration Of Aerolized Cyclosporine As Prophylaxis To
Prevent Acute Rejection After Lung Transplantation
[0055] The following section describes experimental data relating
to administration of aerosolized cyclosporine to a transplant
recipient directly following lung transplantation. The rate of
histological acute rejection for pilot subjects that received
aerosolized cyclosporine early after lung transplantation (average
day 10) was compared to controls that received conventional oral
therapy (tacrolimus, oral cyclosporine, azathioprine and
prednisone). As indicated by the data presented, the administration
of aerosolized cyclosporine directly following transplantation is
capable of preventing acute rejection in the transplant
recipient.
Material And Methods
Subject Population
[0056] The subject demographics appear in Table I below. A total of
three subjects underwent double lung transplantation (DL), one
subject underwent heart-lung transplantation (H-lung), and nine
subjects received either a right (RSL) or left (LSL) single lung
transplantation.
1TABLE I Initial Day of Days Recipient Donor aerosol of Transplant
CMV CMV Baseline cyclosporine follow S Sex Diagnosis Age Type
Status Status n administration up 1 M RE-transplant/ 50 DL positive
negative Csa 31 422 OB 2 F CF 23 DL positive negative FK 11 200 3 F
alpha 1 48 LSL positive negative FK 8 294 antitrypsin re-transplant
4 M emphysema 66 LSL negative positive FK 4 181 5 F RE-transplant/
43 RSL positive negative FK 6 358 OB 6 M emphysema 67 RSL negative
positive FK 4 245 7 F emphysema 58 LSL positive positive FK 9 240 8
M emphysema 57 LSL positive negative FK 7 249 9 M emphysema 66 LSL
negative positive FK 3 210 10 M pulmonary htn 37 H-lung positive
positive FK 9 265 11 M IPF 48 LSL negative positive FK 11 199 12 M
emphysema/ 57 LSL positive positive FK 18 71 re-transplant 13 M
alpha 1 44 DL positive negative FK 16 310 antitrypsin CMV =
cytomeglavirus n = baseline immunosupression s = subject
[0057] In the 13 subjects, administration of aerosolized
cyclosporine occurred, on average, ten days following
transplantation. Twelve of the thirteen subjects received
tacrolimus based immunosuppression. The systemic immunosuppressive
drug regimen consisted of oral tacrolimus (0.03 mg/kg/day), or
cyclosporine (2.5-5.0 mg/kg/day) azathioprine (1-2 mg/kg/day),
prednisone (0.3 mg/kg/day).
[0058] All subjects tolerated the aerosolized cyclosporine therapy.
A dose of 300 mg as administered for ten consecutive days followed
by 300 mg three days per week on Mondays, Wednesday, and Fridays.
Creatinine levels were measured at 30 day intervals during aerosol
cyclosporine administration using routine techniques. There have
been no subjects that have experienced renal insufficiency as a
result of the addition of aerosolized cyclosporine to standard
immunosuppressive drug therapies. The average duration of follow-up
was 245 days. Rejection was monitored by serial transbronchial lung
biopsies performed at 2-3 month intervals in all 13 subjects.
Rejection was considered significant if histology showed greater
than or equal to grade II acute cellular rejection or active
bronchiolitis obliterans according to Yousem, S. A. (1990, J. Heart
Lung Transplant. 12:713-716). The rate of rejection in the 13
treated subjects was compared to a group of controls at six months
after lung transplantation. The rejection rate was substantially
less during follow-up in those subjects treated with aerosolized
cyclosporine (see FIG. 2). In addition, treatment has been
administered for up to five years thus far and none of the treated
subjects, monitored at 2 month intervals since starting aerosolized
cyclosporine administration have developed chronic rejection thus
far.
Bioavailability Assays
[0059] Data regarding pharmacokinetics and bioavailability were
obtained using the techniques described in "Pharmacokinetics and
Bio-Availability of Aerosolized Cyclosporine in Lung Transplant
Recipients"(Vega R. et al., 1998, Resp. and Crit. Care Med.
157:329). Blood concentrations of cyclosporine were determined
using a cyclosporine monoclonal immunoassay (TDX; Abbott
Laboratories), 24 hours following administration of the aerosolized
cyclosporine.
