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Advances in Brief |
Battelle Memorial Institute, Columbus, Ohio 43201 [A. R. D., I. M. G., D. P. H., L. J. S., M. E. P., A. R. I.]; The Ohio State University, Columbus, Ohio 43210 [G. D. S.]; National Cancer Institute, NIH, Bethesda, Maryland 20892 [L. M. D. L., D. W., J. L. M.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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12
weeks, when both these and the high-dose mice began losing weight. The
low-dose mice had nonsignificant reductions of 30%
(P < 0.13) and 16% (P <
0.30) for BaP- and NNK-treated mice, respectively without any evidence
of side effects. For BaP- and NNK-treated mice, numbers of hyperplastic
areas directly correlated to dose level and inversely to tumor number,
suggesting arrested progression. Inhaled mid-dose isotretinoin caused
up-regulation of lung tissue nuclear retinoic acid receptors (RARs)
relative to vehicle-exposed mice, RAR
(3.9-fold vehicle), RARß
(3.3-fold), and RAR
(3.7-fold), suggesting that these receptors may
be useful biomarkers of retinoid activity in this system. The
encouraging results from this pilot study suggest that inhaled
isotretinoin merits evaluation in people at high risk for lung cancer. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In a randomized clinical trial, the oral administration of isotretinoin (1–2 mg/kg/day) was significantly protective against second aerodigestive tumors in a cohort of previously treated head-and-neck cancer patients (2 , 3) . Because of the effectiveness of isotretinoin as a preventative of some forms of cancer, its efficacy as a lung cancer chemopreventive agent is under study in several clinical trials (Protocol IDS: UCHSC-92382, NCI-V94-0506 and CBRG-9208, NCI-V92-0159, NBSG-9208).
Enthusiasm for the use of isotretinoin as a chemopreventive agent has been held back, in part due to the occurrence of debilitating drug side effects associated with the doses used in the M. D. Anderson study (4) , which, based on pharmacokinetic data, provided steady-state blood levels of 100–200 ng/ml. Sixteen of the 49 patients in this head-and-neck chemoprevention trial did not complete the course of therapy. Because the benefits of isotretinoin treatment is reduced after cessation of treatment (3) , the expectation is that chronic drug administration would be required, making the patient compliance issue critical.
To address the toxicity concerns, investigators have contemplated lowering the dosages of the drug, but it is unclear whether such a change would jeopardize the desired therapeutic effect. Oral doses of 1 mg/kg failed to reverse lung metaplasia in smokers (5) . Attempts to lessen the severity of the toxic effects by coadministration of vitamin E are under study in clinical trials (Protocol IDS: MDA-DM-97078, NCI-P98-0132), and clearly additional work is merited in this critical area.
We reasoned that because lung cancer arises in the lung epithelium, direct application to the target cells would improve the therapeutic index. Aerosol inhalation can deposit drug directly on the population of cells caught up in the early phase of cancer, potentially achieving much more efficiency compared with reliance on diffusion from the blood. There are theoretical bases to expect major differences in potency between oral and inhaled retinoids. Some highly lipophilic compounds can be significantly retarded in their clearance from the lung epithelial surface into the blood stream (6 , 7) . Because the reverse also is probably the case, the poor results with oral administration may simply be a case of too little drug reaching the target cells in some parts of the lung. In addition, isotretinoin is avidly bound by serum albumin, limiting its availability for promotion of differentiation and inhibition of proliferation (8) . Direct application to the lung epithelium may avoid much of the protein binding, thus greatly increasing potency at the target site.
Surprisingly, despite longstanding interest in isotretinoin,
information on its in vivo pharmacology as a cancer
preventive agent in animal models is scarce. In one study, p.o.
administered isotretinoin of >300 mg/kg weekly failed to prevent
urethane-induced lung cancer in the A/J mouse model (Ref.
9
; Table 1
). Despite this failure, we felt that direct application to the lung
epithelium merited evaluation. To test this premise, we elected to
expose carcinogen-treated A/J mice to isotretinoin aerosol for this
pilot study.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental Design
Mice received injections of one of three carcinogens and were
exposed by inhalation in groups of 21 to three graded concentrations of
isotretinoin or vehicle for 10 (urethane-treated mice) or 16 (NNK- and
BaP-treated mice) weeks. Forty-six mice treated with each carcinogen
and 46 untreated controls were maintained in cages and were not
exposed. Some of these were sacrificed at intermediate times to
determine the progress of carcinogenesis. At first, exposure was daily
for all doses, but after 12 days it was reduced to twice weekly for the
highest dose because of severe local toxicity and three times weekly
for the middle dose as a precautionary measure (Table 2)
.
