- Email: [email protected]
A double-blind histamine placebo—controlled trial of polymerized whole grass for immunotherapy of grass allergy
A double-blind histamine placebo-controlled trial of polymerized whole grass for immunotherapy of grass allergy Leslie C. Grammer, M.D., Martha A. Shaughnessy, B.S., lrena M. Suszko, B.S., John J. Shaughnessy, Ph.D.,* and Roy Patterson, Chicago,
M.D.
Ill.
Twenty-sti putients were recruited for a study of the safety and ej&acy of immunotherap? with IPG. They M’ere randomly assigned to two groups based on skin test titrations to grass ullergens. One group was treated in a double-blind fashion before the 1982 grass season Ivith 12 bveekly injections totaling approximately 48,000 PNU/. and the other group was treuted bvith I? weekly injections of caramelized glucose histamine placebo. Daily symptom and medicution Jc’ore sheets were completed by all patients each day of the grass seuson. Blocking untibod> rose ninefold in the IPG group (p < 0.007) but wus unchanged in the placebo group. There \L’US no signi&mt change in IgE against rye grass group I in either the IPG or the plucebo group. Symptom-score mean in the IPG group was 217 ? 71 (S.E.M.), statistically lower (p < 0.02) than the meun in the placebo group 496 t 117 (S.E.M.). There were no systemic reactions and only minor local reactions. There was no change in routine laboratory tests in either group. Although two prior studies with grass allergen immunotherapy reported efJicacy, these studies did not use symptom-score analysis. This is the first double-blind, histamine placebo-controlled study of grass immunotherapy that demonstrates efficacy by symptom-score index evaluation. IPG is a safe, clinically effective, and potentially cost-effective therapy for grass pollinosis. (.I
ALLERGYCLINIMMUNOL 72:448-453, 1983.)
Polymerized allergens have been studied for several years in order to improve allergen immunotherapy. Their utility in immunotherapy stems from their having reduced allergenicity and retained immunogenicity when compared to unmodified allergens on a weight basis. ’ Polymerized ragweed totaling approximately 50,000 PNU in a 15injection regimen has been demonstrated to be safe and efficacious in a multiinstitutional trial’ and in a double-blind histamine placebo-controlled trial.:’ Polymerization of a grass pollen mixture has been reported .4 PG was found to have reduced allergenicity compared to monomer grass and was able to absorb
specific IgG from sera of patients treated with unmodified grass pollen allergens. The immune response from PG in humans was shown to be similar to that obtained from equivalent weights of aqueous grass extracts.” Subsequently, we have polymerized each grass pollen individually. Mixtures of these IPGs have demonstrated decreased allergenicity when compared to unmodified grass extracts. Furthermore, IPG has the capability of absorbing specific IgE from sera of untreated grass-sensitive patients and specific IgG from sera of patients treated with aqueous grass extractse6 To test the safety and efficacy of IPG as a preferable alternative to current immunotherapy, a double-blind histamine placebo trial was conducted.
From the Section of Allergy-Immunology, Department of Medicine, Northwestern University Medical School, Chicago, III., and *Hope College, Holland, Mich. Supported by United States Public Health Service grant Al-l 1403 and the Ernest S. Bazley Trust. Received for publication Feb. 1, 1983. Accepted for publication May 16, 1983. Reprint requests: Leslie C. Grammer, M.D., Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611.
MATERIAL Patients
448
AND METHODS
Twenty-six patients with histories typical of grass pollinosis and 4+ prick tests to one or more grass pollen extracts were recruited. These grass pollen extracts were 1: 20 w/v extracts of rye, timothy, redtop, June, orchard, and Bermuda obtained from ALO, Columbus, Ohio. These patients were 21 to 65 years of age, had not received immunotherapy for at least 5 yr, and were otherwise healthy as
VOLUME 72 NUMBER 5, PART 1
Abhrt~~~iation.c
IPG: PNU: PG: ALO: RGGI:
Polymerized TABLE 1. Immunotherapy placebo
uwd
Individually polymerized grass Protein nitrogen units Polymerized grass Allergy Laboratories of Ohio Rye grass group I
Injection
grass immunotherapy schedule
1
1: IO I :I0 I:10 Full strength Full strength Full strength Full strength Full strength
2 3 4
5
for IPG or
Volume (ml)
Dilution
No.
449
PNU per injection
.I ._3
90 210 540 900 1800 3600 5400 7200
.b .I .2 .4 .6 .8
determined by history. physical examination, erythrocyte sedimentation rate, complete blood count with differential leukocyte count, and urinalysis. Cutaneous end point titrations to serial tenfold dilutions of six mixed grasses (ALO) and prick tests to mixed Dematiaceae and Aspergilhs ( 1 : 20; ALO) were performed. Patients were randomized to one of two groups on the basis of mixed grass titration. Three patients failed to complete their injections because of transportation difficulties.
placebo bottle: one corresponding to the placebo for the five-grass mix and one corresponding to the placebo for the six-grass mix.