Aerosol Cyclosporing Radioisotope Deposition Studies
[0060] The deposited dose of aerosolized cyclosporine was measured
in subjects at approximately 60-90 days of administration. A
solution of cyclosporine was mixed with 0.3 ml of saline containing
a radioisotope tracer (.sup.99MTc) and total cyclosporine
deposition in the allograft was quantitated using a previously
validated technique of O'Riordan, T. G. et al., (1992, J. Aerosol.
Med. 5:171-177) and O'Riordan, T. G. et al., (1995, Am. J. Respir.
Crit. Care Med. 151:516-521). All subjects deposited the aerosol
cyclosporine in their transplanted lung.
Histological Evaluation
[0061] Histological diagnosis of lung transplant rejection was made
according to Yousem, S. A. et al. (Working Formulation for the
Standardization of Nomenclature in the Diagnosis of Lung Rejection,
1990, J. Heart Lung Transplant, 12:713-716). The rate of
histological acute rejection events (>grade II) was analyzed
within six months after transplantation.
Results
[0062] None of the subjects to date have developed brochiolitis
obliterans. Two of the subjects died from cytomegalovirus infection
and multi-organ system failure.
[0063] A Kaplan Mayer survival curve in the 13 treated subjects
versus 13 contemporary controls that were matched by type of
transplant and age is shown in FIG. 3. Improvement in survival was
noted in those subjects that received aerosolized cyclosporine
(p=0.014).
Nephotoxicity Is Not Observed When Aerolized Cyclosprine Is Given
To Prevent Rejection Soon After Transplantation
[0064] As a measure of toxicity due to systemic absorption of
cyclosporine following aerosolized inhalation administration,
creatinine levels were compared at initiation of aerosolized
cyclosporine administration in transplant recipients and compared
with a group of matched contemporary controls that received
conventional oral immunosuppressive therapy. The creatinine level
at baseline and after a mean of 190 days in transplant recipients
and 181 days in control subjects did not differ when aerosolized
cyclosporine was added to the immunosuppressive therapeutic regimen
(treatment group and controls baseline 0.91 mg/dl.+-.0.22 versus
1.26 mg/dl.+-.0.71, p=0.532; 1.47.+-.0.43 versus 1.58.+-.0.95,
p=0.93).
Pharmacokinetics Of Aerosol Cyclosporine Given Directly After Lung
Transplantation
[0065] To evaluate pharnacokinetics of cyclosporine when given by
aerosol inhalation, bioavailability studies and radioisotope
deposition studies were performed. Bioavailability studies
demonstrated limited systemic absorption from the lung as compared
to the oral administration, and radioisotope studies showed deep
lung deposition of the aerosol following inhalation.
[0066] Data regarding pharmacokinetics and bioavailability was
obtained during the course of the study (FIG. 1). In this study,
pharmacokinetics and bioavailability of aerosolized cyclosporine,
(300 mg dose) was measured (FIG. 1A). Five transplant recipients
studied, on average, by post-operative day 21 underwent blood
measurements of cyclosporine after inhalation of a 300 mg dose. All
subjects also received oral tacrolimus.
[0067] Two days later, a dose of intravenous cyclosporine (1 mg/kg
over a 4 hour infusion) was administered and blood concentrations
of cyclosporine were determined by monoclonal immunoassay over 24
hours following the infusion (FIG. 1B). Peak concentrations of
cyclosporine occurred within the first two hours after inhalation
and ranged from 140-280 ng/ml (mean 206.2.+-.56.2). Trough
concentrations after 24 hours ranged from 9-44 ng/ml (mean
24.4.+-.14.6). Bioavailability of aerosolized cyclosporine was
9.1%. Absence of high peak levels following inhalation of
aerosolized cyclospore (3-4 fold lower than conventional trough
levels following an oral dose of cyclosporine) may account for
reduced systemic toxicity when cyclosprine is delivered by aerosol
inhalation.
[0068] A study was conducted to measure regional deposition and
absorption of aerosolized cyclosporine following inhalation of a
300 mg dose in subjects given aerosolized cyclosporine early after
transplantation. Regional deposition after a 300 mg dose measured
by radioisotope techniques is shown below in Table II.
