Polyethylene glycol-modified ragweed pollen extract in rhinoconjunctivitis

Polyethylene glycol-modified extract in rhinoconjunctivitis

ragweed pollen

E. F. Juniper, M.C.S.P., R. S. Roberts, M.Tech., L. K. Kennedy, J. O’Connor, R.N., M. Syty-Golda, M.D., J. Dolovich, M.D., F. E. Hargreave, M.D. Hamilton, Ontario, Canada

R.N.,

Sixty-two ragweed-sensitive adult subjects volunteered to take part in a 2-year, placebo-controlled eficacy study of polyethylene glycol (PEG)-mod$ed ragweed extract, in ragweed pollen-induced rhinoconjunctivitis. At the beginning of the study, subjects were stratified according to skin sensitivity to ragweed extract and PEG-modified ragweed and the severity of hay fever in the previous year. There was random allocation of half to active treatment and half to placebo treatment. Before the first ragweed pollen season the 36 most sensitive subjects received 10 weekly injections (group I), and the remaining 26 received six injections (group 2). Before the second season all subjects received 10 injections. Doses increased by half a log concentration each week unless there were adverse reactions. The mean total dose received by group I in year 1 was 385 pg of protein (28.9 pg AgE) and received by group 2 was 218 pg of protein (16.4 pg AgE). In year 2 the mean total dose was 1829 t.Lg (137.2 pg AgE). Sixty-six percent of injections elicited no reaction or a mild local reaction; the remaining injections produced local redness and swelling more than 2 inches in diameter. Four percent of injections produced systemic symptoms. PEG-modtjied ragweed stimulated increases in ragweed spec$c IgG antibody both years, but increases in ragweed speci$c IgE antibody were sign&ant only in group I in year I. The magnitude of the IgG antibody changes was directly related to the total dose injected. At the beginning of the second year, PEG-modified ragweedtreated subjects still had elevated IgG antibody levels. In the first year the subjects who had received 10 PEG-modt$ed ragweed injections improved in symptoms and medications; however, in subjects who received only six injections there was no difference between active and placebo treatment. In year 2 the actively treated group had significantly less symptoms and needed less medication, and the improvement was significantly better than that observed during the first year. The results indicate that PEG-modified ragweed, administered in suficient doses, is effective in reducing symptoms of ragweed-induced rhinoconjunctivitis and that the effect may be additive when it is administered over consecutive years. (J ALLERGY CLINIMMUNOL75.57835,

1985.)

PEG-modified ragweed is an extract in which ragweed pollen has been conjugated with PEG. Studies in rats and mice have indicated that PEG-modified ragweed has two special properties.’ First, there is a From the Department of Medicine, St. Joseph’s Hospital and McMaster University, and the Department of Clinical Epidemiology and Biostatistics, McMaster University, Hamilton, Ontario, Canada. Supported by a grant from Pharmacia, Ltd., Uppsala, Sweden. Received for publication April 2, 1984. Accepted for publication Sept. 4, 1984. Reprint requests: F. E. Hargreave, M.D., Firestone Regional Chest and Allergy Unit, St. Joseph’s Hospital, 50 Charlton Ave. E., Hamilton, Ontario, Canada L8N 4A6.

578

Abbreviation used PEG: Polyethylene

glycol

major reduction in IgE antibody formation. This apparently is a function of a reduction in phagocytosis by the macrophages and by an increased suppressor T activity. Second, the PEG conjugation greatly reduces interaction with preformed antibodies. If the effects are similar in humans, treatment would potentially reduce the level of allergy while eliciting greater protection from the larger doses that would not be tolerated with aqueous extract. This is the first article

Ragweed pollen extract in rhinoconjunctivitis

VOLUhlE 75 NUMBER 5

579

on the immunologic and therapeutic effects and the safety of PEG-modified ragweed injections in ragweed pollen-induced rhinoconjunctivitis.

bumin, the diluent used for the PEG-modified ragweed. Both active and placebo preparations were colorless, clear solutions that could not be differentiated by sight.

