Replacement therapy for alpha-1-protease inhibitor deficiency in PiZ subjects with chronic obstructive lung disease

Replacement Therapy for Alpha-1-Protease Inhibitor Deficiency in Subjects with Chronic Obstructive Lung Disease

E.W. SCHMIDT, M.D. B. RASCHE, Ph.D. W.T. ULMER, M.D. Bochum, Federal Repubfic of Germany

NIKOLAUS KONIETZKO, M.D. MICHAEL BECKER, M.D. Essen, Federal Republic of Germany

J.P. FALLISE, M.D. Wuppertal, Federal Republic of Germany

J. LORENZ, M.D. R. FERLINZ, M.D. Mainz, Federal Republic of Germany

From the BergmannsheilHospital,4630 Bochum, Federal Republicof Germany;the Ruhrlandklinik, 4300 Essen 16, Federal Republicof Germany;the Bayer Pharmaceutical Research Center, 5600 Wuppertai, Federal Republic of Germany;and the Deparlment of Pneumology of the Universityof Mainz, 6500 Mainz, FederalRepublicof Germany. Requests for reprints should be addressed to Dr. N. Konietzko, the Ruhrlandklinik,4300 Essen 16, Federal Republicof Germany.

In a six-month multicenter feasibility and safety study, 20 patients, who all had a congenital deficiency of alpha-l-protease inhibitor (AIPI) of the PjZ phenotype accompanied by a chronic obstructive lung disease, were treated with human-plasma-derived AIPI. A weekly dose of 60 mg/kg, administered intravenously, was shown to be sufficient to "maintain patient serum levels above the threshold limit of 35 percent, the serum level of healthy persons of the MZ phenotype. This is supposed to be the minimal effective level for protection against the elastolytic attack of the lung and, therefore, satisfies one of the most important criteria of feasibility of long-term replacement therapy. The global concentration in serum or bronchiolar lavage fluid AIPI including active and inactivated AIPI was measured immunologically by rate nephelometry and radial immunodiffusion. The functional activity of A1PI, expressed as free inhibitor activity against trypsin and leukocyte elastase, confirmed that the infused AIPI remained mostly in its active form in the circulation. Reported adverse reactions were moderate and did not require alteration to the schedule of the infusions and/or the dose and rate of administration. Antibodies to AIPI as measured by the Ouchterlony method did not develop. Laboratory and physical signs of possible hepatitis virus contamination were not observed. The longterm replacement therapy, therefore, appears to be safe. Alpha-l-protease inhibitor (A1 PI) is a glycoprotein with an overall molecular weight of 51,000 daltons [1]. The mean concentration of A1PI in the serum of healthy adults of the P~M phenotype is 260 mg/dl, if measured by radial immunodiffusion, with upper and lower limits between 200 and 338 mg/dl for the healthy population irrespective of the genetic form [2]. In a population of 120 apparently healthy male and female adults from northern California, the mean value of A1PI as measured by rate nepheIometry appears to be 150 mg/dl, ranging from 85 to 213 mg/dl [3]. Epidemiologic studies had established that persons with the phenotypes of the congenital deficiencies in A1 PI associated with destructive lung diseases (i.e., "null," ZZ, and SZ) had levels 35 percent of normal or less [4]. In contrast, persons with those phenotypes that are not at higher risk had levels above 35 percent of normal. Thus, the 35 percent level seemed to be the threshold above which enough AIPI could diffuse through the alveolar structures and prevent them from elastolytic attack. The 35 percent level of A1PI will be approximately 50 mg/dl if measured by rate nephelometry and 90 mg/dl if measured by radial immunodiffusion. Since a substitution therapy with A1 PI should slow down the destruction of the