2TABLE II REGIONAL DEPOSITION % REGIONAL VOLUME % Left Left Right
Right Stomach Left Left Right Right Subject Date Upper Lower Upper
Lower on Lung Upper Lower Upper Lower 1 8/29/97 18.5 43.7 26.7 11.1
NO 19.2 16.2 31.0 33.5 2 8/29/97 13.1 9.6 28.1 49.2 YES 22.5 26.1
22.0 29.3 3 8/28/97 10.5 34.4 17.4 37.8 YES 18.4 25.6 23.4 32.5 4
8/26/97 13.0 20.1 7.5 59.6 NO 15.6 13.6 35.0 35.9 5 8/26/97 14.3
26.2 24.4 35.1 NO 20.5 24.1 25.1 30.3 6 8/26/98 7.5 12.5 27.7 52.2
YES 16.3 20.6 30.1 32.9
[0069] Thus, the addition of an aerosolized cyclosporine regimen
early after lung transplantation decreased the rate of acute
rejection. In addition, thus far, no subject has developed chronic
rejection, the most tragic complication following lung
transplantation that is the principal cause of morbidity and
mortality after transplantation. In addition, the drug was
tolerated in all subjects and there has been no evidence of
nephrotoxicity as creatinine levels did not differ from subjects.
No subject receiving aerosolized cyclosporine has developed renal
failure.
Example: Administration Of Cyclosporine For Prevention Of Graft
Rejection
[0070] The following section provides an illustration of the
methods and compositions invention. Specifically, a protocol for
administration of aerosolized cyclosporine to a ant recipient and
methods that may be used for following the progress of the treated
are provided.
Chemistry, Manufacturing And Control Procedures For Aerolized
Cyclosporine
[0071] Cyclosporine powder for manufacturing the solution for
nebulization used in this protocol is obtained from Novartis
Pharmaceutical, East Hanover, N.J., (300 mg of cyclosporine powder,
aerosolized in 4.8 ml propylene glycol). Propylene glycol is a
recognized physiologically acceptable solvent which is used as a
vehicle to deliver other aerosolized formulations such as inhaled
pentamidine. New lots of cyclosporine require a purity and identity
check using high performance liquid chromatography. Cyclosporine
for aerosol delivery is prepared in a standard concentration of
62.5 mg/ml. The specific stability of cyclosporine in propylene
glycol has been tested by high pressure liquid chromatographic
assay of cyclosporine against methanolic standards. Reverse phase
C18 chromatography was performed with a mobile phase of 67%
acetonitrile at 1.0 ml/min, column heated at 70.degree. C., with
ultraviolet detection at 214 nm. These tests have indicated that
the preparation is 84% stable at up to 60 days following
preparation. Lots of cyclosporine in propylene glycol which are
>30 days past the date of preparation should be destroyed, and
fresh lots are spot checked to be sure deterioration had not
occurred by chance.
Method Of Aerolized Cyclosporine Administration During Mechanical
Ventilation
[0072] The recipient subject should be on assist control mode and
relaxed. Sedation can be used if necessary. Tidal volume and
frequency can be consistent with conventional settings that were
being used in the intensive care unit for ventilatory support. The
nebulizer with cyclosporine solution is prepared in the usual
manner and the nebulizer is triggered by the ventilator's nebulizer
trigger system (Bennett 7200). The humidifier circuit should be
bypassed during the nebulization, which will increase nebulizer
efficiency by 50%. The nebulization is carried out to dryness.
Delivery Schedule And Dose Of Aerosols
[0073] Recipient subjects are previously exposed to an aerosol of
propylene glycol to assess their tolerance to the aerosol. A small
number of subjects (2-7%) are expected to be intolerant, in which
case a different solvent is employed. Aerosols containing
cyclosporine are given using a commercially available jet nebulizer
(AeroTech II, CIS-US, Bedford, Mass.). Inhalation is performed for
20-30 minutes through a mouthpiece during spontaneous respiration.
A commercially available high efficiency particulate air filter is
used to ensure absence of environmental contamination (AeroStar,
BioSafety Systems, San Diego, Calif.).