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Formulation of Nebulizer Solution
Powdered isotretinoin was dissolved in 100% ethanol plus 0.1%
-tocopherol and 0.1% ascorbyl palmitate to give isotretinoin
concentrations ranging from 0.1 to 10 mg/ml. The formulations were
prepared monthly. The solutions were protected from light and stored at
-5°C until use. UV-visible spectrophotometric verification of the
formulated test article concentration was performed on all batches in
advance of inhalation treatments with the test article solution. Only
formulations within ± 10% of the targeted concentration were
used on study.
Inhalation Exposure
Solutions were aerosolized using a Pari LC-plus nebulizer (Pari,
Richmond, VA). Animals were exposed in nose-only exposure units
designed to provide a fresh supply of the test atmosphere to each
animal, independent from other animals. The exposure units were based
on the design described by Cannon et al.
(17)
. The units consisted of multi-tier modular
sections, each tier containing eight exposure ports located
peripherally around a central delivery plenum.
During exposures, animals were restrained in unstoppered polycarbonate tubes (C&H Technologies, Westwood, NJ) through which a flow of aerosol, 350–500 ml/min per mouse, passed from the chamber. Each tube was tapered at one end to approximately fit the shape of the animal’s head, and the diameter of the cylindrical portion of the cone was such that the animal could not turn in the cone. Each cone was fastened to the inhalation chamber with the nose portion of the cone protruding through a gasket into the chamber, permitting the animal to breathe the test or control atmosphere emanating from within the central plenum.
Aerosol Characterization
To determine aerosol concentrations, measured volumes of aerosol
were drawn through filters, which subsequently were analyzed for
isotretinoin by a UV-visible method. To determine particle size,
aerosol was drawn through Mercer-type cascade impactors (InTox,
Albuquerque, NM) equipped with filters on each stage and a backup
filter. The individual filters were analyzed for isotretinoin and the
MMADs and geometric standard deviations were calculated from the
data using Battelle software.
Quantitation of Lung Lesions
Within 24 h of the last inhalation exposure, animals were
euthanized by i.p. injection of pentobarbital, and their lungs were
removed and fixed in Bouin’s solution or flash frozen for RAR
determination. The lungs were evaluated in a blinded fashion so that
neither carcinogen nor isotretinoin dose levels were known to the
evaluator, who visually counted hyperplastic areas and adenomas on the
lung pleural surface as described previously (18, 19, 20)
. The
significance of the differences between the mean tumor incidence of the
treatment and the control groups was determined using the Mann-Whitney
Rank Sum test (Statmost TM; DataMost Corp., Sandy, Utah).
Biomarkers: RAR Induction
Antibodies.
Polyclonal antibodies to RAR, ß, and (Santa Cruz Biotechnology
Inc., San Francisco, CA) were used with a BM Chemiluminescence Western
Blotting Kit (Mouse/Rabbit; Boehringer Mannheim Corporation,
Indianapolis, IN).
Apparatus and Reagents for Western Blot Analysis.
The X cell II Mini-cell and Blot Module was used with 10% Tris-glycine
gels and transfer buffer and Tris-glycine SDS sample buffer;
Tris-glycine SDS was used as running buffer (NOVEX-NOVEL Experimental
Technology Inc., San Francisco, CA).
Experimental Design.
Five lungs each from the urethane-treated vehicle, low-dose, and
mid-dose animals were snap frozen in liquid nitrogen and stored at
-70°C for determination of RAR, ß, and by Western blot
analysis. In addition, five lungs each from urethane-injected unexposed
mice and untreated control mice were designated for Western blots. The
lungs were homogenized, and the RARs were determined by standard
Western blotting (21
, 22) . Because of deaths early in the
high-dose experiment, no lungs were available for Western blot analysis
for the high-dose exposures, i.e., all tissue was used for
lesion quantitation purposes.
Samples.
Lung tissue was collected, frozen on dry ice, and kept at -70°C
until used. A 500-mg portion was diced in small pieces and homogenized
in 300 µl of cold PBS with a hand-held homogenizer for 2–3 min.
Samples were centrifuged at 500 rpm for 5 min or until the supernatant
was clear. The pellet was suspended in 400 µl of cold buffer A [2
µl of 0.5 M EDTA, 10 µl of 1 mM EGTA, 50
µl of 100 mM phenylmethylsulfonyl fluoride, 10
µl of 1 M DTT, and 10 ml of 10 mM HEPES (pH
7.9) + 10 mM KCl], and left at ice temperature for 15 min.
