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Protective effect of lodoxamide tromethamine on allergen inhalation challenge
Protective effect of lodoxamide tromethamine on allergen inhalation challenge Gavin D. Watt, B.A., Tiet C. Bui, M.D.,* Againdra Robert G. Townley, M.D. Omaha, Neb.
K. Bewtra,
M.D., and
Lodoxamide tromethamine (U-42,585E) is a new drug intended for prophylaxis of mast cell -mediated allergic disease. It is a water-soluble, cromolyn-like agent with demonstrated activity in rat peritoneal mast cell assay, rat percutaneous anaphylaxis (rat PCA) and sensitized rhesus monkey airway system. Ten allergen-sensitive asthmatics were pretreated with lodoxamide (0.01, 0.1, or I .O mg) or placebo, then challenged with serial dilutions of allergen extract. Analysis of allergen dose-response curve parameters shows that pretreatment with lodoxamide offers sign&ant protection against experimental allergen-induced bronchoconstriction. At 0.01 mg, lodoxamide was effective in over half the subjects tested. Administration of lodoxamide by inhalation at doses of 0.1 and I .O mg uniformly allowed subjects to tolerate signi~cantly larger doses of inhaled allergen. Side effects observed at these doses were minimal
To treat allergic asthma, nonsympathomimetic drugs have been developed that act prophylactically by inhibiting the release of mast cell mediators. Cromolyn sodium, which is generally administered as an insufflated powder, is the sole currently available therapeutic agent with this mode of action. Its experimental efficacy has been adequately demonstrated in both animals’ and humans*; however, some asthmatic patients fail to benefit from treatment. Lodoxamide tromethamine (U-42,585E), [N,N’(2-chloro-Scyano-m-phenylene)dioxamic acid, diTHAM salt], is a new drug intended for the prophylaxis of mast cell-mediated allergic disease. Lodoxamide has been demonstrated to have cromolyn-like activity when studied in the rat peritoneal mast cell assay (PCA) model3 and in Ascaris antigen-sensitized rhesus monkeys. 4 Intravenous lodoxamide was some 2,500 times more active than intravenous cromolyn in inhibiting the rat PCA reac-
From Creighton University School of Medicine. Received for publication April 20, 1979. Accepted for publication April 3, 1980. Reprint requests to: Dr. R. G. Townley, Creighton University School of Medicine, 2500 California St., Omaha, NE 68178. *Present address: Department of Pediatrics, University Hospital, Jacksonville, Fla.
Vol. 66, No. 4, pp. 286-294
tion to egg albumin. In vitro, lodoxamide inhibited compound 48/80-induced histamine release and ionophore-induced 45Ca influx with associated histamine release in purified rat peritoneal mast cells. 3 In isolated guinea pig ileum, lodoxamide, like cromolyn, did not antagonize histamine- or SRS-A-induced contraction.” Further, lodoxamide did not stimulate production of adenyl cyclase in rat lung or inhibit the activity of cyclic adenosine monophosphate (CAMP) phosphodiesterase .9 When given intravenously, orally, or intrabronchially by aerosol, lodoxamide significantly inhibited the increased respiratory frequency and decreased tidal volume induced by antigen challenge in Ascaris-sensitized. anesthetized rhesus monkeys.4 Doses as low as 1 pg by aerosol 30 min prior to challenge effectively prevented lung function changes induced by allergen. Thus, in both the rat PCA and sensitized monkey airway systems, lodoxamide was quantitatively more effective than cromolyn by all administration routes tested. We report a study undertaken to determine: (1) the effect of single doses (0.01, 0.1, or 1.O mg) of inhaled lodoxamide on allergen inhalation provocation tests in a group of allergen-sensitive asthmatics; (2) the effect of short-term administration of lodoxamide on pulmonary, hepatic, hematologic, and renal function; and (3) the side effect profile.
0091-6749/80/100286+09$00.90/0
@ 1980 The C. V. Mosby
Co.
VOLUME NUMBER
66 4
Effect of lodoxamide
on allergen
60
37
36 Allergen
O\
33 Dose
I
63 (Cumulative
Inhalallon
133 Units)
183
challenge
287
% z
1 40
FIG. 1 Dose-response curves for subject No. 1, a 30-yr-old woman with moderate asthma. She was challenged with serial dilutions of short ragweed extract at 84,000 protein nitrogen units [PNU]/ml. Curves were fitted by least-squares linear regression where x = dose of allergen in cumulative inhalation units (1 unit = 1 inhalation at 115,000 w/v) and y = natural log of the FEV, percent of saline baseline. Multiple determinations of FEV, were made when response was ~85%. All points were included in the regression. The two smallest allergen doses, 0.25 and 0.75 units for this subject, are not plotted here but were included in the regression. PD,, is the dose of allergen associated with a 20% fall in pulmonary function. Note that the calculated PD,, for the O.l-mg dose exceeds the maximum administered dose (183 inhalation units). To compensate for overestimation of sensitivity when PDs were extrapolated we calculated the area under the dose-response curve for use as an additional treatment-response variable. For PD < maximum dose the area is triangular. For PD > maximum dose the area is trapezoidal, bounded on the right by the maximum dose. The area under the l.O-mg curve is shaded. O, Control sterile In water, In y = 4.62 - 0.031x. r = 0.958, p < 0.001, area = 0.9; q , 0.1 mg lodoxomide, y = 4.65 - 0.0014x, r = 0.945, p < 0.001, area = 26.1; ., 1.0 mg lodoxomide, In y = 4.62 0.0029x, r = 0.954, p < 0.001, area = 10.2
MATERIALS Subjects
AND METHODS
Ten volunteers participated in this study after giving informed consent. The University Human Research Committee approved the study. A physician was in attendance and adequate resuscitative drugs and equipment were available at all testing sessions. We studied five male and five female subjects between the ages of 14 and 29 yr with mild to moderate extrinsic asthma (Table I). Diagnosis of extrinsic asthma was based on the subject’s history, skin-test response, physical examination, and response to allergen inhalation challenge and bronchodilating drugs. Prior to bronchial challenge, subjects did not take cromolyn or bronchodilators for 12 hr or antihistamines for 72 hr. None of the subjects was on longterm cromolyn therapy. Only one subject, No. 6, was taking steroids-beclomethasone dipropionate inhaler (Vanceril), which was withheld 6 hr before testing. The subjects were also asked to abstain from tobacco, coffee, tea, chocolate, and cola beverages for at least 6 hr prior to testing. Before each session, subjects were questioned concerning the above and compliance was affirmed.
Experimental
protocol
Subjects were seen at least five times; during the initial visit (VO) they were screened; the subsequent four visits (Vl , V2, V3, V4) served as drug test days. During a subject’s initial visit we established his suitability for the study and determined baseline values. After the history and physical examination, we performed prick and intradermal (ID) skin tests to a screening battery of 10 common allergens plus a negative saline control and positive histamine control (ID, 0.001 mgiml). Using the extract that induced the largest response, we then titrated with serial dilutions (l/500,000; l/100,000; 1150,000; l/10,000; l/5,000 w/v or 2+ wheal) to determine the initial concentration for the allergen inhalation provocation test? An allergen inhalation challenge after pretreatment with sterile water was performed (single-blind placebo) to establish the baseline allergen threshold dose or “control” value. We performed the allergen inhalation challenges according to a modification of the standardized method described by Chai et al.” Each subject first received five inhalations of saline diluent from the nebulizer as a control, then received sequentially increasing concentrations of allergen aerosol.