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lung by displacing the protease-antiprotease balance in favor of the antiprotease, it was decided to assess whether a long-term substitution with A1 PI would be feasible and safe. METHODS Study Population. The first group of patients (seven men and two women; age range, 35 to 50 years; median, 45 years) was treated at the Ruhrlandklinik in Essen. The second group of patients (six men and two women; age range, 37 to 65 years; median, 47 years) was treated at the Bergmannsheil Hospital in Bochum. The third group included only three patients (two men aged 51 and 62 years, one woman aged 44 years) and was treated in the Pulmonary Department of the University of Mainz. All patients had a congenital deficiency in A1PI of the P~Z phenotype with chronic obstructive lung disease. Demographic data, including sex, age, height~ weight, smoking status (i.e., smoker or exsmoker, and the number of years smoking is indicated), date of the first respiratory difficulty, and date of diagnosis of a congenital deficiency of A1PI of the PbZ phenotype, are • shown in Table I. Four parameters of the lung function (inspiratory vital capacity, residual volume, forced expiratory volume in one second, and airway resistance) at the entry are listed in Table II. All patients were completely informed of the aims, possible advantages, and risks of this study and gave their written consent. Preparation of A1PI. A1PI was supplied by Cutter Biologi-

TABLE I

cal of Miles Inc., Berkeley, California. It is prepared from pooled human plasma of normal donors by a modification of the cold ethanol fractionation method of Cohn. A1PI is extracted from the same plasma fraction as albumin, which, to our knowledge, has never been reported to be associated with any case of hepatitis virus transmission. Each unit of plasma used in the manufacturing process had been found nonreactive for hepatitis B surface antigen. In addition, during the manufacturing process A1PI is heat treated in solution at 60°C for not less than 10 hours in the presence of stabilizers prior to lyophilization. Quantification of A1PI. The quantification of global A1PI was performed using two immunologic methods: rate nepheIometry and radial immunodiffusion. Standards and reagents were supplied by Beckman Instruments Inc., Brea, California, for rate nephelometry, and Behringwerke, Marburg, Federal Republic of Germany, for radial immunodiffusion. Instructions of both manufacturers were strictly observed. The quantification of functional A1 PI was done measuring the free inhibitor activity against trypsin and human leukocyte elastase according to a method described elsewhere [5] and modified by Rasche. Briefly, 0.2 ml of a 1:100 diluted sample was mixed to 0.2 ml trypsin solution (reconstituted as 22.4 units/100 ml, trypsin provided by Merck, Darmstadt, Federal Republic of Germany) and 1.6 ml Tris buffer (pH 7.8, 0.2 M). After an incubation time of five minutes at 30°C, 1 ml of the substrate Bapna (N-banzoyl-l-arginin-4-nitroanilid, 1 mg in 2 ml, Merck, Darmstadt, Federal Republic of Germany) was added to the mixture. The extinction was read spectrophotometrically at 405 nm each minute for five minutes. The concentra-

Individual Data of Each Patient Entered into the Study Sex

Age

Height (cm)

Weight ( k g )

SmokingStatus*

Year of First Complaints

Year of Diagnosis of P~Z Deficiency

Group 1 1 2 3 4 5 6 7 8 9

M M F M M F M M M

46 35 45 37 45 44 45 50 45

170 190 165 171 178 173 181 167 169

~64 56 67 70 68 63 74 61 61

20 12 No 10 10 15 No 20 15

1978 1982 1972 1975 1980 1984 1981 1972 1979

1980 1984 1980 1985 1984 1984 1984 1982 1984

Group 2 1 2 3 4 5 6 7 8

M F M M M M F M

51 37 40 51 53 43 65 42

179 165 177 178 180 174 178 195

80 69 75 70 85 62 86 87

25 12 No 30 30 25 No 20

1979 1980 1975 1955 1983 1980 1960 1978

1981 1984 1978 1974 1984 1980 1981 1980

Group 3 1 2 3

M M F

51 62 44

174 191 172

77 90 60

30 20 No

1975 1975 1982

1984 1982 1984

Patient Number

M -- male; F = female. "Smoking status: No = no smoker; if ex-smoker, number of years of smoking is given.