A 25-µl volume of a 10% solution of NP-40 was added, and the samples
were mixed in a vortex vigorously for 10 s. Samples were
centrifuged at 14,000 rpm for 1 min at 4°C, and the pellet was
treated once more in this fashion. The supernatant was removed, and
25–100 µl of buffer C [4 µl of 0.5 M EDTA, 20 µl of
100 mM EGTA, 20 µl of 0.1 M
phenylmethylsulfonyl fluoride, 2 µl of 1 M DTT, and 2 ml
of 20 mM HEPES (pH 7.9) + 0.4 M NaCl] were
added. Pellets were resuspended by tapping gently on the bottom of the
Eppendorf tube. Samples were rocked vigorously in a bucket of ice on an
orbital shaker for 30 min. Samples were then centrifuged at 14,000 rpm
for 5 min at 4°C. Supernatants were kept frozen at -70°C until
needed. Protein concentrations were determined using the Bradford
method (23)
.
Analysis.
Samples were prepared by adding one part sample buffer to one part
sample and mixing well. For denaturing conditions, samples were heated
at 95°C for 5 min. Five µl of Rainbow Standard and 5 µl of
Biotinylated Molecular Marker were used. Twenty µg of protein per
sample was loaded in each lane. Electrophoresis was performed with the
voltage set at 125 V for 1–1.5 h. Gel transfer was executed at 25 V
for 2 h. The membrane was stained in Ponceau S for 5 min and
destained with one wash of 5% acetic acid. The membrane was washed in
TBS solution until the staining disappeared and then was incubated in 1
ml of blocking solution and 9 ml of TBS for 60 min and with primary
antibody solution overnight (20 µl of primary antibody solution in 1
ml of blocking solution and 9 ml of TBS; dilution of 1:500). The
membrane was washed in PBS-Tween 20 three times for 10 min each, and
then was incubated in 1 ml of blocking solution and 19 ml of TBS along
with 20 µl of antibiotin horseradish peroxidase-linked
antibody and 2 µl of secondary (rabbit antimouse antibody; 1:1000
dilution) for 30 min. The membrane was washed in PBS-Tween 20 four
times for 10 min each. The film was exposed to detect and subsequently
developed for analysis.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tocopherol acetate and
ascorbyl palmitate stabilizers, were 0.21, 0.3, and 1.2%, leading to
minimum droplet sizes (i.e., if all of the ethanol had
evaporated) of 0.38, 0.43, and 0.69 µm, respectively. The minimum
droplet sizes were calculated by assuming a MMAD of 3 µm for the
Pari-LC jet plus nebulizers used in these experiments and using the
relationship dfinal = dorig
x f1/3, where
dfinal is the final diameter,
dorig is the original diameter, and f is the mass
fraction of solute.]
Calculations of Deposited Dose.
Inhaled monodisperse particles having an aerodynamic diameter of 1.09
µm deposit 9.2% in the pulmonary region, with 59.2% total
deposition (24)
. Taking the average mouse weight as
22 g, the respiratory minute volume, calculated as Raabe et
al. (24)
had done, was 2.1 x [mass
(g)]0.75 ml/min (25)
. Assuming the
same deposition efficiency as 1.09-µm monodispersed particles (for
simplicity; the actual values would vary somewhat for these aerosols),
the calculated daily pulmonary doses of isotretinoin for each 45-min
exposure were 0.005, 0.081, and 2 mg/kg per exposure. Calculated
total deposited doses were 0.034, 0.54, and 12.4 mg/kg per exposure
(Table 2)
.
Chemoprevention by Inhaled Isotretinoin.
All comparisons are to the vehicle-exposed control mice unless noted.
Mice exposed to the high isotretinoin dose had substantial reductions
in tumor multiplicity, ranging from 56 to 80% below vehicle controls,
for all three carcinogens (Table 3)
, but during daily exposures for the first 2 weeks, they experienced
excessive toxicity to the snout and forelimbs. These mice lost weight
(Fig. 1)
, and 35% died. Following a 2-day respite, the exposure frequency
was reduced to twice weekly, and the body weights increased to those of
the vehicle-exposed control mice (Fig. 1)
and the lesions resolved,
although two more mice died early in the study (Table 4)
. At the end of exposure, weights were again below those of the vehicle
controls (Fig. 1)
. In light of the significant consequences of local
retinoid toxicity in this model, extrapolation of these results to
humans will be difficult.