MATERIAL Subjects

Study

AND METHODS

Sixty-two ragweed-sensitiveadult subjects,with a history of seasonal rhinoconjunctivitis, volunteered to participate in the study. There were 26 female and 36 male subjects, and agesranged from 21 to 61 yr (mean 40.5). Each subject had a full history obtained by questionnaire, physical examination, ECG, and skin prick tests with 16 common allergen extractsand tenfold serialdilutions of ragweed extract (0.002 to 200 pg) and PEG-modified ragweed (0.02 to 2000 pg). Blood was drawn for complete blood count, differential, platelet count, sedimentation rate, serum creatinine, bilirubin, aspartatetransferase,alanine transferase,alkaline phosphatase, total protein, albumin, glucose, blood urea nitrogen, uric acid, cholesterol, triglycerides, inorganic phosphate, and calcium. Urine was examined for glucose, protein, and ketones. Any subject revealing a positive medical history, other than rhinoconjunctivitis or asthma, or revealing abnormalities in the laboratory screening was excluded from the study. Women of childbearing age were not included unless they were not at risk of pregnancy. All subjects gave informed written consent. Material Short ragweed pollen (Ambrosia elatior) was defatted with ether and extracted 1: 10 with 0.09% NaCl, pH 7.2, for 19 hr. The mixture was filtered, and the filtrate was fractionated on a SephadexG-25 column (PharmaciaFine Chemicals, Uppsala, Sweden) by use of 0.09% NaCl as eluant. The protein fraction was collected and was freezedried. The content of AgE was determined by radial immunodiffusion analysiscalibrated with a standardreference solution and was found to be 7.5% of the protein content. The ragweed allergen extract was modified with mPEG monosuccinate, 3000 MW, with use of the mixed anhydride method.* The mPEG with this method is coupled mainly to the primary amino groups of the proteins. The degree of modification was determined by analyzing the product for its content of mPEG and protein and calculating the ratio of moles of mPEG per weight of protein. The content of mPEG was determined by a nuclear magnetic resonance technique, and the protein content was determined by amino acid analysis.The molecular weight of mPEGsuccinatewas determined by gel filtration on a calibrated Sephacryl S-200 column (PharmaciaFine Chemicals). Two mPEG-modified ragweed preparations were used, one each for the first and second year. The first preparation contained 59% mPEG, 16% protein, and 1.2 mol of mPEGper 1000 gm of protein. The secondpreparation contained 58% mPEG, 14% protein, and 1.5 mol of mPEGper 1000 gm of protein. The allergenic activity of these preparations was 0.2% of the unmodified ragweed allergen extract measured with RAST inhibition. The placebo injections were saline with normal serum al-

design

Subjects were matched in pairs according to the results of the ragweed and PEG-modified ragweed skin prick tests, the recorded severity of their ragweed pollen-induced hay fever the previous year, associatedasthma, gender, and sensitivity to the fungal sporesAlternaria tenuis and Cladosporium (Hormodendrum) that are in the air at the same time as the ragweed pollen. Half the subjectswere randomly assignedto receive PEG-modified ragweed injections both years, and the other half assignedto receiveplacebo therapy (Table I). The selectedsubjects were studied for two consecutive ragweed seasons. The first year, 12 wk before the ragweed pollen season, the subjects were divided into two groups. Each of the 36 subjects with the most severe sensitivity to ragweed, assessedby their skin test results, received 10 injections with the 18 on active treatment startingat 0.2 p,g of protein (0.015 pg AgE). Each of the remaining 26 subjects with milder sensitivity receivedsix injections with the 13 on active treatment starting at 2.0 pg of protein (0.15 pg AgE). The placebo injections were administered in similar volumes to the active treatment. In the secondyear all subjectsreceived the same treatment as they had the previous year, and each subject received 10 injections. Those on active treatment started two tenfold concentrations below the last dose received the previous year. If there was no reaction to this dose, the next dose was increasedtenfold. In both yearsthe injections were administered at or near weekly intervals, and the dose was increasedby half a log concentration each week unless an adversereaction occurred. Subjects recorded all adverse reactions that occurred within 24 hr of each injection. In year 1, if the swelling or rednessof the local reaction was between 1.5 and 2 inches in diameter, the dose of the subsequent injection was not increased.If the reaction was more than 2 inchesin diameter or if systemicsymptoms occurred, the subsequentdose was reduced. In year 2, if the local reaction was 2 to 4 inches in diameter, the dose was maintained, and it was reduced if the reaction was more than 4 inches in diameter. Both years, during the second week in August, a week before the onset of ragweed pollen exposure, the subjects started completing diary cards. Twice each day they scaled the severity (0 = none, 1 = mild, 2 = moderate, and 3 = severe) and duration (1 = a few short episodes, 2 = many episodes, and 3 = continuous) of sneezing, stuffy nose, rhinorrhea, conjunctivitis, and cough. As soon as symptoms became troublesome, they were instructed to take medications in a standardizedmanner. They took 4 mg of chlorpheniramine maleate for rhinitis and conjunctivitis when it was needed up to six times per day. If drowsiness occurred, the dose was held at the maximum that was tolerable. If symptoms were still troublesome, rhinitis was treated with additional 100 p.g of beclomethasone dipro-