Symptom
Immunotherapy
and medication
score
6
7 8-12
schedule
In February 1982. the patients began a series of 12 weekly injections. The IPG schedule is shown in Table I. The placebo schedule was identical. If a patient had a large local reaction, the dose was repeated.
Daily symptom and medication score sheets were completed three times daily by each patient from the first week in May through the first 3 days in July 1982. This permitted quantification (0 to 3) of several symptoms, such as sneezing, nasal discharge, pruritus, and cough. Names of medications and numbers of pills taken were also listed. In the total score, antihistamines were multiplied by 5 since that is a reasonable estimate of reduction in symptom score from the ingestion of one antihistamine. The total score was the summation of medication scores and symptoms from all categories, each of which was given equal weight. Upon completion of the grass season, patients were asked whether or not their symptoms were significantly improved.
Immediate local reactions were recorded as centimeters of induration and erythema. Delayed local reactions occurring at 4 to 24 hr were reported by the patients. If doses had to be repeated because of a large local reaction, additional injections were administered to reach 48,000 PNU or the equivalent volume of placebo.
Polymerization
Laboratory
of individual
grasses
Each of six grass pollens (rye, timothy, redtop, orchard, June, and Bermuda) (ALO) was defatted with ether, extracted with NHdHCOB. precipitated by (NH&SOr, and fractionated by Sephadex G- 15 chromatography, resulting in six different monomer grasses. Polymerization with glutaraldehyde (ALO) occurred at 25” C in 4 hr. Six IPGs were obtained with Sepharose 4B and Sephadex G-200 gel filtration chromatography; molecular weights of each IPG ranged from 200,000 to 20 million daltons. The PNU count per milliliter of each polymer was determined by the microKjeldahl method. Sterility and safety requirements of the Food and Drug Administration Bureau of Biologics were verified by ALO. Since all patients reacted to all six grasses or to all except Bermuda, it was necessary to prepare two different grass polymer mixtures: one containing five grasses and one containing all six grasses.
Histamine
placebo
The caramelized glucose histamine placebo was identical in color to IPG. It was prepared as described by Norman et al.’ with the highest histamine concentration being 0.025 mgiml. One of two different designations was given to each
Quantitation of allergic immunotherapy
reactions
to
tests
Before initiation of the study and at the completion of injections, each patient had a complete blood count, a differential leukocyte count, urinalysis, and a measurement of erythrocyte sedimentation rate.
Immunologic
studies
Blood for immunologic studies was drawn from each patient before the initiation of injections and after the completion of injections. Serum was separated and stored frozen before use. RGGI (National Institutes of Health, Bethesda, Md.) binding by IgE was measured by a previously described polystyrene tube technique.8 Briefly, polystyrene tubes were coated with myeloma IgE. Rabbit anti-human IgE was then added to form an IgE immunosorbent. After a 48 hr incubation with 100 ~1 of patient serum, the tubes were washed, and ‘?-treated RGGI was added in excess. After 48 hr of incubation, tubes were washed and counted. The amount of specific IgE directed against RGGI in 1 ml of serum was calculated. The total serum RGGI-binding capacity was determined by a modification9 of the ammonium sulfate method of Lidd and Farr.‘O Briefly, 100 /LI samples were incubated with 10,
450
Grammer
1000
J. ALLERGY CLIN. IMMUNOL. NOVEMBER 1983
et al
1
900
800 1 700
1
f
000
co 1
500
B
400
42 m Ei
300 :
::
I _,_,_........ ...‘.... ,........ . ,........’ i i_
1
P 200 1
I
I
Pretreatment February 1982
Pretreatment February 1982
Posttreatment May 1982
FIG. 1. Total serum binding activity of RGGI is shown for patients receiving placebo (0) and IPG (01. The vertical bars represent S.E.M., the rise in the IPG group is significant (p < 0.007), and there is no change in the placebo group.
100. and 1000 ng of Y-treated RGGI. RGGI bound to antibody was precipitated with 40% saturated ammonium sulfate, and the amount of RGGI bound by 1 ml of serum in antigen excess was calculated. This is a quantitative estimate of blocking antibody and is expressed as nanograms of RGGI bound per milliliter of serum.
Seasonal
pollen counts
From the first week in May through the 1982, Chicago area pollen counts were days per week by Hinsdale Hospital by slide method. Rotating impaction sampler available.
first week in July obtained several use of a gravity counts were not
RESULTS Patients
There were no statistically significant differences between the two groups by Student’s t test analysis of IgE against RGGI, by log rank test of mixed grass cutaneous end point titration, by chi-square analysis of Bermuda reactivity, or by chi-square analysis of mold reactivity.