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. At the end of exposure, the weights of these
mice were 11% below those of vehicle controls. Other signs of toxicity
were absent, although 3% (2 of 63) of the mice died before the end of
exposure (Table 4)
. [The reduction in body weight is of uncertain
significance. Caloric restriction >10% for a significant portion of
an animal’s lifetime reduces tumorigenesis in some organs
(26)
but increases tumorigenesis in the lung
(27)
.] Hyperplastic areas were significantly elevated in
the NNK- and BaP-treated mice exposed to mid and high doses of
isotretinoin. Total lesions, i.e., hyperplastic areas plus
adenomas, were not affected by treatment in the NNK-induced animals but
were fewer for the urethane-treated animals at the high isotretinoin
dose and for the BaP-treated animals at both the mid and high
isotretinoin doses (Table 3)
.
For the animals receiving low doses of isotretinoin, the numbers of
tumors were not affected by treatment at the 95% confidence level, but
for both the NNK- and BaP-treated mice, trends in line with those of
the mice receiving mid and high doses of isotretinoin were evident for
both tumors and hyperplastic areas (Table 3)
.
For the urethane and BaP treatments, the mice exposed to vehicle had
fewer tumors than the cage control animals (Table 3)
. This phenomenon
was observed to an even greater degree in a chemoprevention study in
A/J mice with aerosolized budesonide, where the control mice were
exposed essentially to air only (28)
, and possibly is
related to the tumorigenesis-inhibiting effect of stress
(29)
, although a contribution from the ethanol vehicle or
the antioxidant excipients, ascorbyl palmitate and -tocopherol,
cannot be ruled out in the studies reported here.
Biomarkers: RAR Induction.
Inhaled mid-dose 13-cis retinoic acid up-regulated lung
tissue RAR by 3.9-fold over solvent, RARß by 3.3-fold, and RAR
by 3.7-fold, showing that these receptors are useful biomarkers of
retinoid activity in this system (Fig. 2)
. An explanation for the apparent increases in RARs in group 1
relative to group 5, which differed only in that the group 1 animals
had received an i.p. injection of urethane 16 weeks prior to sacrifice,
is not available and must await further research to determine the
reproducibility of the relatively small increases. Similarly, more
studies are needed to determine the significance, if any, of the
apparent induction of RARs in the group 2 mice exposed to vehicle
aerosol.
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DISCUSSION |
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Inhalation Exposures to Isotretinoin.
Ethanolic solutions of isotretinoin were aerosolized with particle
sizes calculated to provide substantial pulmonary deposition. The
ethanol was not removed from the exposure air. The inhaled ethanol, as
well as the excipients -tocopherol and ascorbyl palmitate, may have
had an effect on carcinogenesis for the urethane and BaP treatments
because the vehicle-exposed animals had fewer tumors than unexposed
controls. However, the effect in these experiments—20 and 30%
decreased tumor multiplicity for urethane and BaP, respectively—was
less than that observed by others (50%) when BaP-treated control mice
were exposed to essentially air alone (28)
. In any case,
the addition of isotretinoin to the aerosols produced significant
decreases in tumors relative to vehicle-only aerosols.
Lung Tumor Prevention by Inhaled Retinoids.
In this study, we looked at three different doses of isotretinoin
aerosols inhaled daily. The lowest dose was not significantly
effective. The highest dose was associated with lethal toxicity,
presumably due to extensive ulceration of the snout and forearms of the
mice. The assumption was that this was related to the well-known local
toxic effects of retinoids on skin. This apparent local toxic response
resolved with a reduction in dose frequency, and significantly fewer
lung nodules occurred for all three of the carcinogens with this dosing
schedule. In light of the frequent lethal toxicity associated with the
high-dose exposures, however, we restricted our focus to the mid-dose
exposure as being the relevant drug dose.
With the mid dose, significant toxic signs were not observed other than
the weight loss that occurred near the end of the study (Fig. 1)
. The
3% fatality rate in this cohort would not be unusual in an experiment
involving this degree of manipulation of the experimental animals, the
absence of deaths in the low-dose and vehicle-exposed groups possibly
being a statistical fluke. Even in the vehicle controls, there was a
>10% weight difference, relative to unexposed animals, due to the
experimental procedure. The stress of forced aerosol inhalation in
rodents is expected to be very different from voluntary aerosol
inhalation in humans.