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TABLE

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I. Subject

J. ALLERGY CLIN. IMMUNOL. MAY 1995

characteristics

by treatment,

sensitivity

group,

and year

Year 1 Group Active

No.

Age MIF Sensitivity to RW* 0.002 p.g 0.02 f.Lg 0.2 IJG 2.0 IJG 20 M 200 IJS Sensitivity to PEG* 20 I# 200 I-% 2ooo lJ% Severity of hay fevert Severe Moderate/severe Moderate Moderate/light Light Associated asthma Skin sensitivity to Cladosporium (Hormodendrum) or Aitenaria

Vear 2

I

Group

Placebo

Active

II Placebo

Active

Placebo

18 37 10/8

18 40 10/8

13 40 815

13 45 81.5

24 38 14110

25 44 13112

0 3

0 3

0 0

0 0

0 4

0 4

11

11

0

0

14

15

4

4 0 0

0 12 1

5 0 1

3

0 0

0 12 1

6 9 3

6 9 3

0 0 13

0 1 12

5 15 4

8 13 4

2 6 6 4 0 5

3 4 9

0 3 3

0 3

1

5

1 3

2 4

0 2 3 5 3 4

5 4 5

3 3 7 6 4 4

4

6

1

4

5

9

11

1

1

RW = ragweed. *The minimum dose eliciting a 3 mm wheal. TAssessed from the recorded symptoms and medication requirements the previous year.

pionate nasal spray when it was needed up to four times a day, and conjunctivitis was treated with naphazoline HClantazoline phosphate eye drops, one to two drops when drops were needed up to four times a day, and medrysone eye drops, one drop one to four times a day. Symptoms of asthmawere treated with inhaled 200 pg of salbutamol when it was needed up to four times a day. All use of medications was recorded daily in the diary. Both years the nurses administering the injections were the only investigators aware of who was receiving active treatment and in what dose and who was receiving placebo treatment. They did not participate in the assessmentof adversereactions or monitor the self-assessmentby the patient of symptoms and medication required during the ragweed season. This was done by another investigator who was blinded. Rigorous efforts were made to keep the subjects blinded as to which treatment they were receiving. They were not told that they would receive the same treatment the second year. Blood was drawn before and after the course of injections and at the end of the ragweed pollen seasonfor measurement of IgE and IgG antibodies. Ragweed specificIgE was mea-

sured by RAST,’ and IgG was measured by a technique based on the adsorption of IgG to a Sepharoseprotein A complex followed by the addition of test serum and then ‘ZSI-labeledragweed.4 Blood and urine were sent to the laboratory at these times for full screening. In addition blood sampleswere taken after the third and sixth injections for liver enzyme analysis. Daily mean ragweed pollen counts (number per cubic meter of air) were measured throughout the pollen seasons by useof the Hirst (Burkard Manufacturing Co., Ltd., Rickmansworth, Hertfordshire, England) volumetric sporetrap.’ The trap was located on the roof of the Regional Laboratory in Hamilton, Ontario, at a height of 6m above the ground. Analysis Symptom scoreswere expressedas a daily mean for each of the 6 wk of the pollen season (year 1, August 8 to September 18; year 2, August 9 to September 19). Medicationswere expressedas the mean daily tablet requirement for chlorpheniramine maleate and daily number of applicationsfor nasal spray and eye drops for each of the 6-week periods. Within each year the symptom scoresand medi-

VOLUME 75 NUMBER 5

TABLE

II. Percentage

of injections

producing Year

Reaction (inches) 0 to 1.5*

1.5 to 2 >2

Group (“/.I

67.05 15.34 17.61

I

local adverse

Ragweed

pollen

extract

reactions

by sensitivity

in rhinoconjunctivitis

group

581

and year

1 Group I%.)

II

62.82 17.95 19.23

Reaction (inches)