[0074] Aerosolized cyclosporine administration begins on
post-transplant Day 6 and continues dosing daily, for 11
consecutive days. This initial daily dosing, followed by three
times weekly has been successfully used, as described in Section 6,
supra. Following the initial daily dosing, aerosols are
administered on Mondays, Wednesdays, and Fridays for convenience.
In spontaneously ventilated subjects, spirometry will be obtained
prior to and immediately after treatment during the first 3 days
with a Morgan spirometer interfaced with a Medical Graphics Model
1070 pulmonary function analyzer (350 Oak Grove Parkway, St. Paul,
Minn.). Quantitation of deposition of aerosolized cyclosporine
using radionucleotides is calculated on the seventh day of aerosol
administration. Changes in the inhaled dose of aerosolized
cyclosporine from a baseline of 300 mg is dependent on measured
allograft deposition as follows: subjects that deposit between
10-15 mg, increase aerosolized cyclosporine to 400 mg; 5-10 mg,
increase aerosolized cyclosporine to 500 mg. Further increments are
based on subsequent deposition studies. Changes to doses are made
if higher than expected allograft cyclosporine deposition is
measured after a 300 mg dose as follows: 25-30 mg, decrease
aerosolized cyclosporine to 200 mg; 30-35 mg, decrease aerosolized
cyclosporine to 100 mg. In double lung recipients, deposition of
inhaled cyclosporine can vary between the right and left
allografts. In such bilateral transplant recipients, the lung that
deposits the lower cyclosporine concentration is used to make the
necessary dose adjustments.
[0075] Spirometry is performed immediately after inhalation of
aerosolized cyclosporine during the initial 10 days of treatment,
and a reduction in the Forced Expiratory Volume (FEVI) of greater
than 15% on two separate occasions, associated with clinical
symptoms of breathlessness is grounds for reducing the dose of
aerosolized cyclosporine. The nebulizer charge is reduced by 100 mg
per day and spirometry is repeated immediately after the dose. The
minimum dose of aerosolized cyclosporine is 100 mg.
[0076] Symptoms of pharyngeal soreness, cough and breathlessness
may occur in transplant recipients during the course of aerosolized
cyclosporine administration. If intolerable side effects occur, the
dose of the aerosol preparation is reduced or discontinued; but,
the attending physician is encouraged to reinstitute administration
at a later time. Should the recipient's condition change, the
physician can use any clinically indicated intervention that is
appropriate for the given situation, including adjustments of
concomitant treatment with other drugs.
Detection And Grading Of Rejection And Measurement Of Allograft
Function
[0077] Blinded histopathologic interpretation of biopsy specimens
is conducted using the accepted standard grading system of the
International Society for Heart and Lung Transplantation.
Successful prevention of rejection is defined as transbronchial
biopsy with a histologic grade of acute rejection that is
.ltoreq.grade I (Yousem, S. A. et al., 1990, J. Heart Lung
Transplant 12:713-716).
[0078] Spirometry (FVC, FEV.sub.1, FEF25-75) is performed at
baseline and at 6 week intervals throughout treatment. By
establishing baseline spirometric indices for each recipient prior
to aerosolized cyclosporine administration, and comparing these
with values measured during administration, individual regression
lines of the FEV.sub.1, can be calculated for each subject.
Analysis of the rate of decline of the FEV.sub.1 and
histopathological assessment of the allograft allows diagnosis of
chronic rejection.
Cytokine And Chemokine Gene Expression
[0079] Cytokine and chemokine gene expression are measured from
bronchoalveolar lavage cells (BAL) and peripheral blood lymphocytes
(PBL) in treated subjects and the dose of aerosolized cyclosporine
is adjusted accordingly. BAL cells and PBL are isolated immediately
prior to aerosolized cyclosporine administration at approximately
day 7 and at the time of bronchoscopy and cytokine mRNA expression
is determined at baseline. The effects of local enhanced
immunosuppression with aerosolized cyclosporine on the expression
of IL-2, IL-6, IL-10, TGF-.beta., IFN-.gamma., inducible nitric
oxide synthase (iNOS), Granzyme and perform are tested. Cellular
gene expression of the various cytokines are measured at 8-week
intervals, at the time BAL cells are isolated after each protocol
bronchoscopy, and at various time intervals throughout the
treatment period. An increase in expression of the cytokines serves
as an indicator that an increase in the dose of cyclosporine is
required.