288 Watt et al.
J. ALLERGY
TABLE I. Sample
description
and challenge
data Control
Subject I
2 3 4 5* 6 7 8 9 IO 11
Sex
Age (yr)
F F M M F F M M M F F
29 20 14 15 24 26 27 21 22 22 24
Placebo
0.01 mg
Date
TD
FR
Date
TD
FR
Date
TD
FR
7/7 7/25 8/11 8/17 8/30 9/13 9/22 9/20 IO/13 11/7
23 583 633 83 33 43 1,358 53 183 2,033 Not done
49. I 71.3 71.4 51.6 65.0 76.1 69.1 53.3 61.8 77.7
712 I 8/8 8/23 9/7 9/l 9/19 9/29 IO/20 lo/18 II/16 l/31
83 583 833 333 33 13 2,033 33 83 1,335 1
50.0 74.0 77.9 59.8 63.8 78.8 75.6 56.9 79.8 65.6 78.9
7/14 8/10 8/18 8/29 9/15 9/26 IO/I 1 IO/l I 11/8 II/21 l/11
83 1,333 2,333 833 6 333 2.833 33 833 1,835 3
54.5 91.7 76.1 36.7 75.1 75.2 89.1 50.1 66.5 73.5 72.0
Allergen
SR SR A SR SR SR A SR SR SR SR
CLIN. IMMUNOL. OCTOBER 1980
SR: Short ragweed extract (84,000 protein nitrogen units [PNU]/ml at l/10 w/v); A: Alrernaria extract (20,000 PNU/ml at I/ 10 w/v); TD: threshold dose, i.e., last dose of allergen administered in cumulative inhalation units (! unit = 1 inhalation of extract at l/5,000 w/v); FR: mean final response(percent of saline baseline FEV,). *Subject No. 5 was restudied as subject No. 11 becauseshe developed a respiratory infection during this challenge series.
TABLE II. Possible side effects
TABLE III. Delayed
Possible side effects Subject Pretreatment
Yes
No
5
7
0. I mg/ml lodoxamide 0 0.01 mg/ml lodoxamide 3 Single-blind control 3 Double-blind placebo 1 Total 12
11 8 7 10 43
1 mg/ml lodoxamide
Treatment
reactions Reported symptoms
Time (hr after challenge)
Subject/symptom*
1
0.1 mg
4
0.1 mg
Tightness, wheeze Coughing
6
5
Placebo
Tightness, wheeze, Pruritus
8 14
6
Control
6
Placebo 0.1 mg 0.01 mg
Coughing,* tightness, wheeze Coughing* Coughing,* pruritus Pruritus
7
Control
Tightness, wheeze
8
Control I.0 mg
Coughing* Coughing,* tightness, wheeze Coughing*
1/FWNT,S/W,7/LW, 9/L, 11/w 1/HFW,2/H,S/W 5/W,7/T,9/L 5/w
*See text for abbreviations. The initial challenge dose was chosen as five breaths of allergen at a concentration that elicited a 2+ response (wheal, 6 to 8 mm > diluent control) on intradermal skintest titration. Five successive 0.6-see nebulizations of each concentration were administered, using a DeVilbiss No. 42 nebulizer and Rosenthal-French nebulization dosimeter.’ The dosimeter delivers a 0.6-set burst of compressed air at 6 L/min as the subject begins inspiration through the nebulizer. Five minutes after allergen inhalation the subject performed two or more forced expiratory maneuvers to ensure reproducibility of the observed pulmonary function. We recorded only the highest values of the forced expiratory volume in 1 set (FEV,), forced vital capacity (FVC), forced expiratory flow from 25% to 75% of the total volume (FEFasmir,), forced expiratory flow from 200 to 1,200 ml (FEF?,,,,-,,&, and peak expiratory flow rate (PEFR) at each step. The challenge was stopped and the threshold dose determined when the subject sustained a 20% or greater fall in FEV, from the postsaline baseline for 15 min. Signs and symptoms were checked 5 min after each dose of allergen.
I .O mg
0.01 mg
Coughing, tightness, wheeze Tightness, wheeze
6 6
-18 -18 4 8
*See text. At the end of each challenge subjects inhaled 10 breaths of isoproterenol HCI 0.5% to restore pulmonary function to prechallenge level. During the course of the screening visit (VO) we informed all subjects that drug-related side effects might occur, specifically headache, tiredness, flushing and warmth, lightheadedness, dizziness, nausea and stomach cramps, and urgency and burning of the urethra. After each treatment and throughout the challenge, we asked the subjects, “How do you feel?” and recorded their responses.
VOLUME NIJMBER
Effect of lodoxamide
66 4
0.1 mg
1.0 mg
Date
TD
FR
7/1Y 8/I? 8/25 8/22 9126 9/28 IO/4 10/22 1O/27 1l/l4 I/24
183 1,333 2,333 833 8 53 2,833 78 833 3,335 8
80.7 87.2 88.4 39.3 73.5 83.3 86.6 50.4 73.7 82.0 14.2
Date
7/26 8/l 8/16 8/31 9122 9/2 1 IO/14 9/27 1l/IO 1215 l/9
TD
FR
L33 1,333 2,333 333 6 83 2,833 83 833 4,335 3
68.3 82.6 93.7 77.0 76.6 78.4 95.3 67.3 40.4 97.7 73.8
on allergen challenge
289
The dose at each step of the challenge (x-axis) was considered as the cumulative number of inhalation units through that step (1 inhalation unit = 1 inhalation of allergen extract at 1: 5,000 w/v concentration). Response was
considered as Percent of saline baseline. These response values (y-axis) were further transformed by taking the natural logarithm (In). Dose-response curves were fitted by the least-squares method. Parameters of the regression equa-
tions were used to generatethree responsevariables: slope (“reactivity”),
provocative dose (“sensitivity”).
and area
under the dose-responsecurve. A set of typical dose-responsecurves is shown in Fig. 1. The conclusions and examples presented here will deal with FEV,, which was the challenge criterion variable. Most analysesuse the provocative doseassociatedwith a 20% fall in FEV, (FEV, PD.&. We also examined curve parameters for the other four PFs, which all showed the samegeneral trends with respectto treatmentsand subjects.
Test days
RESULTS
On test days (VI, V2, V3, and V4) baseline pulmonary functions were measured, and if a subject’s pretreatment FEV, was within 20% of his VO baseline, he was pretreated 30 min prior to allergen inhalation challenge. All subjects inhaled 1 ml of aerosolized pretreatment solution (sterile water “placebo,” 0.01, 0.1 or 1.Omg lodoxamide). Treatments were administered in random order, double-blind fashion. On test days we gave somesubjectsan initial dose basedon the control challenge equivalent to the cumulative doseof several initial challenge steps(e.g., 3 inhalations of 1: 5,000 w/v = sum of dosesfrom 5 x 1: 500,000 through 5 x I : 10,000) so that the last challenge dose would be administered 60 to 90 min after drug pretreatment. Two steps beyond the control threshold dose was chosen as the maximum doseend point to reducethe incidence and severity of delayed reactions. To evaluate the effect of short-term administration of iodoxamide by inhalation on cardiovascular, hepatic, hematologic, and renal function, we recordedelectrocardiogram and vital signs, and obtained samplesfor urinalysis, SMA12, and complete blood count before the allergen challenge on the control day (VO) and following the allergen challenge on the last visit (V4).
During the study, which was begun in July, 1977, and completed in January, 1978, we performed 54 allergen inhalation challenges after pretreating the 10 subjects by inhalation with either single-blind control (10 challenges) or double-blind placebo and 0.01, 0.1, or 1.0 mg of lodoxamide (44 challenges). We observed a >20% fall in FEVl in 45 challenges, <20% drop in FEV, in 9 challenges. One challenge (subject 6, V3) was terminated at the subject’s request after a drop in FEVl of 18.4%. Subject 5 developed a respiratory infection after the control and placebo treatments; her drug-treated provocative doses were significantly less than placebo and control. The placebo and drug-treated challenges were repeated 4 mo later; this second set of results is shown as subject 11. For comparative analysis of treatments and placebo, only this second set of results, as subject 11, is included.
Analysis of allergen inhalation challenge dose-response curves Each challenge generated dose-responsedata for five pulmonary functions (PFs)-FEV,, FVC, FEF2j-iS, FEL-1 ,mo,and PEFR-which were measured and calculated on a Cardio-Pulmonary Instruments 200 spirometer and 560 digitizer. During the testing the concentration of
allergen extract, number of inhalations, and the largest PF values observed were recorded at each step. Information from the data sheets was then encoded on punch cards for computer use. Calculations were made on an IBM 1130 computer and a Monroe 1860 programmable calculator, using locally written programsand the IBM SSP (Scientific Subroutine Package).