Despite the reduction in inhaled dose frequency taken as a precaution
against potential local nasal toxicity, the mid dose was still
associated with a significant reduction in the number of lung nodules
for both of the tobacco-related carcinogens, BaP and NNK. This finding
is even more significant when considering the amount of drug that was
required to achieve this effect. For example, over most of the study,
the mid-dose level, including extrapulmonary dose, was <0.5% of an
oral dose used in the previously discussed in vivo
experiments (Table 1)
; based on pulmonary dose alone (Table 2)
, the
dosage was <0.15% during the first 2 weeks and <0.06% during the
remainder of the experiment. By all accounts, these comparisons suggest
remarkable drug potency for the inhaled aerosol.
The finding that a modest dose of inhaled retinoid is both tolerated and efficacious supports the contention that lung therapy by inhalation is the preferred route of lung delivery for dealing with the airway-confined phase of a disease process, as has been reported with certain agents used to treat pulmonary infections (30) and corticosteroids for cancer prevention (28 , 31) . An obvious application for the inhalation approach is the use of retinoids as lung cancer chemopreventive agents.
Hyperplasia and Total Lesions: Mode of Action.
The preliminary data for the NNK-treated mice suggest that isotretinoin
does not eliminate initiated cells but inhibits their progression to
the tumor stage: hyperplastic areas inversely correlated with tumors,
whereas total lesions, i.e., hyperplastic areas plus
adenomas, remained relatively constant (Table 3)
. A similar increase in
hyperplastic areas occurred in the BaP-treated mice, but in this case,
total lesions decreased, suggesting that initiated cells were either
eliminated or were constrained to microscopic clusters.
BaP and NNK are putative major carcinogens in tobacco smoke (32) , and thus are the most relevant of the carcinogens used in this study. The similarities between the dominant molecular lesions caused by BaP and NNK—BaP causes G-C to T-A transversions in the first nucleotide and NNK causes G-C to A-T transitions in the second nucleotide, both in codon 12 (33) —argues against a substantial biological difference among cells initiated by the two carcinogens.
In contrast to the NNK- and BaP-treated animals, tumor multiplicity in
the urethane-treated mice was decreased only at the high isotretinoin
dose, the meaning of which is obfuscated by associated toxicity, and
there was no effect on numbers of hyperplastic areas (Table 3)
. The
total number of lesions was markedly reduced in the high-dose animals,
suggesting elimination of initiated cells or restriction of clonal
expansion to microscopic lesions. Like NNK and BaP, urethane, an
ethylating agent, mutates K-ras, but at codon 61 instead of
codon 12 (33)
. Morphological differences in tumors also
occur. The fractions of tumors classified as solid tumors were 78 and
88% for BaP- and NNK-induced tumors, respectively, but only 57% for
urethane-induced tumors (34)
. It is interesting to
speculate that these differences contribute to the varied responses to
inhaled isotretinoin, but there appears to be no supportive data in the
literature.
Retinoid Toxicity at Efficacious Doses.
Although the high dose with the twice-weekly schedule was only 6% of a
nontoxic oral dose (Table 1)
, it was associated with weight loss toward
the end of the study (Fig. 1
and Table 4
). For the NNK- and BaP-treated
mice, this dose was essentially no more efficacious than the much
smaller mid dose (Table 3)
. Perhaps surprisingly, the mid inhaled dose
at only 0.4% of a nontoxic oral dose also caused weight loss in mice
exposed for >10 weeks. Examination of the weight data over the
duration of the experiment (data from BaP-treated mice in Fig. 1
; data from NNK- and urethane-treated mice not shown) confirms
the late onset of the weight loss.
There are at least two possible explanations for this finding:
(a) the total dose may have been higher than calculated as a
result of uptake through the skin of the exposed snout; and
(b) local toxicity may have occurred in the respiratory
tract. It seems unlikely that sufficient isotretinoin could have been
absorbed through the skin to produce systemic toxicity, nor would such
a conclusion be supported by numerous inhalation studies in mice with
aerosols of other compounds. This leaves local toxicity as a possible
explanation. Microscopic examination of tissues for pathological
changes was neither planned nor carried out for this pilot efficacy
study; however, gross examination of the lungs revealed no differences
between control and treated lungs except for the differences in numbers
of tumors and hyperplastic areas. Given that the pulmonary dose is
calculated to be only 13% of the total deposited dose and would be
distributed over 640 cm2
(35)
,
pulmonary toxicity seems unlikely.
In contrast to the large surface of the lung, the upper respiratory
tract, mostly nasal mucosa, has a surface area of only 3
cm2
(36)
but receives 87% of the
deposited dose. Coupling this high dose with the ease with which
rodents develop debilitating nasal lesions (37
, 38)
, we
suggest that an explanation for the weight loss in the mid-dose mice is
local nasal toxicity, which developed to significant levels after 10
weeks of exposure.