[0080] Unseparated BAL cells and Ficoll-Hypaque isolated PBMC are
snap frozen before and after a short stimulation of one hour with
phytohemagglutinin (PHA). Stimulation with PHA permits detection of
the presence of IL-2 mRNA in unseparated BAL cells from rejecting
allografts but does not stimulate up-regulation of IL-2 in BALs
from transplant recipients during quiescence or in naive PBL cells.
A similar experience was reported by J. Andersson et al., (1994,
Immunology 83:16-24) who found that preactivated T-cells, after a
short course of stimulation (two hours, anti-CD3), exhibited
intracellular cytokine production, while naive cells required
24-hour stimulation.
[0081] Cytokine gene expression is qualitatively measured by
application of RT-PCR. Total RNA is extracted from unstimulated BAL
cells and peripheral blood lymphocytes using the RNAzol B modified
method (Chirgwin, J. M. et al., 1979, Biochemistry 18:5294) The RNA
concentration is determined by spectrophotometry. The complementary
DNA (CDNA) was synthesized by transcription from RNA in the
presence of human placental RNA-ase inhibitor, Immol/L
deoxynucleoside triphosphates, oligonucleotide deoxythymidine
primer, murine leukemia virus reverse transcriptase and reverse
transcriptase buffer. The RT PCR of the resulting CDNA is performed
according to well established protocols known in the art. Aliquots
of the CDNA are amplified using primers specific for cytokines
measured.
[0082] Amplification is carried out for 30 cycles on a Perkin-Elmer
Cetus Model 480 thermal cycler (Norwalk, Conn.). As an internal
control for quality and potential degradation of RNA, all RNA
samples are assessed for the constitutive gene .beta.-actin
cyclophilin. For negative controls, PCR amplification is performed
with sterile water substituted for cDNA. PCR products are analyzed
by electrophoresis in 2% agarose gels and visualized by ethidiurn
bromide staining. RT-PCR is carried out in the presence of
.sup.32P-deoxycytidine triphosphate labeled primers. The product of
the amplification is electrophoresed on an 8% polyacrylamide gel
that is dried and submitted to autoradiography. The amounts of
radioactivity incorporated in the PCR product are then counted with
a .beta.-scanner.
[0083] The results are expressed as a ratio of cytokine to actin,
and cytokine to cyclophilin, expression. The cytokine to actin
ratio is determined at the time of initiation and during
aerosolized cyclosporine administration. Changes in cytokine gene
expression over time are correlated by linear regression with the
dose of aerosolized cyclosporine deposited in the allograft and the
grade of histologic inflammation associated with acute
rejection.
Reduction Of Maintenance Doses Of Prednisone And Tacrolimus
[0084] In many instances, aerosolized cyclosporine is administered
with other immunosuppressive drugs, such as prednisone,
azathioprine, tacrolimus, as well as oral cyclosporine. Systemic
(oral) immunosuppression is gradually reduced for subjects to whom
aerosolized cyclosporine is administered that are free of
histologic rejection. Should surveillance biopsies fail to show
significant rejection (.ltoreq.grade I acute rejection) on two
consecutive occasions, prednisone doses are reduced from 0.3
mg/kg/day to 0.2 mg/kg/day until two additional biopsies are free
of rejection, and then the dose drops to 0. 1 mg/kg/day. The
prednisone dose remains at this level unless rejection occurs at
which time it is increased to 0.3 mg/kg/day to begin the cycle
again. After completion of the prednisone taper, tacrolimus blood
levels are gradually reduced by approximately 5 ng/ml at 4-month
intervals to maintain blood levels at a minimum of 7.5-10 ng/ml.
Should two consecutive biopsies show significant rejection during
tacrolimus taper (acute rejection.gtoreq.grade 2 or active
obliterative bronchiolitis), tacrolimus blood levels are increased
to the previous baseline (15- 20ng/ml).