Possible
side effects (Table II)
We administered treatment (drug or sterile water) a total of 55 times to 10 people. Five subjects reported symptoms after 8 of 34 drug treatments: there were six reports of flushing (F) and/or warmth (W), four of headache (H), two of lightheadedness (L), and one of nausea, urgency, and urethral burning (N). One subject reported warmth after a placebo treatment. On control days after pretreatment with sterile water (single-blind), 3 of 10 subjects reported symptoms: 1 subjected reported warmth, 1 headache, and 1 tiredness (T). There was no statistically significant difference (p = 0.22) between drug and placebo treatments in the occurrence of side effects. Symptoms reported within 1 hr after pretreatment may have been due to (1) drug treatment (side ef-
290
J. ALLERGY
Watt et al.
TABLE IV. FEV,PD: Analysis
of variance
[In(PD20 Subject I
2 3 4 6 7 8 9 10 II
Mean SD
r=-87 n= IO
Source of variation
p< 0.01
‘00 0
0.2
0.4
0.6
Slope: Skin Test
0.8
Titration
I.0
l-2
Curve
FIG. 2. Positive correlation of intradermal skin-test titration with reactivity to placebo-pretreated allergen challenge (v = -5.5x + 8.6). Thus, individuals who are more reactive to injected allergen (wheal size increases rapidly with increasing dose increments) are also more reactive to inhaled allergen. Note that as the slope approaches zero the negative natural logarithm (-In) increases.
fects), (2) allergen challenge, or (3) exercise of the test. Symptoms, when reported, were generally mild. Delayed reactions (Table Ill)
due to inhaled
allergen
Delayed reactions, manifesting as coughing, wheezing, tightness, and pruritus, appeared within 24 hr after challenge and mostly within the first 4 to 8 hr. Subjects reported 7 episodes of delayed reactions after 33 challenges pretreated with inhaled lodoxamide, 1 case out of 11 double-blind placebo challenges, and 4 out of 10 single-blind control placebo challenges. Pretreatment with lodoxamide did not significantly affect the observed proportion of delayed reactions (p = 0.26). During allergen challenge subjects 6 and 8 were troubled with persistent coughing, which continued for several days. Some subjects reported typical, short-term episodes of tightness and wheezing (see Table III). Subject 6 reported 2 instances of pruritus on the day following challenge after treat-
CLIN. IMMUNOL OCTOBER 1980
+ l)]
Placebo
0.01 mg
0.1 mg
1.0 mg
3.44 6.02 6.70 5.01 2.41 7.51 2.71 4.44 6.61 0.57 4.54 2.24
3.54 8.03 7.63 5.59 4.08 8.60 2.60 6.13 7.31 1.20 5.47* 2.52
5.29 7.52 8.29 5.54 5.54 8.37 3.43 6.50 8.29 2.00 6.08? 2.17
4.45 7.27 8.52 5.67 4.43 8.95 3.87 5.46 9.22
Mean square
F
P
21.82 4.73 0.27
82.2 17.8
df
Subjects
9
Treatments Residual
3 27
I .25 5.91t
2.57
<
Critical value for Dunnet’s test (two-sided): *for p < 0.05, x must be greater than 5.45. tfor p < 0.01, x must be greater than 5.70.
ment with 0.1 and 0.01 mg lodoxamide. Subject 5 reported wheezing and tightness at 8 hr and pruritus at 14 hr after her control challenge. Correlation
of control
and placebo
An allergen inhalation challenge was administered as part of the screening visit (VO). We have designated this single-blind challenge as the “control. ” Double-blind, sterile water-pretreated challenges occurred in random order during the course of the four “study ’ ’ visits (VI to V4); we designated these as “placebo. ” FEV, PD2,, from the control and placebo visits did not differ significantly and were highly correIated (r = 0.898, p < 0.01). Allergen challenge in this sample was a reproducible procedure that yielded results amenable to statistical analysis. As may be seen in Figs. 2 and 3, the sample can be divided into two subgroups: subjects 2, 3, 7, and 10 tolerated much larger provocative doses than did subjects 1, 4, 5, 6, 8, and 9. Nothing in particular characterized the two groups except slope of the skin-test titration curve, which was correlated significantly with In slope of the placebo dose-response curve (r = 0.869, p i 0.01, Fig. 2). In this scattergram, subjects 2, 3, 7, and 10 formed a tight cluster away from the rest of the group. Subjects 3 and 7 were challenged with serial dilutions of Alternaria extract; all other subjects received serial dilutions of short ragweed extract.
VOLUME NUMBER
66 4
Effect of lodoxamide
on allergen
challenge
291
$633
ia I833 2833 1833 I333 633
333
\I
83
16 /0 33
v) .?I 5 .: z 5g
63
3.3
3.8 0.3 0.05 Control
Placebo
1.01 mg
0 Img
I Omg
FIG. 3. Effects of pretreatment with sterile water (control, placebo) and lodoxamide tromethamine (0.01, 0.1, or 1.0 mg) on response to allergen inhalation challenge as measured by the provocative dose at 80% of saline baseline for FEV, (PD,,). PDs were calculated in inhalation units (1 unit = 1 inhalation of allergen extract at 115,000 w :v) shown in the right-hand scale, then transformed as the natural log [In(PD + l)] to normalize the distribution. The means k 1 SEM shown are for 10 subjects except control where n = 9. Numbered lines correspond to individual subjects. Subject No. 5 was restudied as subject No. 11 because she developed a respiratory infection. Values for No. 5 are excluded from the means.
Comparison challenges
of placebo
with
drug-treated
A two-factor (randomized block) analysis of variance (ANOVA) with subjects as blocks and doses of lodoxamide (0.0, 0.01, 0.1, or 1.O mg) as treatments was performed. Dunnet’s tests (two-tailed) was used to test the null hypotheses of no difference between placebo and drug-treated means for the three variables (slope, PD, area), for four PFs (FEV,, FEFZ5-r5, FEL- I ,zootPEFR). PD and area were not normally distributed in this sample (e.g., for placebo FEV, PD,,,:mean + SD ~407.7 5 586.2,median = 83.4, range, 0.77 to 1831.8 inhalation units). For statistical analysis, PD and area were transformed as In (x + 1); slope was considered as In slope. Examination of Table IV and associated F values (variance ratios) show significant effects due to both the blocks (inter-
subject variation) and the treatments (drug effects). In this table all drug-treated means were significantly greater than placebo (p < 0.05 for 0.01 mg; p < 0.01 for 0.1 and 1.0 mg). FEVr area and PD were the only variables that showed 0.01 mg lodoxamide to be significantly different from placebo. By a two-tailed test, the 0.01 mg means generally approached significance for other PFs and parameters, and may well be considered different under less stringent criteria (Fig. 4). There was no significant qualitative difference among the three treatment means when tested nonparametrically with Friedmann’s ANOVA by ranks (e.g., X2 = 3.8, df = 2, p = 0.15 for FEV,, InPD,,) . Although we administered a single-blind control and a double-blind placebo, the results were not significantly different and it seemed reasonable to test
292
Watt et al.