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T A B L E II

Parameters of Lung Function at the Entry

Patienl Number

IVC (liters)

RV (liters)

FEV1 (liters/second)

Raw (water/liter/second)

Group 1 1 2 3 4 5 6 7 8 9

4,600 2,700 1,700 4,200 2,900 2,100 4,000 2,700 2,600

3,300 6,200 3,700 4,900 7,400 6,200 4,500 7,100 5,900

1,700 900 900 1,000 800 1,000 2,000 1,000 900

3.0 5.9 5.5 3.8 6.3 4.5 2.4 5.5 5.9

Group 2 1 2 3 4 5 6 7 8

2,200 ND ND 4,200 2,200 2,800 3,300 ND

6,300 ND ND 6,300 6,900 7,200 6,100 ND

850 ND ND 1,200 900 1,000 1,100 ND

4.9 6.0 10.6 4.0 5.8 8.0 3.6 5.5

Group 3 1 2 3

3,600 3,400 3,200

4,700 10,900 4,300

1,100 900 1,350

ND ND ND

IVC = inspiratory vital capacity; RV = residual volume; FEV1 = forced expiratory volume in one second; Raw

tion in mU/ml was then given by the formula: extinction of the trypsin solution alone minus extinction of the serum multiplied by the conversion factor. The conversion factor is calculated as five multiplied by the dilution of serum -~ divided by 6.6. The quantification of the free inhibitor activity against human leukocyte elastase was performed as follows: 0.1 ml of a 1:100 diluted serum sample was mixed to 0.1 ml leukocyte elastase (kindly provided by Professor Hochstrasser, Munich, Federal Republic of Germany) dissolved in Hepes buffer (pH 8.0, 0.1 M) in a 1 mg leukocyte elastase:l ml Hepes buffer proportion; 1.8 ml Hepes buffer (pH 8.0, 0.1 M) was added to this mixture. After an incubation time of five minutes at 30°C, 0.1 ml substrate Sapna (Suc-L-Ala-L-Ala-L-Val-p-nitroanilid, Bachem Chemie, Switzerland) was added and the extinction was measured spectrophotometrically at 405 nm each minute during five minutes. The concentration in mU/ml was then given by the formula: extinction of the leukocyte elastase solution alone minus extinction of the serum multiplied by the conversion factor. The conversion factor is calculated by five multiplied by the dilution of serum -~ divided by 0.0962. Study Design. In this noncontrolled, observational study performed in three German centers, the feasibility and safety of a long-term replacement therapy for A1 PI was evaluated. The capacity of a single weekly infusion to maintain the A1 PI level above 35 percent of the normal value of persons with the MM phenotype was adopted as the main criterion of efficacy. In the evaluation of safety, routine tests of hematologic, clotting, renal, hepatic, and electrolytic functions were performed. Additionally, lung function tests (inspiratory capacity, forced expiratory volume in one second, residual volume, and airway resistance) were undertaken if the health condition of

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=

airway resistance; ND = not done.

the patients permitted them. Time points of all investigations are given in Table III. Based upon a previous experience [6], an initial dosage of 60 mg/kg was administered each week with the possibility to augment this if the 35 percent level had not been reached after one month. Patients were hospitalized for the first week of the trial. They attended their respective investigational centers for all laboratory and physical examinations. At dates where only an infusion was scheduled, however, this was sometimes administered by the family physician who had been previously perfectly informed on the aims of the study. The statistical analysis of this study was carried out by an independent institution (Institut f~r Numerische Statistik, Cologne, Federal Republic of Germany), which analyzed only the data from the group 1 and group 2 patients, since group 3, consisting of three patients only, was considered too small and could have caused inconsistent influences on the data. In those last patients, only safety data will be reported. RESULTS

Feasibility (global AIPI). Thirty minutes after the end of the first infusion of 60 mg/kg A1PI, the concentration of this compound in patients of group 1 had risen from 18.1 __ 3.2 mg/dl (mean basal level before therapy) to 221.2 -+ 35.4 mg/dl.and in patients of group 2 from 28 _+ 11 mg/dl to 328 +_ 51 mg/dl. After this peak, the concentration decreased steadily to 48.3 -+ 5.9 mg/dl (for group 1) and 96.0 -- 17 mg/dl (for group 2). Those values were determined just before the second infusion seven days later. Shortly before the last scheduled infusion six

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T A B L E III

Time Points of Laboratory and Physical Investigations Day 1 Belore First Infusion