Posttreatment May 1982
FIG. 2. Results are shown for IgE against RGGI RGGll before and after treatment in the IPG group the placebo group (0). The vertical bars represent There is no statistically significant change in either
Quantitation
of allergic
(/gE-a(0) and S.E.M. group.
reactions
Two of 10 patients receiving IPG had at least one immediate local reaction characterized by erythema and induration; one patient had erythema alone. Totals of 43 cm erythema and 23 cm induration were recorded when data from all patients who received IPG were combined. This averaged 4 mm erythema and 2 mm induration per injection. One patient receiving IPG had one large late local reaction that required one additional injection. There were no systemic reactions to IPG. Two of 13 patients receiving histamine placebo had at least one immediate local reaction consisting of induration and erythema whereas three patients had erythema alone. In total, placebo-treated patients had 44 cm erythema and 1.5 cm induration; this averaged 3 mm erythema and 0.1 mm induration per injection. One patient receiving histamine placebo had one large local reaction that required one additional injection. Immunologic
studies
Fig. 1 demonstrates changes in total serum antibody against RGGI after IPG and placebo injections. In the IPG patients, there was a statistically significant (p < 0.007 paired t test) ninefold increase in blocking antibody whereas there was no change in placebo-
VOLUME 72 NUMBER 5. PART 1
Polymerized
grass
immunotherapy
451
100
Season
Total=260
90 1
p=o.33 0.10 0.10 cl.140.13 0.03 0.02 0.07 0.23 1
June
May
2
WEEKS FIG. 3. Pollen counts method several days The units are pollen
were obtained by the gravity per week and totaled for each grains per square centimeter.
slide week.
pollen counts
Pollen counts were totaled for each week in May and June 1982 and are shown in Fig. 3. Symptom/medication
4
1
2
3
4
5
June WEEKS
treated patients. Change of IgE against RGGI with injections in the IPG and placebo groups is illustrated in Fig. 2. There was no statistically significant change in IgE against RGGI in the IPG-treated patients or in patients given placebo. Statistical analyses were performed by use of the log transformations of the data. Seasonal
3
MY
score indices
Graphic representation of average daily symptom/medication scores in IPG- and placebo-treated groups is shown in Fig. 4. Two weeks demonstrate that the symptom-score indices of the IPG group were significantly lower by Mann-Whitney U test than those of the placebo control group. A graph of the total seasonal symptom scores is shown in Fig. 5. The first vertical bar is representative of 1 S.E.M. for the IPG group whereas the second bar represents S.E.M. for the placebo group. Thep value determined by Mann-Whitney U test or by Student’s t test was less than 0.05, indicating that symptom scores in the IPG group were significantly lower than those of the placebo group. Of the 10 patients in the IPG group, nine reported improved symptoms. This was in contrast to the placebo patients of whom only
FIG. 4. Average for IPG patients and S.E.M. are
daily symptom-score indices are plotted (0) and placebo patients (0). The p value shown for each point plotted.
three of 13 patients reported improvement. Statistical analysis by Fisher’s exact test reveals this to be a significant difference at the 0.01 level. Laboratory
tests
Compared to results before injections, there were no differences in complete blood count with differential leukocyte count, erythrocyte sedimentation rate, or urinalysis in either group after injections. DISCUSSION
This clinical trial was designed to evaluate safety, therapeutic efficacy, and immunogenicity of IPG. From the aspect of safety, an important finding in this study was the absence of immediate-type systemic reactions in spite of the large dose, 48,000 PNU, given in a short time period of 12 wk. Anaphylaxis is well recognized as the major risk factor for patients receiving unmodified allergen injections. This risk is substantial, 8% to 30% per patient course ,‘I in spite of multiple dilutions and slowly increasing doses that characterize the usual clinical practice of allergy. Thus when the absence of anaphylaxis is added to the rapid dose schedule of this study, the advantages of IPG over usual grass pollen immunotherapy are obvious. Because the number of injections required for the course of immunotherapy is significantly reduced, there should be an associated reduction in such costs
452
Grammer
J. ALLERGY CLIN. IMMUNOL NOVEMBER 1983
et al
I I
200 1
p=o.o2
.