J. ALLERGY
CLIN. IMMUNOL OCTOBER 1980
8
! 6
r
f
-
P .Ol 0.1 I.0 Slope
L
P .Ol 0.1 I.0 Provocative Dose
P ,Ol 0.1 I.0 Area
FIG. 4. Comparison of effect of inhaled pretreatment on parameters of dose-response curves. Pretreatments were sterile water (P) or lodoxamide tromethamine (0.01, 0.1, or 1.0 mg). For parameter units In is the natural logarithm. Bar heights are means for IO subjects + 1 SEM. All drug-treated means are significantly greater than placebo (see text). TABLE
V. Relative
effect
of treatment
Ratio* Lodoxamide (ms)
zt dose
Lodoxamide (ms)
dose
Subject
0.01
0.1
1.0
0.01
0.1
1.0
I 2 3 4 6 7 8 9 10 Increase No change Decrease
0.1 41.3 3.5 I.0 2.4 3.8 -0.2 100.4 0.8 6 2 1
7.2 22.2 8.8 0.9 12.3 2.6 1.9 156.0 3.3 8 1 0
2.3 16.0 11.8 1.2 3.7 6.2 4.0 40.4 9.5 9 0 0
1.13 14.20 4.18 1.79 2.28 4.02 -1.47 75.97 0.65 5 4 0
3.85 8.09 6.38 1.73 5.14 3.38 2.25 92.50 2.99 8 1 0
2.54 6.12 7.18 1.90 2.97 5.00 4.28 46.43 1.95 7 2 0
PDt: Provocative dose for a drug treatment; PDc: control provocative dose; PDp: placebo provocative dose; PDu: mean of PDc and PDp. *Ratio = PDt - PDpl 1PDc - PDp) tZ = PDt - PDu/SD of PDu.
treatment values considering what was known about untreated variation. Table V shows two comparisons. The left half of the table shows the ratio of the difference between placebo and treated PDs to the difference between placebo and control PDs (raw PD values were used here) as ratio = (PDt - PDp)/ (PDc - PDp(. After treatment with 0.01 mg, six sub-
jects increased (ratio > l), two stayed the same (0 < ratio < 1) and one decreased (ratio < 0); for 0.1 mg, eight increased, one showed no change, and none decreased; for 1.O mg, all nine increased. The right half of the table uses as its criterion how far away in standard deviation units a treated value is from the untreated mean; a significant change is defined as Z > 1.96, i.e., p < 0.05 (two tails). For 0.01 mg, five subjects increased significantly and four showed no significant change; for 0.1 mg, eight increased and one had no change; for 1.O mg, seven increased and two had no change. Subject 4, who showed no change for all three treatments, had the largest control/placebo variance. Fig. 5 shows the protective effect of lodoxamide as measured by the percent decrease from the baseline following allergen challenge. The mean decrease in FEV, after allergen challenge was 33% whereas the mean decrease following lodoxamide was only 16%, 3%, or 7.5% at 0.01, 0.1, or 1.O mg, respectively. Thus, at the O.l-mg dose, all subjects showed protection and the degree of protection was essentially complete (over 90%) for the allergen threshold dose that was administered following placebo. We also examined the possibility that lodoxamide may act as a bronchodilator. This was done by comparing the pretreatment saline baseline FEV, with the FEV, observed at 5 and 30 min after treatment. The mean increase 30 min after placebo was 4.0 + 1.7%, afterO.Olmgwas3.9 t 2.5%afterO.lmgwas3.6 k 1.O%, and after 1.Omg was 3.7 k 1.5%. These values are not significantly different by ANOVA.
VOLUME NUMBER
Effect of lodoxamide
66 4
on allergen
challenge
293
DISCUSStON Administration of lodoxamide tromethamine by inhalation at doses of 0.1 and 1.O mg uniformly allowed subjects to tolerate significantly larger doses of inhaled allergen extract as measured by the slope, PD, and area under the challenge dose-response curve. Lodoxamide at 0.01 mg appeared to be effective in over half the subjects tested. Lodoxamide aerosol was nonirritating and did not act immediately as a bronchodilator. Although lodoxamide at all the doses tested was generally significantly more effective than placebo, there was no correlation with dosage. Five subjects showed maximum protection at 1.O mg and four subjects at 0.1 mg. Animal studies have shown a biphasic dose-response curve for both lodoxamide and cromolyn; thus, it is possible that because of individual variation in optimal dose requirements, peak effectiveness may be seen at smaller than maximal dose. Examination of the means in Fig. 3 suggests that the most universally effective dose was 0.1 mg, and that individuals who were more allergen sensitive were also more drug sensitive in that their drug curves were shifted to the left of the less sensitive group (e.g., compare subjects 1, 6, and 9 with 3, 7, and 10). Lodoxamide permitted the test subjects to tolerate considerably more allergen. Inspection of Fig. 3 shows that after placebo, the geometric mean provocative dose was about 90 inhalation units, whereas after 0.1 mg of lodoxamide it was about 450. Although the increase from placebo of the mean amount of allergen tolerated was about fivefold, some subjects tolerated up to 23 times as much allergen (e.g., subject 6 in Fig. 3). The degree of protection with lodoxamide was considerably greater than what we have reported previously for cromolyn sodium.** 7-g For example, a similar population of 14 subjects with allergic bronchial asthma showed a mean decrease of 37% in their FEV after allergen challenge pretreated with placebo cromolyn sodium.7 Following cromolyn sodium, 20 mg by Spinhaler, the mean decrease in FEV, was 23% and three of the subjects showed no protection; thus, the degree of protection was only 62% following cromolyn sodium vs 90% for lodoxamide. Neither lodoxamide nor cromoiyn sodium has shown any appreciable immediate bronchodilator action, although cromolyn sodium has been reported to inhibit phosphodiesterase. lo Cromolyn sodium did not protect against histamine or methacholine bronchial challenges .*a i-g Similar studies have not been done with lodoxamide. Reported side effects, chiefly flushing and a subjective feeling of warmth, were generally mild and
0 1 At
0. I PlCCIbO 0.01 FEV, Response (MeonfS.E.M.,n=lOl Lorl Allsrqen Oo3r Of Placebo
I.0 Challenge
FIG. 5. Effect of lodoxamide on allergen inhalation challenge as measured by the percent decrease in FEV, at the last dose of the placebo-treated challenge. Each subject’s set of dose-response curves is thus considered at a single value for dose of allergen.
were lessened with repeated administrations. Delayed reactions occurred at about the same rate for drugtreated and untreated challenges; however, after drug treatment subjects were able to tolerate substantially larger doses of allergen. Thus, it is not possible to rule out lodoxamide’s ability to prevent or alter delayed responses. Lodoxamide tromethamine is a new antiallergy drug that is rapidly transported across mast cell membranes. Lodoxamide stabilized mast cell membranes by inhibiting Ca++ influx .3*4 In animal studies lodoxamide has been demonstrated to be more potent than cromolyn. Johnson et al. have demonstrated oral activity in monkeys and rats.“, 4 We have shown that lodoxamide, a water-soluble, cromolyn-like agent, administered by aerosol, protects against experimental allergen-induced bronchoconstriction. The authors gratefully acknowledge the technical assistance of Kathleen Burke, Kathleen Paziak, Dianne Hert, and Rose Garred, and the time and effort expended by the volunteer subjects.Lodoxamide was obtained from The Upjohn Company through the auspices of H. Johnson, A. Sheridan, and C. Cross, whose contributions to the study were indispensable. REFERENCES 1. Cox JSG: Disodium cromoglycate (FPL 670 I’lntal’): A specific inhibitor of reaginic antibody-antigen mechanisms. Nature 216: 1328, 1967.
294 Watt et al.
2. Kolotkin B, Lee C, Townley R: Duration and specificity of sodium cromolyn on allergen inhalation challenge in asthmatics. J ALLERGYCLIN IMMUNOL53:288, 1974. 3. Johnson H, Van Hout C, Wright J: Inhibition of allergic reactions by cromoglycate and by a new anti-allergy drug U42,585E. 1. Activity in the rat. Int Arch Allergy Appl Immuno1 56~416, 1978. 4. Johnson H, Van Hout C, Wright J: Inhibition of allergy reactions by cromoglycate and by a new anti-allergy drug U42,585E. II. Activity in primates against aerosolized Ascaris ~nurn antigen. Int Arch Allergy Appl Immunol56:487, 1978. 5. Chai H, Farr R, Froehlich LA, Mathison DA, McLean JA, Rosenthal RR, Sheffer AL, Spector SL, Townley RG: Standardization of bronchial inhalation challenge procedures. J ALLERGY CLIN IMMUNOL 56:323, 1975.
J. ALLERGY
CLIN. IMMUNOL. OCTOBER 1980
6. Dunnet CW: Multiple comparison procedures for comparing several treatments with a control. J Am Stat Assn 50:1096. 1955. 7. Ryo UY, Kang B, Townley RG: Cromolyn therapy in patients with bronchial asthma. JAMA 236:927, 1976. 8. Kang B, Townley R, Lee CK, Kolotkin BM: Bronchial sensitivity to histamine before and after sodium cromoglycate in bronchial asthma. Br Med J 1:867, 1976. 9. Townley RG: Pharmacological blocks to mediator release: Clinical applications. Adv Asthma Allergy 2(3):7, 1975. 10. Taylor WA, Francis DH, Sheldon D: Anti-allergic actions of disodium cromoglycate and other drugs known to inhibit cyclic 3’,5’-nucleotide phosphodiesterase. Int Arch Allergy Appl Immunol 47:175, 1974.