Monitor For Immunosuppressive Drug Toxicity
[0085] Throughout the course of treatment recipient subjects are
monitored monthly for evidence of toxicity due to immuno
suppression. One or more of the following variables are monitored
in each subject: 1) serum creatinine; 2) blood pressure; 3) tremor,
headache, paresthesia, confusion and psychiatric disorders, such as
depression and anxiety; 4) nausea, dysphagia, constipation,
vomiting, gastritis, gastric and duodenal ulcer, oral moniliasis,
diarrhea; 5) hirsutism and gingival hyperplasia; 6) hepatic
dysftinction; 7) diabetes mellitus, gout, and hypercholesterolemia;
8) osteoporosis by quantitating bone mineral density by bone
density scan, stress fractures by radioisotopic bone scan if
clinically indicated, arthritis and arthralgias, muscle pain and
myopathy, and 9) post-transplant lymphoproliferative disease and
other neoplasms. Should the subject show evidence of toxicity, the
dose of aerosolized cyclosporine will be adjusted accordingly.
Monitor For Infection Within And Outside Of The Allograft
[0086] The number of infectious complications, including pneumonia,
emphyema, sinusitis, septicemia, abscesses, and urinary tract,
viral, pulmonary and systemic fungal, and skin and wound infections
that occur during the treatment are also monitored.
[0087] Peripheral blood samples are collected pretransplant
(baseline), 2 weeks posttransplant, every 2 months, and when
recipients are evaluated for infection and rejection. The response
to the following three different types of stimuli can be assayed:
1) stimulation by recall antigens (RA) (TT 4 .mu.g/ml, CMV 1:200
dilution) to determine the function of CD4+T cells responding to
nominal antigen presented by autologous APCs; 2) stimulation by a
pool of MHC disparate cells to assess the response of T-h cells
(CD4+and CD8+) to direct presentation of alloantigen (ALLO), 3) and
the polyclonal stimulation of T cells by mitogens PHA and
conconavalin A mitogen (ConA).
Aerosol Studies: Measuring Drug Deposition Using Radionucleides
[0088] Total aerosol deposition in the subject is measured using a
mass balance technique. Using this method, the amount of
radioactivity inhaled by the subject and the amount exhaled are
measured using filters. In the case of a small particle nebulizer
such as the AeroTech II (Cis-Us, Bedford, Mass.) (used to deliver
aerosolized cyclosporine), the dose deposited in the recipient
subject is near equivalent to the total lung dose, because
pharyngeal/laryngeal deposition is minimal. The difference between
these two measurements is the amount deposited in the subject. The
advantage of this approach is that it avoids the use of attenuation
coefficients which may be difficult to interpret in the context of
non-uniform aerosol deposition. The radioactive exposure is
equivalent to typical x-rays of the ribs (100-200 millirads).
[0089] A recipient subject, wearing nose clips, inhales a nebulized
radioactive solution from a typical nebulizer circuit. A low
resistance absolute filter is attached to the expiratory part of
the nebulizer. This filter is designated as the "exhalation
filter". However, in addition to capturing all the particles that
are exhaled by the subject, it will also capture those particles
that are produced by the nebulizer during the expiratory phase of
respiration, i.e., particles that were never inhaled. These latter
particles are referred to as "the leakage" of the nebulizer. In
order to determine the amount inhaled and "the leakage", a
calibration run is necessary that necessitates duplicating the
subject's breathing pattern. The output of the subject's nebulizer
(.mu.Ci/min) is determined by interposing a filter between the
nebulizer and the subject's mouth ("inspiratory filter") and
capturing the aerosol that would be inhaled. Aerosol produced by
the nebulizer, but not inhaled into the inspiratory filter (i.e.,
during expiration) is captured on the "leakage filter." The
inspiratory filter captures all of the particles that would have
been inhaled by the subject. During the calibration run, because
there is no exhalation of particles from the subject, all the
particles on the filter at the expiratory port are the equivalent
of the amount that would have been "leaked." The amount of
radioactivity on the leakage filter is subtracted from the amount
on the subject's exhalation filter (from the subject's original
treatment run) to give the amount truly exhaled. After decay
correction, the amount deposited is calculated as the difference
between the amount of cyclosporine inhaled and the amount exhaled.
The same nebulizer is used for the treatment and the calibration
run since significant inter-nebulizer variability may occur. VEmon
represents an estimate of minute ventilation (VE) used to monitor
breathing pattern during aerosol delivery. It consists of minute
ventilation plus the gas used to run the nebulizer during
expiration, and serves as an indicator that the breathing pattern
was controlled during both the calibration (left) and treatment
runs (right) (Smaldone GC, Dickinson G, 1992, Chest 101:
82-87.)