Year 2 (%I

0 to 2* 2 to 4 >4

68.33 20.42 11.25

*Maximum diameter of local redness and swelling recorded within 24 hr of each injection.

cation requirements for the actively treated and placebotreated subjectswere compared by use of unpaired t tests. Since ragweed pollen counts vary from year to year, it is not possibleto make direct comparison between years. However, by comparing the within-subject changes between the 2 yr in the actively treated and placebo-treated groups, it was possible to determine whether there had been a further improvement in the PEG-modified ragweed-treated subjects. This was examined by use of chi-squared analysis. Changes in antiragweed IgE and IgG antibody levels were examined by use of unpaired t tests. Although the same technique was used to assaythe IgG both years, the analysis was carried out at two centers. Different reference sera and different methods of expressingthe resultswere used; therefore, direct comparison between the 2 yr could not be made. Linear regressionanalysis was used to relate the total dose of PEG-modified ragweed receivedto immunologic changes and the occurrence of adverse reactions to preinjection information. Differences were considered significant at p < 0.05 (two tailed).

RESULTS Sixty-two subjects started the study, and all but two finished the course of injections the first year. One subject in the placebo-treated group was excluded, after one injection, as a result of an acute episode of tachybrady arrhythmia of unknown cause. The second subject, on active treatment in group 1, received only six injections as she was unable, for personal reasons, to attend the clinic for the final four injections. She completed her diary during the ragweed season and was included in the efficacy analysis. Two subjects, one on active treatment and the other on placebo treatment, moved from the area after the course of injections and did not complete diary cards. Forty-nine of the original subjects volunteered to participate the second year. Six of the remaining subjects had moved from the area, three had other illnesses, three did not like injections, and one was pregnant. There were 25 of the 49 subjects in the placebo-treated group and 24 in the active-treatment group. All completed the full course of injections and were monitored during the ragweed season. In year 1 the mean total dose of PEG-modified

ragweed received by group 1 was 385 kg (28.9 pg AgE), range 3.02 to 218.62 pg (0.23 to 16.4 pg AgE), and received by group 2 was 218 p.g (16.4 kg AgE), range 6.00 to 888.0 Fg (0.45 to 66.6 pg AgE). In year 2, when all subjects received 10 injections, the mean total dose was 1829 kg (137.1 pg AgE), range 10.26 to 12,066 p,g (0.77 to 905.0 Fg AgE). The frequency of local redness and swelling occurring within 24 hr of injections is illustrated in Table II. In year 1 systemic symptoms occurred after 2.3% of injections in group 1 and after 3.8% in group 2. These were generally mild and included hives, rhinoconjunctivitis, drowsiness, nausea, aching body, and depression. Two subjects required 10 mg of hydroxyzine HCl for hives, one was administered 0.3 ml of adrenalin for hives, and one was administered 50 mg of dimethydrinate for nausea. There was no difference in the frequency of occurrence of adverse reactions between the two treatment groups. The second year systemic symptoms occurred after 5.8% of injections and were the same type as the previous year. Three subjects required treatment. One had severe total body hives and required 0.3 ml of adrenalin three times, 250 mg of hydrocortisone sodium succinate, 10 mg of hydroxyzine HCL four times a day, and 30 mg of prednisone per day for 3 days. The other two required 4 mg of chlorpheniramine maleate for rhinitis. The rate of systemic reactions per injection was 0.04. In both years the severity of adverse reactions and doses at which they occurred did not demonstrate any relationship to initial skin sensitivity to ragweed (p = 0.32), PEG-modified ragweed (p = O-59), or to the initial IgE antibody level (p = 0.45). At the beginning of the first year, the IgE and IgG antibody levels did not differ between the actively treated and placebo-treated subjects both in group 1 (p = 0.83 and p = 0.32, respectively) and in group 2 (p = 0.28 andp = 0.10, respectively [Table III]). Both years injections of PEG-modified ragweed stimulated an increase in IgE, but when the changes were compared with changes in the placebo-treated group, this only reached significance in group 1 the first year