K. Bewtra,
M.D., and
Lodoxamide tromethamine (U-42,585E) is a new drug intended for prophylaxis of mast cell -mediated allergic disease. It is a water-soluble, cromolyn-like agent with demonstrated activity in rat peritoneal mast cell assay, rat percutaneous anaphylaxis (rat PCA) and sensitized rhesus monkey airway system. Ten allergen-sensitive asthmatics were pretreated with lodoxamide (0.01, 0.1, or I .O mg) or placebo, then challenged with serial dilutions of allergen extract. Analysis of allergen dose-response curve parameters shows that pretreatment with lodoxamide offers sign&ant protection against experimental allergen-induced bronchoconstriction. At 0.01 mg, lodoxamide was effective in over half the subjects tested. Administration of lodoxamide by inhalation at doses of 0.1 and I .O mg uniformly allowed subjects to tolerate signi~cantly larger doses of inhaled allergen. Side effects observed at these doses were minimal
To treat allergic asthma, nonsympathomimetic drugs have been developed that act prophylactically by inhibiting the release of mast cell mediators. Cromolyn sodium, which is generally administered as an insufflated powder, is the sole currently available therapeutic agent with this mode of action. Its experimental efficacy has been adequately demonstrated in both animals’ and humans*; however, some asthmatic patients fail to benefit from treatment. Lodoxamide tromethamine (U-42,585E), [N,N’(2-chloro-Scyano-m-phenylene)dioxamic acid, diTHAM salt], is a new drug intended for the prophylaxis of mast cell-mediated allergic disease. Lodoxamide has been demonstrated to have cromolyn-like activity when studied in the rat peritoneal mast cell assay (PCA) model3 and in Ascaris antigen-sensitized rhesus monkeys. 4 Intravenous lodoxamide was some 2,500 times more active than intravenous cromolyn in inhibiting the rat PCA reac-
From Creighton University School of Medicine. Received for publication April 20, 1979. Accepted for publication April 3, 1980. Reprint requests to: Dr. R. G. Townley, Creighton University School of Medicine, 2500 California St., Omaha, NE 68178. *Present address: Department of Pediatrics, University Hospital, Jacksonville, Fla.
Vol. 66, No. 4, pp. 286-294
tion to egg albumin. In vitro, lodoxamide inhibited compound 48/80-induced histamine release and ionophore-induced 45Ca influx with associated histamine release in purified rat peritoneal mast cells. 3 In isolated guinea pig ileum, lodoxamide, like cromolyn, did not antagonize histamine- or SRS-A-induced contraction.” Further, lodoxamide did not stimulate production of adenyl cyclase in rat lung or inhibit the activity of cyclic adenosine monophosphate (CAMP) phosphodiesterase .9 When given intravenously, orally, or intrabronchially by aerosol, lodoxamide significantly inhibited the increased respiratory frequency and decreased tidal volume induced by antigen challenge in Ascaris-sensitized. anesthetized rhesus monkeys.4 Doses as low as 1 pg by aerosol 30 min prior to challenge effectively prevented lung function changes induced by allergen. Thus, in both the rat PCA and sensitized monkey airway systems, lodoxamide was quantitatively more effective than cromolyn by all administration routes tested. We report a study undertaken to determine: (1) the effect of single doses (0.01, 0.1, or 1.O mg) of inhaled lodoxamide on allergen inhalation provocation tests in a group of allergen-sensitive asthmatics; (2) the effect of short-term administration of lodoxamide on pulmonary, hepatic, hematologic, and renal function; and (3) the side effect profile.
0091-6749/80/100286+09$00.90/0
@ 1980 The C. V. Mosby
Co.
VOLUME NUMBER
66 4
Effect of lodoxamide
on allergen
60
37
36 Allergen
O\
33 Dose
I
63 (Cumulative
Inhalallon
133 Units)
183
challenge
287
% z
1 40
FIG. 1 Dose-response curves for subject No. 1, a 30-yr-old woman with moderate asthma. She was challenged with serial dilutions of short ragweed extract at 84,000 protein nitrogen units [PNU]/ml. Curves were fitted by least-squares linear regression where x = dose of allergen in cumulative inhalation units (1 unit = 1 inhalation at 115,000 w/v) and y = natural log of the FEV, percent of saline baseline. Multiple determinations of FEV, were made when response was ~85%. All points were included in the regression. The two smallest allergen doses, 0.25 and 0.75 units for this subject, are not plotted here but were included in the regression. PD,, is the dose of allergen associated with a 20% fall in pulmonary function. Note that the calculated PD,, for the O.l-mg dose exceeds the maximum administered dose (183 inhalation units). To compensate for overestimation of sensitivity when PDs were extrapolated we calculated the area under the dose-response curve for use as an additional treatment-response variable. For PD < maximum dose the area is triangular. For PD > maximum dose the area is trapezoidal, bounded on the right by the maximum dose. The area under the l.O-mg curve is shaded. O, Control sterile In water, In y = 4.62 - 0.031x. r = 0.958, p < 0.001, area = 0.9; q , 0.1 mg lodoxomide, y = 4.65 - 0.0014x, r = 0.945, p < 0.001, area = 26.1; ., 1.0 mg lodoxomide, In y = 4.62 0.0029x, r = 0.954, p < 0.001, area = 10.2
MATERIALS Subjects
AND METHODS
Ten volunteers participated in this study after giving informed consent. The University Human Research Committee approved the study. A physician was in attendance and adequate resuscitative drugs and equipment were available at all testing sessions. We studied five male and five female subjects between the ages of 14 and 29 yr with mild to moderate extrinsic asthma (Table I). Diagnosis of extrinsic asthma was based on the subject’s history, skin-test response, physical examination, and response to allergen inhalation challenge and bronchodilating drugs. Prior to bronchial challenge, subjects did not take cromolyn or bronchodilators for 12 hr or antihistamines for 72 hr. None of the subjects was on longterm cromolyn therapy. Only one subject, No. 6, was taking steroids-beclomethasone dipropionate inhaler (Vanceril), which was withheld 6 hr before testing. The subjects were also asked to abstain from tobacco, coffee, tea, chocolate, and cola beverages for at least 6 hr prior to testing. Before each session, subjects were questioned concerning the above and compliance was affirmed.
Experimental
protocol
Subjects were seen at least five times; during the initial visit (VO) they were screened; the subsequent four visits (Vl , V2, V3, V4) served as drug test days. During a subject’s initial visit we established his suitability for the study and determined baseline values. After the history and physical examination, we performed prick and intradermal (ID) skin tests to a screening battery of 10 common allergens plus a negative saline control and positive histamine control (ID, 0.001 mgiml). Using the extract that induced the largest response, we then titrated with serial dilutions (l/500,000; l/100,000; 1150,000; l/10,000; l/5,000 w/v or 2+ wheal) to determine the initial concentration for the allergen inhalation provocation test? An allergen inhalation challenge after pretreatment with sterile water was performed (single-blind placebo) to establish the baseline allergen threshold dose or “control” value. We performed the allergen inhalation challenges according to a modification of the standardized method described by Chai et al.” Each subject first received five inhalations of saline diluent from the nebulizer as a control, then received sequentially increasing concentrations of allergen aerosol.