Only toxicology studies with histopathology included will provide
definitive explanations for the phenomenon of weight loss at these low
doses, but if our suggestion that upper respiratory tract toxicity is
to blame is correct, the observation is probably not relevant to the
safety of inhaled retinoids in people. For pulmonary toxicology studies
except for neoplasia, nasal toxicity in rodents, which are obligate
nose breathers, from inhalants is usually not considered relevant for
evaluating possible effects in humans unless human exposure will
include the nasal cavity. For example, local nasal toxicity would not
be a concern with a chemopreventive agent administered by oral
inhalation because this route of administration skips drug transit
across the nasal cavity. Moreover, the dose-response curve for efficacy
appears to have already plateaued at the mid dose (Table 3)
, suggesting
that the dose could be lowered to a point between the low dose and the
mid dose without sacrificing efficacy.
Limitations of the A/J Mouse Model: The Possible Role of
Inflammation in Lung Cancer.
A special limitation of mouse inhalation models is that with the nature
of the drug delivery system, a major fraction of the total administered
drug will deposit on the snout and in the upper respiratory tract.
Without delivering drug via a tracheotomy, there is no other
alternative. Therefore, an artifact of this model is inefficient drug
delivery to the deep lung. In humans, where much more efficient
pulmonary drug delivery devices exist, the fraction of the drug that is
impacted in and around the snout in the mouse would be expected to
travel directly into the pulmonary airway. This improved drug delivery
efficiency would greatly reduce the potential for local toxicity.
For some drugs, the high extrapulmonary deposition in the mouse model
might confound interpretations regarding the effectiveness of the
pulmonary route of drug delivery. In the case of isotretinoin, we
suggest that the extrapulmonary-deposited drug probably is not germane
because it would either be swallowed or absorbed into the blood stream
in much lower amounts than ineffective oral doses (Table 1)
and so is
unlikely to have contributed significantly to efficacy.
We used an animal model for evaluation of efficacy. Animal models for human lung cancer are widely accepted (10 , 11) , but like all preclinical models, the A/J mouse model is imperfect. With the A/J model, mice treated with complete carcinogens do not develop lung inflammation and the attendant rapid cell proliferation that is common in human lung disease. The contribution of inflammation to aerodigestive cancerization is becoming more evident, and this may be, in part, how retinoids effect their chemopreventive benefit in humans.
A connection between the inflammation-associated enzyme COX-2 and retinoid pathways is suggested by the fact that the Ras/extracellular signal-regulated kinase signaling pathway appears to play a role in the regulation of COX-2 expression. Human non-small cell lung cancer cell lines with mutations in K-ras have high expression levels of COX-2, and inhibition of ras activity in these cell lines decreases COX-2 expression (39) . Rat intestinal epithelial cells and fibroblasts transfected with H-ras overexpress COX-2, whereas inhibitors of extracellular signal-regulated kinase ameliorate this response (40) . We and others have found COX activity to be potentially significant in aerodigestive cancers (41, 42, 43) . A high percentage of murine and human lung adenocarcinomas have a mutated ras gene and a constitutively activated ras signaling pathway (13) , which may explain the high levels of COX-2 seen in some lung tumors. RARß is known to interfere with the ras signaling pathway by inhibiting the function of the activator protein transcription factor (44) . An expected result of this interference would be the down-regulation of COX-2 expression, which may play a role in the decreased tumorigenesis seen in the A/J lung cancer model following isotretinoin inhalation. Such an effect of RARs on COX-2 expression is supported by published data showing that retinoids inhibit the epidermal growth factor (i.e., ras)-induced transcription of COX-2 in human oral squamous carcinoma cells (45) .
Through time, the development of in vivo models that more
closely mirror the actual process of carcinogenesis in humans would be
highly desirable. For the pilot evaluations discussed here, we believe
the A/J mouse model is adequate as long as its shortcomings are
acknowledged. In future experiments, the local aerosol drug dose in and
on the snout can be reduced through modifications of the exposure
system to prevent facial exposure and by reducing the particle diameter
to 0.3 µm, which will increase the pulmonary-to-total dose ratio
to 64% (24)
.
Induction of RARs.