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Ill. IgG and IgE levels

by treatment,

sensitivity

group,

and year

W Year Group

PEG-modified ragweed Preinjection Postinjection Post-RW season Placebo Preinjection Postinjection Post-RW season

bE

l*

Year

1

Group

2

Year

2*

Group

1

1

Group

2

Year

2

55.5 A 59.2 184.4 + 232.0 257.6 k 356.9

19.6 -t- 15.1 88.2 L 113.0 104.2 k 137.0

0.283 k 0.513 1.766 -+ 2.503 1.169 2 1.065

39.6 ? 42.0 48.7 2 48.4 55.9 +- 50.3

21.7 t 27.5 27.8 2 38.1 41.6 t 54.3

26.9 k 33.1 34.0 L 42.0 36.2 5 44.8

38.9 +- 29.1 38.4 k 29.7 47.7 k 38.1

59.1 k 71.6 52.2 k 55.5 64.5 k 73.9

0.066 -+ 0.101 0.076 k 0.114 0.086 t 0.130

36.7 Ifr 31.4 32.7 k 25.4 68.2 -+ 86.0

12.2 5 12.0 10.7 ? 10.8 21.6 k 24.7

27.8 t 30.2 27.9 + 38.2 35.7 k 40.0

RW = ragweed; means f SD. *IgG was measured at separate centers in years that the 2 yr cannot be compared directly.

TABLE IV. Change in regweed ragweed pollen season

specific

1 and 2. Different

reference

IgE and IgG levels

PEG-modified

ragweed

sera and different

after injection

methods

of expressing

treatment

and the

Placebo

the results

mean

Difference

I@ Preinjection-postinjection Year 1 Group 1 Group 2 Year 2 Postinjection-post-RW season Year 1 Group 1 Group 2 Year 2

9.1 -t 16.1 6.0 k 16.6 7.1 e 26.5

-4.0 2 11.8 - 1.5 k 4.0 0.2 k 27.4

p = 0.014* p = 0.137 p = 0.37

7.2 k 15.1 13.8 + 31.0 2.3 e 12.4

35.5 t 70.0 10.9 k 16.1 7.7 t 31.8

p = 0.159 p = 0.770 p = 0.438

128.9 +- 226.9 68.6 k 110.5 1.48 k 2.46

-0.5 r 11.2 -6.9 k 27.3 0.01 * 0.06

p = 0.032* p = 0.032* p = 0.008*

73.2 + 235.7 16.0 -r- 31.5 -0.60 k 1.77

9.3 k 14.9 12.3 k 34.6 0.01 2 0.05

p = 0.281 p = 0.787 p = 0.106

W Preinjection-postinjection Year 1 Group 1 Group 2 Year 2 Postinjection-post-RW season Year 1 Group 1 Group 2 Year 2 RW = ragweed; means 2 SD. *Statistical significance at p < 0.05.

(p = 0.014) (Table IV). Injections of PEG-modified ragweed also stimulated increases in IgG; when the increases were compared with placebo-treated group levels, the increase reached significance both in year 1 (group 1 p = 0.032, group 2p = 0.032) and also in year 2 (p = 0.008). Both years the increases in IgG were linearly related to the total dose of PEGmodified ragweed injected (p < 0.0001). No such

relationship was demonstrated for IgE. At the beginning of the second year, the IgG antibody levels in the subjects previously treated with PEG-modified ragweed were still higher than the levels in the placebo-treated group (p = 0.043), but there was no difference in IgE levels (p = 0.49). During the ragweed pollen season changes in IgG and IgE antibody levels in the actively treated subjects did not reach

Ragweed

VOLUME 75 NUMBER 5

YEAR

I GROUP

I

YEAR

I

GROUP

pollen

extract

in rhinoconjunctivitis

583

I,

i : 2

COUGH

0.5 r

FIG. 1. Mean daily symptom (closed circles) and placebo

FIG. 2. Mean circles) and

daily medication placebo (open

0.5

r

scores for each (open circles).

requirements circles).

COUGH 0.5

of the

for each

significance either year. However, in the two placebotreated groups the first year, the increases in IgE antibody reached significance (p = 0.04 andp = 0.02, respectively), but in the second year the changes were not significant. Symptom scores recorded during the ragweed pollen season are illustrated in Fig. 1. In year 1 the subjects in group 1 receiving PEG-modified ragweed consistently recorded less severity and duration of all symptoms; however, because of the large betweensubject variance, these overall changes did not reach statistical significance. The largest differences between the actively treated and placebo-treated subjects

six study

r

weeks.