288 Watt et al.
J. ALLERGY
TABLE I. Sample
description
and challenge
data Control
Subject I
2 3 4 5* 6 7 8 9 IO 11
Sex
Age (yr)
F F M M F F M M M F F
29 20 14 15 24 26 27 21 22 22 24
Placebo
0.01 mg
Date
TD
FR
Date
TD
FR
Date
TD
FR
7/7 7/25 8/11 8/17 8/30 9/13 9/22 9/20 IO/13 11/7
23 583 633 83 33 43 1,358 53 183 2,033 Not done
49. I 71.3 71.4 51.6 65.0 76.1 69.1 53.3 61.8 77.7
712 I 8/8 8/23 9/7 9/l 9/19 9/29 IO/20 lo/18 II/16 l/31
83 583 833 333 33 13 2,033 33 83 1,335 1
50.0 74.0 77.9 59.8 63.8 78.8 75.6 56.9 79.8 65.6 78.9
7/14 8/10 8/18 8/29 9/15 9/26 IO/I 1 IO/l I 11/8 II/21 l/11
83 1,333 2,333 833 6 333 2.833 33 833 1,835 3
54.5 91.7 76.1 36.7 75.1 75.2 89.1 50.1 66.5 73.5 72.0
Allergen
SR SR A SR SR SR A SR SR SR SR
CLIN. IMMUNOL. OCTOBER 1980
SR: Short ragweed extract (84,000 protein nitrogen units [PNU]/ml at l/10 w/v); A: Alrernaria extract (20,000 PNU/ml at I/ 10 w/v); TD: threshold dose, i.e., last dose of allergen administered in cumulative inhalation units (! unit = 1 inhalation of extract at l/5,000 w/v); FR: mean final response(percent of saline baseline FEV,). *Subject No. 5 was restudied as subject No. 11 becauseshe developed a respiratory infection during this challenge series.
TABLE II. Possible side effects
TABLE III. Delayed
Possible side effects Subject Pretreatment
Yes
No
5
7
0. I mg/ml lodoxamide 0 0.01 mg/ml lodoxamide 3 Single-blind control 3 Double-blind placebo 1 Total 12
11 8 7 10 43
1 mg/ml lodoxamide
Treatment
reactions Reported symptoms
Time (hr after challenge)
Subject/symptom*
1
0.1 mg
4
0.1 mg
Tightness, wheeze Coughing
6
5
Placebo
Tightness, wheeze, Pruritus
8 14
6
Control
6
Placebo 0.1 mg 0.01 mg
Coughing,* tightness, wheeze Coughing* Coughing,* pruritus Pruritus
7
Control
Tightness, wheeze
8
Control I.0 mg
Coughing* Coughing,* tightness, wheeze Coughing*
1/FWNT,S/W,7/LW, 9/L, 11/w 1/HFW,2/H,S/W 5/W,7/T,9/L 5/w
*See text for abbreviations. The initial challenge dose was chosen as five breaths of allergen at a concentration that elicited a 2+ response (wheal, 6 to 8 mm > diluent control) on intradermal skintest titration. Five successive 0.6-see nebulizations of each concentration were administered, using a DeVilbiss No. 42 nebulizer and Rosenthal-French nebulization dosimeter.’ The dosimeter delivers a 0.6-set burst of compressed air at 6 L/min as the subject begins inspiration through the nebulizer. Five minutes after allergen inhalation the subject performed two or more forced expiratory maneuvers to ensure reproducibility of the observed pulmonary function. We recorded only the highest values of the forced expiratory volume in 1 set (FEV,), forced vital capacity (FVC), forced expiratory flow from 25% to 75% of the total volume (FEFasmir,), forced expiratory flow from 200 to 1,200 ml (FEF?,,,,-,,&, and peak expiratory flow rate (PEFR) at each step. The challenge was stopped and the threshold dose determined when the subject sustained a 20% or greater fall in FEV, from the postsaline baseline for 15 min. Signs and symptoms were checked 5 min after each dose of allergen.
I .O mg
0.01 mg
Coughing, tightness, wheeze Tightness, wheeze
6 6
-18 -18 4 8
*See text. At the end of each challenge subjects inhaled 10 breaths of isoproterenol HCI 0.5% to restore pulmonary function to prechallenge level. During the course of the screening visit (VO) we informed all subjects that drug-related side effects might occur, specifically headache, tiredness, flushing and warmth, lightheadedness, dizziness, nausea and stomach cramps, and urgency and burning of the urethra. After each treatment and throughout the challenge, we asked the subjects, “How do you feel?” and recorded their responses.
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0.1 mg
1.0 mg
Date
TD
FR
7/1Y 8/I? 8/25 8/22 9126 9/28 IO/4 10/22 1O/27 1l/l4 I/24
183 1,333 2,333 833 8 53 2,833 78 833 3,335 8
80.7 87.2 88.4 39.3 73.5 83.3 86.6 50.4 73.7 82.0 14.2
Date
7/26 8/l 8/16 8/31 9122 9/2 1 IO/14 9/27 1l/IO 1215 l/9
TD
FR
L33 1,333 2,333 333 6 83 2,833 83 833 4,335 3
68.3 82.6 93.7 77.0 76.6 78.4 95.3 67.3 40.4 97.7 73.8
on allergen challenge
289
The dose at each step of the challenge (x-axis) was considered as the cumulative number of inhalation units through that step (1 inhalation unit = 1 inhalation of allergen extract at 1: 5,000 w/v concentration). Response was
considered as Percent of saline baseline. These response values (y-axis) were further transformed by taking the natural logarithm (In). Dose-response curves were fitted by the least-squares method. Parameters of the regression equa-
tions were used to generatethree responsevariables: slope (“reactivity”),
provocative dose (“sensitivity”).
and area
under the dose-responsecurve. A set of typical dose-responsecurves is shown in Fig. 1. The conclusions and examples presented here will deal with FEV,, which was the challenge criterion variable. Most analysesuse the provocative doseassociatedwith a 20% fall in FEV, (FEV, PD.&. We also examined curve parameters for the other four PFs, which all showed the samegeneral trends with respectto treatmentsand subjects.
Test days
RESULTS
On test days (VI, V2, V3, and V4) baseline pulmonary functions were measured, and if a subject’s pretreatment FEV, was within 20% of his VO baseline, he was pretreated 30 min prior to allergen inhalation challenge. All subjects inhaled 1 ml of aerosolized pretreatment solution (sterile water “placebo,” 0.01, 0.1 or 1.Omg lodoxamide). Treatments were administered in random order, double-blind fashion. On test days we gave somesubjectsan initial dose basedon the control challenge equivalent to the cumulative doseof several initial challenge steps(e.g., 3 inhalations of 1: 5,000 w/v = sum of dosesfrom 5 x 1: 500,000 through 5 x I : 10,000) so that the last challenge dose would be administered 60 to 90 min after drug pretreatment. Two steps beyond the control threshold dose was chosen as the maximum doseend point to reducethe incidence and severity of delayed reactions. To evaluate the effect of short-term administration of iodoxamide by inhalation on cardiovascular, hepatic, hematologic, and renal function, we recordedelectrocardiogram and vital signs, and obtained samplesfor urinalysis, SMA12, and complete blood count before the allergen challenge on the control day (VO) and following the allergen challenge on the last visit (V4).
During the study, which was begun in July, 1977, and completed in January, 1978, we performed 54 allergen inhalation challenges after pretreating the 10 subjects by inhalation with either single-blind control (10 challenges) or double-blind placebo and 0.01, 0.1, or 1.0 mg of lodoxamide (44 challenges). We observed a >20% fall in FEVl in 45 challenges, <20% drop in FEV, in 9 challenges. One challenge (subject 6, V3) was terminated at the subject’s request after a drop in FEVl of 18.4%. Subject 5 developed a respiratory infection after the control and placebo treatments; her drug-treated provocative doses were significantly less than placebo and control. The placebo and drug-treated challenges were repeated 4 mo later; this second set of results is shown as subject 11. For comparative analysis of treatments and placebo, only this second set of results, as subject 11, is included.
Analysis of allergen inhalation challenge dose-response curves Each challenge generated dose-responsedata for five pulmonary functions (PFs)-FEV,, FVC, FEF2j-iS, FEL-1 ,mo,and PEFR-which were measured and calculated on a Cardio-Pulmonary Instruments 200 spirometer and 560 digitizer. During the testing the concentration of
allergen extract, number of inhalations, and the largest PF values observed were recorded at each step. Information from the data sheets was then encoded on punch cards for computer use. Calculations were made on an IBM 1130 computer and a Monroe 1860 programmable calculator, using locally written programsand the IBM SSP (Scientific Subroutine Package).