01 IPG
PLACEBO
GROUP
FIG. 5. Total seasonal symptom are plotted for the IPG group
scores (0) and
(mean placebo
GROUP
and S.E.M.) group (01.
as total injection fee, total office visit fee for immunotherapy monitoring, pay withheld for time off from work for injections, baby-sitter charges, and parking fees. Thus this form of therapy has the potential of being more cost-effective than conventional aqueous extract therapy. Local reactions were minimal. There was only one instance in 120 injections of a large late local reaction that required repetition of a dose. In each group, there was one local reaction that required a repeat injection so that the groups were not distinguishable as to frequency of significant local reactions. There were no changes in any of the laboratory parameters measured, further corroborating the safety of immunotherapy with IPG. Significant improvement in symptom and medication scores was demonstrated when placebo and IPG patients were compared by Mann-Whitney U test. Further evidence for efficacy is that 90% of IPG patients reported improvement whereas only 23% of placebo patients reported improvement, a statistically significant difference by Fisher’s exact test. Two other double-blind placebo-controlled studies of grass pollinosis have been reported: one with usual aqueous grass extract” and one with glutaraldehyde-treated tyrosine adsorbed grass extract .‘a In each, efficacy of immunotherapy was demonstrated; both relied on chi-square analysis of the nominal answer to “Were your symptoms improved?” Thus this clinical trial is the first double-blind placebo-controlled trial to dem-
onstrate efficacy of grass pollen immunotherapy on the basis of symptom and medication scores. In this trial, there was only a modest rise in IgE directed against RGGI. However, after 48,000 PNU of IPG, blocking antibody against RGGI rose ninefold, averaging 591 ng RGGI bound/ml. This result is comparable to results previously reported in which larger cumulative doses of monomeric antigen were used.” There was no change in either immunologic parameter in placebo-treated patients. This double-blind histamine placebo-controlled trial demonstrates efficacy, safety, and immunogenicity of IPG. High levels of blocking antibody were induced in treated patients, and they tolerated IPG well with no systemic reactions, no changes in usual laboratory parameters, and fewer injections than would be given in conventional immunotherapy with weight-equivalent amounts of allergen. Moreover. statistically, symptom/medication scores of IPGtreated patients were significantly lower than those of placebo-treated patients. The efficacy and safety of polymerized pollen allergens with both ragweed and grass pollen preparations have now been demonstrated. Polymerized preparations of other inhalant allergens should be equally safe and effective provided that there is demonstration of reduced allergenicity and retained antigenicity. Furthermore, two major pollen allergens in the United States should be available for immunotherapy in polymerized form.
REFERENCES 1. Patterson R, Suszko IM, Bacal E, Zeiss CR, Kelly JF. Pruzansky JJ: Reduced allergenicity of high molecular weight ragweed polymers. J ALLERGY CLIN IMMUNOL 63:47, 1979. 2. Hendrix SG, Patterson R, Zeiss CR, et al: A multi-institutional trial of polymerized whole ragweed for immunotherapy of ragweed allergy. J ALLERGY CLIN IMMUNOL 66:486, 1980. 3. Grammer LC, Zeiss CR, Suszko IM, Shaughnessy MA, Patterson R: A double-blind placebo controlled trial of polymerized whole ragweed for immunotherapy of ragweed allergy. J ALLERGY CLIN IMMUNOL 69:494, 1982. JJ, Zeiss CR, Metzger J. 4. Patterson R, Suszko IM, Pruzansky Roberts M: Polymerization of mixtures of grass allergens. J ALLERGY CLIN IMMLJNOL 59314, 1977. 5. Her&ix SG, Patterson R, Zeiss CR, Suszko IM: The immune response in humans and rabbits to monomeric and polymeric grass allergens. J Clin Immunol 2:lO. 1982. 6. Patterson R, Suszko IM, Grammer LC, Shaughnessy MA: Polymerization of individual species of grass pollen allergens. J ALLERGY CLIN IMMUNOLOGY 72:129, 1983. 7. Norman PS, Winkenwerder WL, Lichtenstein LM: Immunotherapy of hay fever with ragweed antigen E: comparison with whole pollen extract and placebo. J ALLERGY 4293, 1968. 8. Zeiss CR, Pruzansky JJ, Patterson R, Roberts M: A solid phase
VOLUME 72 NUMBER 5, PART 1
radioimmunoassay for the quantitation of human reaginic antibody against ragweed antigen E. J Immunol 110~414, 1973. 9. Zeiss CR. Metzger WJ, Levitz D: Quantitative relationship between IgE antibodies and blocking antibodies specific for antigen E in patients given immunotherapy with ragweed antigen E. Clin Exp Immunol 28~250, 1977. 10. Lidd D. Farr RS: Primary interaction between ‘:r’I labeled ragweed pollen and antibodies in the sera of humans and rabbits. J ALLERCYCLIN IMMUNOL 33:45, 1962.
Polymerized
I1
grass
immunotherapy
453
Levy DA: Hazards and adverse reactions associated with administration of allergenic extracts. In Brede HD, Going H, editors: Regulatory control and standardization of allergenic extracts. New York, 1980, Gustav Fischer Verlag, p. 178. 12. Frankland AW, Augustin R: Grass pollen antigens effective in treatment. Clin Sci 23:95, 1962. 13. Miller ACML., Tees EC: A metabolisable adjuvant: clinical trial of grass pollen-tyrosine adsorbate. Clin Allergy 4:49, 1974.