RARs were investigated as biomarkers because their genes contain
retinoic acid response elements and as such are likely to be
up-regulated soon after exposure to retinoids, i.e., they
are first-order dependence genes (46)
. The
induction of all three RARs was at least 3-fold in lungs exposed to mid
levels of isotretinoin. Only the urethane-treated mice were examined in
this pilot study, but these probably represent the other treatment
groups for this determination because all mice were exposed to the same
aerosols. The induction of the RARs in the mid-dose mice correlated
with efficacy in the BaP- and NNK-treated mice and may not only provide
biomarkers for exposure but may also be a part of the mechanism for the
efficacy of inhaled isotretinoin. The up-regulation of lung RARs by
inhaled isotretinoin occurs across species, as reported in a companion
study,3
which also indicates that oral administration induces liver, but
not lung RARs, and that administration by inhalation induces only lung,
and not liver receptors.
Implications of Improving the Therapeutic Index of Retinoid
Administration.
The clinical trials to unequivocally establish the chemopreventive
benefit of oral isotretinoin are likely to be completed in the near
future. Even if positive, however, long-term compliance is expected to
be a major issue because of the significant frequency of debilitating
side effects. Even if changing the route of administration of retinoids
only decreased the side effect profile, this would make the drug much
more interesting to contemplate for broad clinical utility. Moreover,
because the role of retinoids in maintaining optimal bronchial
epithelial differentiation has been extensively studied, other general
beneficial effects of retinoic acids on epithelia have been well
documented. For example, in a study using a rodent model of chronic
obstructive pulmonary disease, there was a suggestion that
retinoids can reverse parenchymal lung injuries associated with
compromised respiratory function (47)
. Indeed, if
there is a causal relationship, this benefit may contribute to the
cancer-preventive effects because individuals suffering from chronic
obstructive pulmonary disease and other smoking-related diseases are at
increased risk for developing lung cancer (48)
.
Chemoprevention of Lung Cancer in Smokers.
Although the role of retinoid biology is thought to be pivotal in the
process of tobacco-induced carcinogenesis, attempts to use retinoids as
lung cancer chemopreventive agents have been problematic as shown by
clinical trials (49, 50, 51)
. In two of three large
ß-carotene studies, there were a greater number of lung cancers
occurring in smokers on the ß-carotene arm. In a more recent
randomized control trial of 13-cis retinoic acid in stage I
resected non-small cell lung cancer, during the first evaluation of the
trial, there was a question about the time to disease recurrence in
smokers receiving active drug (52)
. Although the trial was
already completed in regard to accrual, the smokers still on retinoid
were instructed to stop treatment until the validity of the correlation
was sorted out. The speculation of smokers being harmed by efforts to
chemoprevent lung cancer is conceptually plausible. The presence of
ongoing carcinogenic damage in the setting of the airway of a smoker
receiving drug involves complex scenarios in which adverse outcomes are
possible. Clearly, this same approach in former smokers represents a
more favorable opportunity to document an objective benefit of
chemoprevention. In light of this situation, perhaps the most
responsible way to proceed is to carefully study the benefit of
aerosolized chemoprevention in parallel cohorts based on smoking
status. A clinical trial that formally evaluates the outcomes of
aerosolized retinoid chemoprevention as a function of smoking status is
needed to address this important issue.
In this preliminary analysis of the pulmonary delivery of isotretinoin by inhalation, there was evidence of efficacy at weekly pulmonary doses as low as 0.25 mg/kg and suggested efficacy at doses as low as 0.04 mg/kg in reducing the pulmonary carcinogenicity of the tobacco carcinogens NNK and BaP in A/J mice. Because pulmonary drug delivery deposits drug directly on the tumor compartment, efficacy can be achieved at low doses: mid and low weekly pulmonary doses were <2 and <0.3%, respectively, of the highest recommended weekly oral dose of isotretinoin for acne treatment (Accutane 55; Roche). The results reported here, however, are all of the more encouraging because they probably were produced by the <10% of the inhaled aerosol that deposited in the lung as the extrapulmonary dose was probably too low to have a systemic effect. This suggests that an improved therapeutic index can be achieved in humans by more selectively delivering retinoid chemopreventive agents to deep lung tissue using aerosols. Further work with this approach, both preclinically and in the clinic, is justified to validate the true benefit of this important new chemoprevention delivery approach.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Battelle, 505 King Avenue, Columbus, OH 43201-2693;
Phone: (614) 424-4644; Fax: (614) 424-3268; E-mail: Dahla{at}Battelle.org 
2 The abbreviations used are: BaP,
benzo[a]pyrene; NNK,
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; MMAD, mass median
aerodynamic diameter; RAR, retinoic acid receptor; TBS, Tris-buffered
saline; COX, cyclooxygenase.