COUGH

PEG-modified

of the 6 wk. PEG-modified

ragweed

ragweed

(closed

occurred during the middle weeks of the study when the ragweed pollen counts were at their highest. Therefore, each week was examined individually. The severity and duration of nose and eye symptoms were significantly lower in the active-treatment group in week 3 (p = 0.046), and the severity of eye and nose symptoms (p = 0.042) and the duration of all symptoms (p = 0.050) were lower in week 4. In group 2 no differences were observed between the actively treated and placebo-treated subjects both overall and for individual weeks. Medications used during the ragweed pollen season are illustrated in Fig. 2. In year 1 the group 1 subjects

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J. ALLERGY CLIN. IMMUNOL. MAY 1985

No drug-related changes were observed in the blood and urine analyses.

AUBUST

FIG. 3. Daily ragweed Canada; 1982 (closed

pollen circles)

DATE

SEPTEMBER

counts in Hamilton, Ontario, and 1981 (open circles).

required less antihistamines, nasal spray, and eye drops, but this only reached overall significance for the eye drops (p = 0.019). When individual weeks were examined for the antihistamines and nasal spray, the differences did not reach significance. In group 2 no differences in medications were observed between the PEG-modified ragweed-treated and placebo-treated subjects. The second year, the PEG-modified ragweedtreated subjects recorded significantly less overall symptom severity (p = 0.007) and duration (p = 0.047). The mean severity and duration of sneezing was less (p = 0.004) as were the mean severity and duration of eye symptoms (p = 0.006) and the mean severity of nose symptoms (p = 0.030). Each week from weeks 2 to 6, all nose and eye symptoms demonstrated significant improvement except week 5 in which only the decreased severity of sneezing was significant. Although the use of medications was less, it only reached significance for nose spray and eye drops during week 6 (p = 0.014 and p = 0.025, respectively). The number of days when the pollen count was more than 50 grains per cubic meter of air was higher in year 2 than in year 1 (p = 0.015) (Fig. 3). Within subject analysis between the 2 yr demonstrated that there was a further improvement in symptoms in the PEG-modified ragweed-treated subjects the second year (p = 0.043), but no further changes were observed in the medication requirements (p > 0.05).

DISCUSSION The results of this study indicate that PEG-modified ragweed, administered in sufficiently high doses, is effective in reducing symptoms of ragweed polleninduced rhinoconjunctivitis. In this study the side effects of the injections were generally mild and required little treatment. The injections induced significant increases in ragweed specific IgG antibodies, but the increases in IgE antibodies were less pronounced. No toxicologic changes were observed. The increases in IgG antibodies induced by PEGmodified ragweed demonstrated the same trend as those observed after aqueous injections in man’-* and those induced by PEG-modified ragweed in mice.’ However, the changes in IgE antibodies were slightly different; they demonstrated neither the decreases demonstrated in mice’ nor the increases characteristic of aqueous6-8and modified ragweed extract9 injections in man. This would suggest that the features of PEGmodified allergen observed in experimental animals are also operative in allergic humans but to a lesser degree. The elevated IgG antibody levels at the beginning of the second year would suggest that annual courses of injections may have an additive effect. In this study improvements in symptoms were observed the second year, but, with the present study design, it could not be determined how much was due to the larger doses administered the second year and how much was due to an additive effect of treatment during 2 yr. The local and systemic side effects from the injections were generally mild, and they were well tolerated by the subjects. Only one subject required intensive treatment, and in both years it was for total body hives. The previous injection the second year had induced symptoms that the subject had mistaken for sunburn and had not reported. Ragweed specific IgE levels and skin prick test results in this subject were not different from the results in other subjects. The number of systemic symptoms per injection was similar to those reported for glutaraldehyde-modified-ragweed pollen-tyrosine adsorbate, alum precipitate,” and aqueous injections.” However it is recognized that the potencies and circumstances may not be comparable. The routine blood and urine analysis demonstrated no abnormal trends supporting the safety of the treatment. Although rigorous efforts were made to keep the subjects blinded as to which treatment they were receiving, some who had severe reactions guessed that they had received active treatment. No formal ques-