M.D.
Ill.
Twenty-sti putients were recruited for a study of the safety and ej&acy of immunotherap? with IPG. They M’ere randomly assigned to two groups based on skin test titrations to grass ullergens. One group was treated in a double-blind fashion before the 1982 grass season Ivith 12 bveekly injections totaling approximately 48,000 PNU/. and the other group was treuted bvith I? weekly injections of caramelized glucose histamine placebo. Daily symptom and medicution Jc’ore sheets were completed by all patients each day of the grass seuson. Blocking untibod> rose ninefold in the IPG group (p < 0.007) but wus unchanged in the placebo group. There \L’US no signi&mt change in IgE against rye grass group I in either the IPG or the plucebo group. Symptom-score mean in the IPG group was 217 ? 71 (S.E.M.), statistically lower (p < 0.02) than the meun in the placebo group 496 t 117 (S.E.M.). There were no systemic reactions and only minor local reactions. There was no change in routine laboratory tests in either group. Although two prior studies with grass allergen immunotherapy reported efJicacy, these studies did not use symptom-score analysis. This is the first double-blind, histamine placebo-controlled study of grass immunotherapy that demonstrates efficacy by symptom-score index evaluation. IPG is a safe, clinically effective, and potentially cost-effective therapy for grass pollinosis. (.I
ALLERGYCLINIMMUNOL 72:448-453, 1983.)
Polymerized allergens have been studied for several years in order to improve allergen immunotherapy. Their utility in immunotherapy stems from their having reduced allergenicity and retained immunogenicity when compared to unmodified allergens on a weight basis. ’ Polymerized ragweed totaling approximately 50,000 PNU in a 15injection regimen has been demonstrated to be safe and efficacious in a multiinstitutional trial’ and in a double-blind histamine placebo-controlled trial.:’ Polymerization of a grass pollen mixture has been reported .4 PG was found to have reduced allergenicity compared to monomer grass and was able to absorb
specific IgG from sera of patients treated with unmodified grass pollen allergens. The immune response from PG in humans was shown to be similar to that obtained from equivalent weights of aqueous grass extracts.” Subsequently, we have polymerized each grass pollen individually. Mixtures of these IPGs have demonstrated decreased allergenicity when compared to unmodified grass extracts. Furthermore, IPG has the capability of absorbing specific IgE from sera of untreated grass-sensitive patients and specific IgG from sera of patients treated with aqueous grass extractse6 To test the safety and efficacy of IPG as a preferable alternative to current immunotherapy, a double-blind histamine placebo trial was conducted.
From the Section of Allergy-Immunology, Department of Medicine, Northwestern University Medical School, Chicago, III., and *Hope College, Holland, Mich. Supported by United States Public Health Service grant Al-l 1403 and the Ernest S. Bazley Trust. Received for publication Feb. 1, 1983. Accepted for publication May 16, 1983. Reprint requests: Leslie C. Grammer, M.D., Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611.
MATERIAL Patients
448
AND METHODS
Twenty-six patients with histories typical of grass pollinosis and 4+ prick tests to one or more grass pollen extracts were recruited. These grass pollen extracts were 1: 20 w/v extracts of rye, timothy, redtop, June, orchard, and Bermuda obtained from ALO, Columbus, Ohio. These patients were 21 to 65 years of age, had not received immunotherapy for at least 5 yr, and were otherwise healthy as
VOLUME 72 NUMBER 5, PART 1
Abhrt~~~iation.c
IPG: PNU: PG: ALO: RGGI:
Polymerized TABLE 1. Immunotherapy placebo
uwd
Individually polymerized grass Protein nitrogen units Polymerized grass Allergy Laboratories of Ohio Rye grass group I
Injection
grass immunotherapy schedule
1
1: IO I :I0 I:10 Full strength Full strength Full strength Full strength Full strength
2 3 4
5
for IPG or
Volume (ml)
Dilution
No.
449
PNU per injection
.I ._3
90 210 540 900 1800 3600 5400 7200
.b .I .2 .4 .6 .8
determined by history. physical examination, erythrocyte sedimentation rate, complete blood count with differential leukocyte count, and urinalysis. Cutaneous end point titrations to serial tenfold dilutions of six mixed grasses (ALO) and prick tests to mixed Dematiaceae and Aspergilhs ( 1 : 20; ALO) were performed. Patients were randomized to one of two groups on the basis of mixed grass titration. Three patients failed to complete their injections because of transportation difficulties.
placebo bottle: one corresponding to the placebo for the five-grass mix and one corresponding to the placebo for the six-grass mix.