Possible
side effects (Table II)
We administered treatment (drug or sterile water) a total of 55 times to 10 people. Five subjects reported symptoms after 8 of 34 drug treatments: there were six reports of flushing (F) and/or warmth (W), four of headache (H), two of lightheadedness (L), and one of nausea, urgency, and urethral burning (N). One subject reported warmth after a placebo treatment. On control days after pretreatment with sterile water (single-blind), 3 of 10 subjects reported symptoms: 1 subjected reported warmth, 1 headache, and 1 tiredness (T). There was no statistically significant difference (p = 0.22) between drug and placebo treatments in the occurrence of side effects. Symptoms reported within 1 hr after pretreatment may have been due to (1) drug treatment (side ef-
290
J. ALLERGY
Watt et al.
TABLE IV. FEV,PD: Analysis
of variance
[In(PD20 Subject I
2 3 4 6 7 8 9 10 II
Mean SD
r=-87 n= IO
Source of variation
p< 0.01
‘00 0
0.2
0.4
0.6
Slope: Skin Test
0.8
Titration
I.0
l-2
Curve
FIG. 2. Positive correlation of intradermal skin-test titration with reactivity to placebo-pretreated allergen challenge (v = -5.5x + 8.6). Thus, individuals who are more reactive to injected allergen (wheal size increases rapidly with increasing dose increments) are also more reactive to inhaled allergen. Note that as the slope approaches zero the negative natural logarithm (-In) increases.
fects), (2) allergen challenge, or (3) exercise of the test. Symptoms, when reported, were generally mild. Delayed reactions (Table Ill)
due to inhaled
allergen
Delayed reactions, manifesting as coughing, wheezing, tightness, and pruritus, appeared within 24 hr after challenge and mostly within the first 4 to 8 hr. Subjects reported 7 episodes of delayed reactions after 33 challenges pretreated with inhaled lodoxamide, 1 case out of 11 double-blind placebo challenges, and 4 out of 10 single-blind control placebo challenges. Pretreatment with lodoxamide did not significantly affect the observed proportion of delayed reactions (p = 0.26). During allergen challenge subjects 6 and 8 were troubled with persistent coughing, which continued for several days. Some subjects reported typical, short-term episodes of tightness and wheezing (see Table III). Subject 6 reported 2 instances of pruritus on the day following challenge after treat-
CLIN. IMMUNOL OCTOBER 1980
+ l)]
Placebo
0.01 mg
0.1 mg
1.0 mg
3.44 6.02 6.70 5.01 2.41 7.51 2.71 4.44 6.61 0.57 4.54 2.24
3.54 8.03 7.63 5.59 4.08 8.60 2.60 6.13 7.31 1.20 5.47* 2.52
5.29 7.52 8.29 5.54 5.54 8.37 3.43 6.50 8.29 2.00 6.08? 2.17
4.45 7.27 8.52 5.67 4.43 8.95 3.87 5.46 9.22
Mean square
F
P
21.82 4.73 0.27
82.2 17.8
df
Subjects
9
Treatments Residual
3 27
I .25 5.91t
2.57
<
Critical value for Dunnet’s test (two-sided): *for p < 0.05, x must be greater than 5.45. tfor p < 0.01, x must be greater than 5.70.
ment with 0.1 and 0.01 mg lodoxamide. Subject 5 reported wheezing and tightness at 8 hr and pruritus at 14 hr after her control challenge. Correlation
of control
and placebo
An allergen inhalation challenge was administered as part of the screening visit (VO). We have designated this single-blind challenge as the “control. ” Double-blind, sterile water-pretreated challenges occurred in random order during the course of the four “study ’ ’ visits (VI to V4); we designated these as “placebo. ” FEV, PD2,, from the control and placebo visits did not differ significantly and were highly correIated (r = 0.898, p < 0.01). Allergen challenge in this sample was a reproducible procedure that yielded results amenable to statistical analysis. As may be seen in Figs. 2 and 3, the sample can be divided into two subgroups: subjects 2, 3, 7, and 10 tolerated much larger provocative doses than did subjects 1, 4, 5, 6, 8, and 9. Nothing in particular characterized the two groups except slope of the skin-test titration curve, which was correlated significantly with In slope of the placebo dose-response curve (r = 0.869, p i 0.01, Fig. 2). In this scattergram, subjects 2, 3, 7, and 10 formed a tight cluster away from the rest of the group. Subjects 3 and 7 were challenged with serial dilutions of Alternaria extract; all other subjects received serial dilutions of short ragweed extract.
VOLUME NUMBER
66 4
Effect of lodoxamide
on allergen
challenge
291
$633
ia I833 2833 1833 I333 633
333
\I
83
16 /0 33
v) .?I 5 .: z 5g
63
3.3
3.8 0.3 0.05 Control
Placebo
1.01 mg
0 Img
I Omg
FIG. 3. Effects of pretreatment with sterile water (control, placebo) and lodoxamide tromethamine (0.01, 0.1, or 1.0 mg) on response to allergen inhalation challenge as measured by the provocative dose at 80% of saline baseline for FEV, (PD,,). PDs were calculated in inhalation units (1 unit = 1 inhalation of allergen extract at 115,000 w :v) shown in the right-hand scale, then transformed as the natural log [In(PD + l)] to normalize the distribution. The means k 1 SEM shown are for 10 subjects except control where n = 9. Numbered lines correspond to individual subjects. Subject No. 5 was restudied as subject No. 11 because she developed a respiratory infection. Values for No. 5 are excluded from the means.
Comparison challenges
of placebo
with
drug-treated
A two-factor (randomized block) analysis of variance (ANOVA) with subjects as blocks and doses of lodoxamide (0.0, 0.01, 0.1, or 1.O mg) as treatments was performed. Dunnet’s tests (two-tailed) was used to test the null hypotheses of no difference between placebo and drug-treated means for the three variables (slope, PD, area), for four PFs (FEV,, FEFZ5-r5, FEL- I ,zootPEFR). PD and area were not normally distributed in this sample (e.g., for placebo FEV, PD,,,:mean + SD ~407.7 5 586.2,median = 83.4, range, 0.77 to 1831.8 inhalation units). For statistical analysis, PD and area were transformed as In (x + 1); slope was considered as In slope. Examination of Table IV and associated F values (variance ratios) show significant effects due to both the blocks (inter-
subject variation) and the treatments (drug effects). In this table all drug-treated means were significantly greater than placebo (p < 0.05 for 0.01 mg; p < 0.01 for 0.1 and 1.0 mg). FEVr area and PD were the only variables that showed 0.01 mg lodoxamide to be significantly different from placebo. By a two-tailed test, the 0.01 mg means generally approached significance for other PFs and parameters, and may well be considered different under less stringent criteria (Fig. 4). There was no significant qualitative difference among the three treatment means when tested nonparametrically with Friedmann’s ANOVA by ranks (e.g., X2 = 3.8, df = 2, p = 0.15 for FEV,, InPD,,) . Although we administered a single-blind control and a double-blind placebo, the results were not significantly different and it seemed reasonable to test
292
Watt et al.
J. ALLERGY
CLIN. IMMUNOL OCTOBER 1980
8
! 6
r
f
-
P .Ol 0.1 I.0 Slope
L
P .Ol 0.1 I.0 Provocative Dose
P ,Ol 0.1 I.0 Area
FIG. 4. Comparison of effect of inhaled pretreatment on parameters of dose-response curves. Pretreatments were sterile water (P) or lodoxamide tromethamine (0.01, 0.1, or 1.0 mg). For parameter units In is the natural logarithm. Bar heights are means for IO subjects + 1 SEM. All drug-treated means are significantly greater than placebo (see text). TABLE
V. Relative
effect
of treatment
Ratio* Lodoxamide (ms)
zt dose
Lodoxamide (ms)
dose
Subject
0.01
0.1
1.0
0.01
0.1
1.0
I 2 3 4 6 7 8 9 10 Increase No change Decrease
0.1 41.3 3.5 I.0 2.4 3.8 -0.2 100.4 0.8 6 2 1
7.2 22.2 8.8 0.9 12.3 2.6 1.9 156.0 3.3 8 1 0
2.3 16.0 11.8 1.2 3.7 6.2 4.0 40.4 9.5 9 0 0
1.13 14.20 4.18 1.79 2.28 4.02 -1.47 75.97 0.65 5 4 0
3.85 8.09 6.38 1.73 5.14 3.38 2.25 92.50 2.99 8 1 0
2.54 6.12 7.18 1.90 2.97 5.00 4.28 46.43 1.95 7 2 0
PDt: Provocative dose for a drug treatment; PDc: control provocative dose; PDp: placebo provocative dose; PDu: mean of PDc and PDp. *Ratio = PDt - PDpl 1PDc - PDp) tZ = PDt - PDu/SD of PDu.