[0090] The mass balance technique measures the dose deposited in
the subject. To determine the regional distribution of the dose
(right vs. left lung or central airways versus lung periphery),
gamma camera imaging of deposited radioactivity is needed. When
particles are inhaled by a subject, they will either be exhaled or
be retained (deposited). In healthy subjects, a uniform deposition
pattern within the lungs indicates that most of the particles have
deposited in small peripheral airways or alveoli by means of
gravitational sedimentation. A common non-uniform pattern seen in
healthy subjects due to inertial impaction, is the peri-hilar
pattern in which particles have deposited predominantly in the
typical uniform and non-uniform deposition patterns. The aerosol
deposition patterns are superimposed on an outline of the whole
lung, which was generated using a Xenon (.sup.133Xe) equilibrium
scan. .sup.133Xe is a gas with a long half-life (5.3 days) which,
when breathed to equilibrium measures regional lung volume.
[0091] Therefore, using regions of interest based on the .sup.133Xe
image, it is possible to facilitate comparison between serial
studies in the same subject, or make intersubject comparisons of
the distribution of deposited particles in the lung and airways
(Iacono et al., Am. J. Respir. Crit. Care Med. 55:1690-1698).
Following a .sup.133Xe equilibrium scan, using a computer in series
with a gamma camera, regions are drawn around each lung which is
called the whole lung zone and another pair of regions are drawn
which centered over the large central airways comprising 33% of the
entire lung area, is called the central zone. The area remaining
after the central zone is deducted from the whole lung zone and is
designated the peripheral zone. To describe the regional pattern of
deposition of an inhaled aerosol, labeled with .sup.99mTc, and
allow intersubject comparisons, the ratio between central (C) and
peripheral (P) lung counts (C/P) is calculated in a manner which
normalized for differences in relative lung thickness by dividing
the C/P .sup.99mTc counts by the C/P 133Xe counts. The ratio
defined the specific C/P ratio (sC/P). Using the resulting sC/P
values, a ratio of 1.0 indicates equal deposition in all lung
regions. Because the central lung region outlines both central
airways and the lung parenchyma surrounding them, an sC/P ratio of
unity reflects predominantly alveolar deposition. Increasing
deposition in the proximal airways results in increasing sC/P
ratios greater than unity. Therefore, determination of the sC/P
ratio allows quantification of initial deposition patterns and
comparisons between subjects.
Aerosol Cyclosporine Kinetic Studies
[0092] Cyclosporine pharmacokinetic studies may be performed at,
for example, Week 12 in recipient subjects. After obtaining a
baseline 3.0 ml blood sample, the subject receives 1.0 mg/kg
cyclosporine intravenously as a 4 hour infusion. Additional blood
samples are drawn at 2.0, 4.0, 5.0, 6.0, 12.0, 18.0 and 24 hours
after initiation of the infusion. On day number 2 of the study,
after the final 24.0 hour sample is obtained, a 300 mg dose of
aerosolized cyclosporine or placebo is given, with blood samples
taken at 0.25, 0.5, 0.75, 1.0, 2.0, 4.0, 6.0, 8.0, 12.0, 18.0, and
24.0 hours after initiation of the aerosolized dose. Samples are
analyzed for both cyclosporine and tacrolimus. The intravenous
cyclosporine dosage allows the calculation of cyclosporine total
body clearance, elimination half-life and volume of distribution.
Having those results, one can accurately calculate the amount of
cyclosporine that was absorbed from the aerosolized dose in those
subjects on the active drug, as well as calculate the absorption
rate constant for drug deposited in the lungs.
[0093] The present invention is not to be limited in scope by the
specific embodiments described herein which are intended as single
illustrations of individual aspects of the invention, and
functionally equivalent methods and components are within the scope
of the invention. Indeed, various modifications of the invention,
in addition to those shown and described herein will become
apparent to those skilled in the art from the foregoing description
and accompanying drawings. Such modifications are intended to fall
within the scope of the claims. Various publications are cited
herein, the contents of which are hereby incorporated, by
reference, in their entireties.
* * * * *