Symptom
Immunotherapy
and medication
score
6
7 8-12
schedule
In February 1982. the patients began a series of 12 weekly injections. The IPG schedule is shown in Table I. The placebo schedule was identical. If a patient had a large local reaction, the dose was repeated.
Daily symptom and medication score sheets were completed three times daily by each patient from the first week in May through the first 3 days in July 1982. This permitted quantification (0 to 3) of several symptoms, such as sneezing, nasal discharge, pruritus, and cough. Names of medications and numbers of pills taken were also listed. In the total score, antihistamines were multiplied by 5 since that is a reasonable estimate of reduction in symptom score from the ingestion of one antihistamine. The total score was the summation of medication scores and symptoms from all categories, each of which was given equal weight. Upon completion of the grass season, patients were asked whether or not their symptoms were significantly improved.
Immediate local reactions were recorded as centimeters of induration and erythema. Delayed local reactions occurring at 4 to 24 hr were reported by the patients. If doses had to be repeated because of a large local reaction, additional injections were administered to reach 48,000 PNU or the equivalent volume of placebo.
Polymerization
Laboratory
of individual
grasses
Each of six grass pollens (rye, timothy, redtop, orchard, June, and Bermuda) (ALO) was defatted with ether, extracted with NHdHCOB. precipitated by (NH&SOr, and fractionated by Sephadex G- 15 chromatography, resulting in six different monomer grasses. Polymerization with glutaraldehyde (ALO) occurred at 25” C in 4 hr. Six IPGs were obtained with Sepharose 4B and Sephadex G-200 gel filtration chromatography; molecular weights of each IPG ranged from 200,000 to 20 million daltons. The PNU count per milliliter of each polymer was determined by the microKjeldahl method. Sterility and safety requirements of the Food and Drug Administration Bureau of Biologics were verified by ALO. Since all patients reacted to all six grasses or to all except Bermuda, it was necessary to prepare two different grass polymer mixtures: one containing five grasses and one containing all six grasses.
Histamine
placebo
The caramelized glucose histamine placebo was identical in color to IPG. It was prepared as described by Norman et al.’ with the highest histamine concentration being 0.025 mgiml. One of two different designations was given to each
Quantitation of allergic immunotherapy
reactions
to
tests
Before initiation of the study and at the completion of injections, each patient had a complete blood count, a differential leukocyte count, urinalysis, and a measurement of erythrocyte sedimentation rate.
Immunologic
studies
Blood for immunologic studies was drawn from each patient before the initiation of injections and after the completion of injections. Serum was separated and stored frozen before use. RGGI (National Institutes of Health, Bethesda, Md.) binding by IgE was measured by a previously described polystyrene tube technique.8 Briefly, polystyrene tubes were coated with myeloma IgE. Rabbit anti-human IgE was then added to form an IgE immunosorbent. After a 48 hr incubation with 100 ~1 of patient serum, the tubes were washed, and ‘?-treated RGGI was added in excess. After 48 hr of incubation, tubes were washed and counted. The amount of specific IgE directed against RGGI in 1 ml of serum was calculated. The total serum RGGI-binding capacity was determined by a modification9 of the ammonium sulfate method of Lidd and Farr.‘O Briefly, 100 /LI samples were incubated with 10,
450
Grammer
1000
J. ALLERGY CLIN. IMMUNOL. NOVEMBER 1983
et al
1
900
800 1 700
1
f
000
co 1
500
B
400
42 m Ei
300 :
::
I _,_,_........ ...‘.... ,........ . ,........’ i i_
1
P 200 1
I
I
Pretreatment February 1982
Pretreatment February 1982
Posttreatment May 1982
FIG. 1. Total serum binding activity of RGGI is shown for patients receiving placebo (0) and IPG (01. The vertical bars represent S.E.M., the rise in the IPG group is significant (p < 0.007), and there is no change in the placebo group.
100. and 1000 ng of Y-treated RGGI. RGGI bound to antibody was precipitated with 40% saturated ammonium sulfate, and the amount of RGGI bound by 1 ml of serum in antigen excess was calculated. This is a quantitative estimate of blocking antibody and is expressed as nanograms of RGGI bound per milliliter of serum.
Seasonal
pollen counts
From the first week in May through the 1982, Chicago area pollen counts were days per week by Hinsdale Hospital by slide method. Rotating impaction sampler available.
first week in July obtained several use of a gravity counts were not
RESULTS Patients
There were no statistically significant differences between the two groups by Student’s t test analysis of IgE against RGGI, by log rank test of mixed grass cutaneous end point titration, by chi-square analysis of Bermuda reactivity, or by chi-square analysis of mold reactivity.