treatment values considering what was known about untreated variation. Table V shows two comparisons. The left half of the table shows the ratio of the difference between placebo and treated PDs to the difference between placebo and control PDs (raw PD values were used here) as ratio = (PDt - PDp)/ (PDc - PDp(. After treatment with 0.01 mg, six sub-
jects increased (ratio > l), two stayed the same (0 < ratio < 1) and one decreased (ratio < 0); for 0.1 mg, eight increased, one showed no change, and none decreased; for 1.O mg, all nine increased. The right half of the table uses as its criterion how far away in standard deviation units a treated value is from the untreated mean; a significant change is defined as Z > 1.96, i.e., p < 0.05 (two tails). For 0.01 mg, five subjects increased significantly and four showed no significant change; for 0.1 mg, eight increased and one had no change; for 1.O mg, seven increased and two had no change. Subject 4, who showed no change for all three treatments, had the largest control/placebo variance. Fig. 5 shows the protective effect of lodoxamide as measured by the percent decrease from the baseline following allergen challenge. The mean decrease in FEV, after allergen challenge was 33% whereas the mean decrease following lodoxamide was only 16%, 3%, or 7.5% at 0.01, 0.1, or 1.O mg, respectively. Thus, at the O.l-mg dose, all subjects showed protection and the degree of protection was essentially complete (over 90%) for the allergen threshold dose that was administered following placebo. We also examined the possibility that lodoxamide may act as a bronchodilator. This was done by comparing the pretreatment saline baseline FEV, with the FEV, observed at 5 and 30 min after treatment. The mean increase 30 min after placebo was 4.0 + 1.7%, afterO.Olmgwas3.9 t 2.5%afterO.lmgwas3.6 k 1.O%, and after 1.Omg was 3.7 k 1.5%. These values are not significantly different by ANOVA.
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DISCUSStON Administration of lodoxamide tromethamine by inhalation at doses of 0.1 and 1.O mg uniformly allowed subjects to tolerate significantly larger doses of inhaled allergen extract as measured by the slope, PD, and area under the challenge dose-response curve. Lodoxamide at 0.01 mg appeared to be effective in over half the subjects tested. Lodoxamide aerosol was nonirritating and did not act immediately as a bronchodilator. Although lodoxamide at all the doses tested was generally significantly more effective than placebo, there was no correlation with dosage. Five subjects showed maximum protection at 1.O mg and four subjects at 0.1 mg. Animal studies have shown a biphasic dose-response curve for both lodoxamide and cromolyn; thus, it is possible that because of individual variation in optimal dose requirements, peak effectiveness may be seen at smaller than maximal dose. Examination of the means in Fig. 3 suggests that the most universally effective dose was 0.1 mg, and that individuals who were more allergen sensitive were also more drug sensitive in that their drug curves were shifted to the left of the less sensitive group (e.g., compare subjects 1, 6, and 9 with 3, 7, and 10). Lodoxamide permitted the test subjects to tolerate considerably more allergen. Inspection of Fig. 3 shows that after placebo, the geometric mean provocative dose was about 90 inhalation units, whereas after 0.1 mg of lodoxamide it was about 450. Although the increase from placebo of the mean amount of allergen tolerated was about fivefold, some subjects tolerated up to 23 times as much allergen (e.g., subject 6 in Fig. 3). The degree of protection with lodoxamide was considerably greater than what we have reported previously for cromolyn sodium.** 7-g For example, a similar population of 14 subjects with allergic bronchial asthma showed a mean decrease of 37% in their FEV after allergen challenge pretreated with placebo cromolyn sodium.7 Following cromolyn sodium, 20 mg by Spinhaler, the mean decrease in FEV, was 23% and three of the subjects showed no protection; thus, the degree of protection was only 62% following cromolyn sodium vs 90% for lodoxamide. Neither lodoxamide nor cromoiyn sodium has shown any appreciable immediate bronchodilator action, although cromolyn sodium has been reported to inhibit phosphodiesterase. lo Cromolyn sodium did not protect against histamine or methacholine bronchial challenges .*a i-g Similar studies have not been done with lodoxamide. Reported side effects, chiefly flushing and a subjective feeling of warmth, were generally mild and
0 1 At
0. I PlCCIbO 0.01 FEV, Response (MeonfS.E.M.,n=lOl Lorl Allsrqen Oo3r Of Placebo
I.0 Challenge
FIG. 5. Effect of lodoxamide on allergen inhalation challenge as measured by the percent decrease in FEV, at the last dose of the placebo-treated challenge. Each subject’s set of dose-response curves is thus considered at a single value for dose of allergen.
were lessened with repeated administrations. Delayed reactions occurred at about the same rate for drugtreated and untreated challenges; however, after drug treatment subjects were able to tolerate substantially larger doses of allergen. Thus, it is not possible to rule out lodoxamide’s ability to prevent or alter delayed responses. Lodoxamide tromethamine is a new antiallergy drug that is rapidly transported across mast cell membranes. Lodoxamide stabilized mast cell membranes by inhibiting Ca++ influx .3*4 In animal studies lodoxamide has been demonstrated to be more potent than cromolyn. Johnson et al. have demonstrated oral activity in monkeys and rats.“, 4 We have shown that lodoxamide, a water-soluble, cromolyn-like agent, administered by aerosol, protects against experimental allergen-induced bronchoconstriction. The authors gratefully acknowledge the technical assistance of Kathleen Burke, Kathleen Paziak, Dianne Hert, and Rose Garred, and the time and effort expended by the volunteer subjects.Lodoxamide was obtained from The Upjohn Company through the auspices of H. Johnson, A. Sheridan, and C. Cross, whose contributions to the study were indispensable. REFERENCES 1. Cox JSG: Disodium cromoglycate (FPL 670 I’lntal’): A specific inhibitor of reaginic antibody-antigen mechanisms. Nature 216: 1328, 1967.
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2. Kolotkin B, Lee C, Townley R: Duration and specificity of sodium cromolyn on allergen inhalation challenge in asthmatics. J ALLERGYCLIN IMMUNOL53:288, 1974. 3. Johnson H, Van Hout C, Wright J: Inhibition of allergic reactions by cromoglycate and by a new anti-allergy drug U42,585E. 1. Activity in the rat. Int Arch Allergy Appl Immuno1 56~416, 1978. 4. Johnson H, Van Hout C, Wright J: Inhibition of allergy reactions by cromoglycate and by a new anti-allergy drug U42,585E. II. Activity in primates against aerosolized Ascaris ~nurn antigen. Int Arch Allergy Appl Immunol56:487, 1978. 5. Chai H, Farr R, Froehlich LA, Mathison DA, McLean JA, Rosenthal RR, Sheffer AL, Spector SL, Townley RG: Standardization of bronchial inhalation challenge procedures. J ALLERGY CLIN IMMUNOL 56:323, 1975.
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6. Dunnet CW: Multiple comparison procedures for comparing several treatments with a control. J Am Stat Assn 50:1096. 1955. 7. Ryo UY, Kang B, Townley RG: Cromolyn therapy in patients with bronchial asthma. JAMA 236:927, 1976. 8. Kang B, Townley R, Lee CK, Kolotkin BM: Bronchial sensitivity to histamine before and after sodium cromoglycate in bronchial asthma. Br Med J 1:867, 1976. 9. Townley RG: Pharmacological blocks to mediator release: Clinical applications. Adv Asthma Allergy 2(3):7, 1975. 10. Taylor WA, Francis DH, Sheldon D: Anti-allergic actions of disodium cromoglycate and other drugs known to inhibit cyclic 3’,5’-nucleotide phosphodiesterase. Int Arch Allergy Appl Immunol 47:175, 1974.