VOLUME 75 NUMBER 5

tion was posed; however, from general comments it was apparent that all the placebo-treated group and most of those on active treatment were still uncertain as to which group they were in even at the end of the second year. This lack of total double-blindness could have led to some bias in the self-assessment of symptom scores and medication requirements. However, in view of the very few subjects involved, it is most unlikely to account totally for the large significant differences in efficacy demonstrated between active and placebo treatment. In this study the subjects were stratified carefully according to previous ragweed pollen-induced symptoms and skin prick sensitivity to aqueous ragweed extract and PEG-modified ragweed. This care ensured comparable levels of sensitivity in the active- and placebo-treatment groups (Table I). All the same it is not possible to anticipate individual pollen exposure, subjective interpretation of symptoms, and tolerance of symptoms before requiring medications. Therefore, the between-subject variance for both symptoms and medications was large. This large variance and the small number of subjects may have accounted for the lack of significant results in group 1 the first year (Fig. 1). The fact that the results reached significance the second year, with only 25 subjects in each treatment group, emphasizes the efficacy of PEG-modified ragweed. Symptomatic benefit appeared to be greater the second year than the first. This may be attributable to higher doses of antigen the second year. An alternative consideration is that there may have been a carry-over effect from the first year. This is suggested by the higher IgG levels in the actively treated group at the beginning of year 2. In conclusion PEG-modified ragweed injections, administered in sufficiently high doses, safely reduces the severity of ragweed pollen-induced rhinoconjunctivitis. To determine whether PEG-modified ragweed elicits a reaction rate or an immunologic and clinical response that is superior to established ther-

Ragweed pollen extract in rhinoconjunctivitis

585

apies, further studies comparing PEG-modified ragweed with aqueous or other modified ragweed extracts are necessary. We thank the subjects for their participation, the Regional Laboratory in Hamilton, Ontario, for the pollen counts, Mrs. D. Thompson and her staff for making up the solutions of PEG-modified ragweed, Mrs. S. Evans for immunologic analysis, and Mrs. L. Whitely and Word Processingfor typing the manuscript. We are grateful to Schering for supplies of Chlortripolon, Glaxo Canada Ltd., for Beconase inhalers, and to Cooper Vision Ltd.. for Vasacon A eyedrops. REFERENCES 1. Lee WY, Sehon AH: Suppression of reaginic antibodies. Immunol Rev 41:200, 1978 2. Wie S, Wie CW, Lee WY, Filion LG, Sehon AH, Akerblom EB: Suppression of reaginic antibodies with modified allergens. III. Preparation of tolerogenic conjugates of common allergens with monomethoxypolyethylene glycols of different molecular weight by the mixed anhydride method. Int Arch Allergy Appl Immunol 64:84, 1981 3. Ceska M, Eriksson R, Varga JM: Radioimmunosorbent assay of allergens. J ALLERGY CLIN IMMUNOL 49: I, 1972 4. Foucard J, Johansson SGO: Immunological studies in vitro and in vivo of children with pollenosis given immunotherapy with an aqueous and a glutaraldehyde-treated tyrosine-adsorbed grass pollen extract. Clin Allergy 6:429, 1976 5. Chatterjee J, Hargreave FE: Atmospheric pollen and fungal spores in Hamilton in 1972, estimated by a Hirst automatic volumetric spore trap. Can Med Assoc J 110:659. 1974 6. Norman PS: An overview of immunotherapy: implications for the future. J ALLERGY CLIN IMMUNOL 65:87, 1980 7. Evans R, Pence H, Kaplan H, Rocklin RE: The effect of immunotherapy on humoral and cellular responses in ragweed hay fever. J Clin Invest 57:1378, 1976 8. Levy DA, Lichtenstein LM, Goldstein EO, Ishizaka K: Immunologic and cellular changes accompanying the therapy of pollen allergy. J Clin Invest 50:360, 1971 9. Cockcroft DW, Cuff MT, Tar10 SM, Dolovich J, Hargreave FE: Allergen injection therapy with glutaraldehyde-modifedragweed pollen-tyrosine adsorbate. J ALLERGY CLIN IMMUNOL 60156, 1977 10. Norman PS, Winkenwerder WL, Lichtenstein LM: Trials of alum-precipitated pollen extracts in the treatment of hay fever. J ALLERGY CLIN IMMUNOL .50:3 1, 1972