Posttreatment May 1982
FIG. 2. Results are shown for IgE against RGGI RGGll before and after treatment in the IPG group the placebo group (0). The vertical bars represent There is no statistically significant change in either
Quantitation
of allergic
(/gE-a(0) and S.E.M. group.
reactions
Two of 10 patients receiving IPG had at least one immediate local reaction characterized by erythema and induration; one patient had erythema alone. Totals of 43 cm erythema and 23 cm induration were recorded when data from all patients who received IPG were combined. This averaged 4 mm erythema and 2 mm induration per injection. One patient receiving IPG had one large late local reaction that required one additional injection. There were no systemic reactions to IPG. Two of 13 patients receiving histamine placebo had at least one immediate local reaction consisting of induration and erythema whereas three patients had erythema alone. In total, placebo-treated patients had 44 cm erythema and 1.5 cm induration; this averaged 3 mm erythema and 0.1 mm induration per injection. One patient receiving histamine placebo had one large local reaction that required one additional injection. Immunologic
studies
Fig. 1 demonstrates changes in total serum antibody against RGGI after IPG and placebo injections. In the IPG patients, there was a statistically significant (p < 0.007 paired t test) ninefold increase in blocking antibody whereas there was no change in placebo-
VOLUME 72 NUMBER 5. PART 1
Polymerized
grass
immunotherapy
451
100
Season
Total=260
90 1
p=o.33 0.10 0.10 cl.140.13 0.03 0.02 0.07 0.23 1
June
May
2
WEEKS FIG. 3. Pollen counts method several days The units are pollen
were obtained by the gravity per week and totaled for each grains per square centimeter.
slide week.
pollen counts
Pollen counts were totaled for each week in May and June 1982 and are shown in Fig. 3. Symptom/medication
4
1
2
3
4
5
June WEEKS
treated patients. Change of IgE against RGGI with injections in the IPG and placebo groups is illustrated in Fig. 2. There was no statistically significant change in IgE against RGGI in the IPG-treated patients or in patients given placebo. Statistical analyses were performed by use of the log transformations of the data. Seasonal
3
MY
score indices
Graphic representation of average daily symptom/medication scores in IPG- and placebo-treated groups is shown in Fig. 4. Two weeks demonstrate that the symptom-score indices of the IPG group were significantly lower by Mann-Whitney U test than those of the placebo control group. A graph of the total seasonal symptom scores is shown in Fig. 5. The first vertical bar is representative of 1 S.E.M. for the IPG group whereas the second bar represents S.E.M. for the placebo group. Thep value determined by Mann-Whitney U test or by Student’s t test was less than 0.05, indicating that symptom scores in the IPG group were significantly lower than those of the placebo group. Of the 10 patients in the IPG group, nine reported improved symptoms. This was in contrast to the placebo patients of whom only
FIG. 4. Average for IPG patients and S.E.M. are
daily symptom-score indices are plotted (0) and placebo patients (0). The p value shown for each point plotted.
three of 13 patients reported improvement. Statistical analysis by Fisher’s exact test reveals this to be a significant difference at the 0.01 level. Laboratory
tests
Compared to results before injections, there were no differences in complete blood count with differential leukocyte count, erythrocyte sedimentation rate, or urinalysis in either group after injections. DISCUSSION
This clinical trial was designed to evaluate safety, therapeutic efficacy, and immunogenicity of IPG. From the aspect of safety, an important finding in this study was the absence of immediate-type systemic reactions in spite of the large dose, 48,000 PNU, given in a short time period of 12 wk. Anaphylaxis is well recognized as the major risk factor for patients receiving unmodified allergen injections. This risk is substantial, 8% to 30% per patient course ,‘I in spite of multiple dilutions and slowly increasing doses that characterize the usual clinical practice of allergy. Thus when the absence of anaphylaxis is added to the rapid dose schedule of this study, the advantages of IPG over usual grass pollen immunotherapy are obvious. Because the number of injections required for the course of immunotherapy is significantly reduced, there should be an associated reduction in such costs
452
Grammer
J. ALLERGY CLIN. IMMUNOL NOVEMBER 1983
et al
I I
200 1
p=o.o2
.