3 D. L. Wang, M. Marko, A. R. Dahl, K. J. Engelke,
I. M. Grossi, D. P. Houchens, L. J. Scovell, M. E. Placke, A. R.
Imondi, G. D. Stoner, R. Dedrick, J. L. Mulshine, and L. M. De Luca.
Topical delivery of 13-cis retinoic acid by inhalation
upregulates expression of lung but not liver retinoic acid receptors,
submitted for publication.
Received 3/ 6/00; revised 5/ 1/00; accepted 5/15/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
IIb/ßIII integrin expression and function in subpopulations of murine tumors. Exp. Cell Res., 201: 23-32, 1992.[CrossRef][Medline]
anthracene in mice. J. Cancer Res. Clin. Oncol., 121: 393-396, 1995.[CrossRef][Medline]
-Tocopherol and ß-carotene supplements and lung cancer incidence in the -tocopherol, ß-carotene cancer prevention study: effects of base-line characteristics and study compliance. J. Natl. Cancer Inst., 88: 1560-1570, 1996.This article has been cited by other articles:
|
|
 |
|
  H. Fu, J. He, F. Mei, Q. Zhang, Y. Hara, S. Ryota, R. A. Lubet, R. Chen, D.-R. Chen, and M. You Lung Cancer Inhibitory Effect of Epigallocatechin-3-Gallate Is Dependent on Its Presence in a Complex Mixture (Polyphenon E) Cancer Prevention Research, June 1, 2009; 2(6): 531 - 537. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Sharma, P. Gao, and V. E. Steele The chemopreventive efficacy of inhaled oltipraz particulates in the B[a]P-induced A/J mouse lung adenoma model Carcinogenesis, August 1, 2006; 27(8): 1721 - 1727. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-J. Lehmler, A. Fortis-Santiago, D. Nauduri, and P. M. Bummer Interaction of long-chain nicotinates with dipalmitoylphosphatidylcholine J. Lipid Res., March 1, 2005; 46(3): 535 - 546. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. W. Wattenberg, T. S. Wiedmann, and R. D. Estensen Chemoprevention of Cancer of the Upper Respiratory Tract of the Syrian Golden Hamster by Aerosol Administration of Difluoromethylornithine and 5-Fluorouracil Cancer Res., April 1, 2004; 64(7): 2347 - 2349. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. C. Winterhalder, F. R. Hirsch, G. K. Kotantoulas, W. A. Franklin, and P. A. Bunn Jr Chemoprevention of lung cancer--from biology to clinical reality Ann. Onc., February 1, 2004; 15(2): 185 - 196. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. C. Zhan, L. J. Gudas, D. Bok, R. Rando, D. M. Nanus, and S. K. Tickoo Differential Expression of the Enzyme That Esterifies Retinol, Lecithin:Retinol Acyltransferase, in Subtypes of Human Renal Cancer and Normal Kidney Clin. Cancer Res., October 15, 2003; 9(13): 4897 - 4905. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. H. Dragnev, D. Stover, and E. Dmitrovsky Lung Cancer Prevention: The Guidelines Chest, January 1, 2003; 123(1_suppl): 60S - 71S. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J L Mulshine and R A Smith Lung cancer * 2: Screening and early diagnosis of lung cancer Thorax, December 1, 2002; 57(12): 1071 - 1078. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Lippman, J. J. Lee, D. D. Karp, E. E. Vokes, S. E. Benner, G. E. Goodman, F. R. Khuri, R. Marks, R. J. Winn, W. Fry, et al. Randomized Phase III Intergroup Trial of Isotretinoin to Prevent Second Primary Tumors in Stage I Non-Small-Cell Lung Cancer J Natl Cancer Inst, April 18, 2001; 93(8): 605 - 618. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. L. Wang, M. Marko, A. R. Dahl, K. S. Engelke, M. E. Placke, A. R. Imondi, J. L. Mulshine, and L. M. De Luca Topical Delivery of 13-cis-Retinoic Acid by Inhalation Up-Regulates Expression of Rodent Lung but not Liver Retinoic Acid Receptors Clin. Cancer Res., September 1, 2000; 6(9): 3636 - 3645. [Abstract] [Full Text]
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, vehicle control (daily
exposure); •, low dose (daily exposure); 
, mid dose (daily
exposures first 12 days, then three times weekly); 
, high dose
(daily exposures first 12 days, then twice weekly); *, 9 of 21
animals sacrificed moribund.