01 IPG
PLACEBO
GROUP
FIG. 5. Total seasonal symptom are plotted for the IPG group
scores (0) and
(mean placebo
GROUP
and S.E.M.) group (01.
as total injection fee, total office visit fee for immunotherapy monitoring, pay withheld for time off from work for injections, baby-sitter charges, and parking fees. Thus this form of therapy has the potential of being more cost-effective than conventional aqueous extract therapy. Local reactions were minimal. There was only one instance in 120 injections of a large late local reaction that required repetition of a dose. In each group, there was one local reaction that required a repeat injection so that the groups were not distinguishable as to frequency of significant local reactions. There were no changes in any of the laboratory parameters measured, further corroborating the safety of immunotherapy with IPG. Significant improvement in symptom and medication scores was demonstrated when placebo and IPG patients were compared by Mann-Whitney U test. Further evidence for efficacy is that 90% of IPG patients reported improvement whereas only 23% of placebo patients reported improvement, a statistically significant difference by Fisher’s exact test. Two other double-blind placebo-controlled studies of grass pollinosis have been reported: one with usual aqueous grass extract” and one with glutaraldehyde-treated tyrosine adsorbed grass extract .‘a In each, efficacy of immunotherapy was demonstrated; both relied on chi-square analysis of the nominal answer to “Were your symptoms improved?” Thus this clinical trial is the first double-blind placebo-controlled trial to dem-
onstrate efficacy of grass pollen immunotherapy on the basis of symptom and medication scores. In this trial, there was only a modest rise in IgE directed against RGGI. However, after 48,000 PNU of IPG, blocking antibody against RGGI rose ninefold, averaging 591 ng RGGI bound/ml. This result is comparable to results previously reported in which larger cumulative doses of monomeric antigen were used.” There was no change in either immunologic parameter in placebo-treated patients. This double-blind histamine placebo-controlled trial demonstrates efficacy, safety, and immunogenicity of IPG. High levels of blocking antibody were induced in treated patients, and they tolerated IPG well with no systemic reactions, no changes in usual laboratory parameters, and fewer injections than would be given in conventional immunotherapy with weight-equivalent amounts of allergen. Moreover. statistically, symptom/medication scores of IPGtreated patients were significantly lower than those of placebo-treated patients. The efficacy and safety of polymerized pollen allergens with both ragweed and grass pollen preparations have now been demonstrated. Polymerized preparations of other inhalant allergens should be equally safe and effective provided that there is demonstration of reduced allergenicity and retained antigenicity. Furthermore, two major pollen allergens in the United States should be available for immunotherapy in polymerized form.
REFERENCES 1. Patterson R, Suszko IM, Bacal E, Zeiss CR, Kelly JF. Pruzansky JJ: Reduced allergenicity of high molecular weight ragweed polymers. J ALLERGY CLIN IMMUNOL 63:47, 1979. 2. Hendrix SG, Patterson R, Zeiss CR, et al: A multi-institutional trial of polymerized whole ragweed for immunotherapy of ragweed allergy. J ALLERGY CLIN IMMUNOL 66:486, 1980. 3. Grammer LC, Zeiss CR, Suszko IM, Shaughnessy MA, Patterson R: A double-blind placebo controlled trial of polymerized whole ragweed for immunotherapy of ragweed allergy. J ALLERGY CLIN IMMUNOL 69:494, 1982. JJ, Zeiss CR, Metzger J. 4. Patterson R, Suszko IM, Pruzansky Roberts M: Polymerization of mixtures of grass allergens. J ALLERGY CLIN IMMLJNOL 59314, 1977. 5. Her&ix SG, Patterson R, Zeiss CR, Suszko IM: The immune response in humans and rabbits to monomeric and polymeric grass allergens. J Clin Immunol 2:lO. 1982. 6. Patterson R, Suszko IM, Grammer LC, Shaughnessy MA: Polymerization of individual species of grass pollen allergens. J ALLERGY CLIN IMMUNOLOGY 72:129, 1983. 7. Norman PS, Winkenwerder WL, Lichtenstein LM: Immunotherapy of hay fever with ragweed antigen E: comparison with whole pollen extract and placebo. J ALLERGY 4293, 1968. 8. Zeiss CR, Pruzansky JJ, Patterson R, Roberts M: A solid phase
VOLUME 72 NUMBER 5, PART 1
radioimmunoassay for the quantitation of human reaginic antibody against ragweed antigen E. J Immunol 110~414, 1973. 9. Zeiss CR. Metzger WJ, Levitz D: Quantitative relationship between IgE antibodies and blocking antibodies specific for antigen E in patients given immunotherapy with ragweed antigen E. Clin Exp Immunol 28~250, 1977. 10. Lidd D. Farr RS: Primary interaction between ‘:r’I labeled ragweed pollen and antibodies in the sera of humans and rabbits. J ALLERCYCLIN IMMUNOL 33:45, 1962.
Polymerized
I1
grass
immunotherapy
453
Levy DA: Hazards and adverse reactions associated with administration of allergenic extracts. In Brede HD, Going H, editors: Regulatory control and standardization of allergenic extracts. New York, 1980, Gustav Fischer Verlag, p. 178. 12. Frankland AW, Augustin R: Grass pollen antigens effective in treatment. Clin Sci 23:95, 1962. 13. Miller ACML., Tees EC: A metabolisable adjuvant: clinical trial of grass pollen-tyrosine adsorbate. Clin Allergy 4:49, 1974.