Investigation Blood status* Blood coagulationt Clinical chemistry t A1PI Hepatitis serology § Lung function t~ Chest x ray and ECG

4 24 30 Minutes Hours Hours

x x x x

x x

x x

x

x x

Day 2

Day 4

Day 7

x

x x x x

x

x

x x x x

x

x

x

x

x

Month Month Month Month Month Month 1 2 3 4 5 6 x x x x x

x x x x x

x x x x x x

x x x x x

x x x x x

x x x x x x x

ECG = electrocardiographic examination. *Blood status means erythrocyte sedimentation rate, hemoglobin, hematocrit, erythrocytes, leukocytes, thrombocytes, differential blood count. tBIood coagulation includes prothrombin time, partial thromboplastin time, plasma thrombin time, fibrin split products (if plasma thrombin time prolonged), fibrinogen. tClinical chemistry includes sodium, chlorine, potassium, glucose, urea, uric acid, creatinine, total proteins, immunoglobulins G, M, and A, bilirubin, lactate dehydrogenase, serum glutamic pyruvic transaminase, serum glutamic oxaloacetic transaminase, alkaline phosphatase, cholesterol, triglycerides, cbmplement C3. §Hepatitis serology includes hepatitis B surface antigen, anti-hepatitis B surface antigen. ~lLung function includes inspiratory capacity, residual volume, forced expiratory volume in one second, and airway resistance.

1

iiit

:it:t B

30

4h

24h

2d

4d

7d

lm

2m

3m

months later, the values for groups 1 and 2 were 86.9 26.3 and 101.0 - 23 mg/dl, respectively. At six months, all patients but one were above the 35 percent level of normal persons as shown in F i g u r e s 1 and 2. F e a s i b i l i t y (functional A1PI). The free inhibitor activities were measured at the same time points as above. Thirty minutes after the end of the first infusion of A1PI, the free inhibitor activity against leukocyte elastase in patients of group 1 had risen from 1 , 5 2 5 - 275 rnU/ml (mean basal level before therapy) to 11,856 +-2,518 mU/ml and in patients of group 2 from 1,543 +- 554 mU/ml to 10,647 _ 1,050 mU/ml. After this peak, the free inhibi-

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4m

5m

bm

Figure 1. Laser nephelometry. Global A 7PI in the nine patients of group 1. Concentration in plasma (mg/dl) measured shortly before the first infusion (B) and then at times indicated (' = minutes, h = hours, d = days, m = months) after the first infusion.

tor activity against leukocyte elastase decreased steadily to 3,374 +_ 770 mU/ml (for group 1) and 2,977 +-955 mU/ml (for group 2). Those values were determined just before the infusion seven days later (Figure 3). The free inhibitor activity against trypsin followed a Similar pattern (Figure 4). Since the free inhibitor activities increased to a level twice the basal value, it was concluded that the compounds remained, at least partly, in their active form after infusion. Safety. Only three events were reported during the trial. In group 2, one female patient experienced many hematomalike spots of 1 to 2 cm diameter in her arms and legs.

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ON ALPHA-1-ANTITRYPSIN

DEFICIENCY--SCHMIDT

ET AL

4°t "°1

\

\

I I

r ,oo,//_ ................... Figure2. Radialimmunodiffusion. G,obal AIPI in the eight patients of group 2. Same explanation as for Figure 1. ' = minutes, h = hours, d = days, m = months,

50F

;

0

. 30

]]

. 4h

14,000~

. . 24h 2d

.

"~

. 4d

. 7d

. lm

2m

3m

4m

5m

5m J

x

12,000J 10, 0008. 000b, 000-

~

4,000Figure 3. Concentration of free inhibitor against leukocyte elastase (mU/ml) in patients of group 1 (x) and group 2 (+). Mean values with SD. ' = minutes, h = hours, d = days.

2,0000

i

B

This phenomenon occurred once after the third infusion, was not accompanied by an alteration of the coagulation parameters (prothrombin time, partial thromboplastin time, plasma thrombin time, fibrinogen), and disappeared spontaneously. This patient has now received the compound for more than 20 months (September 1985 through May 1987) without any further event of this nature. In group 3, one patient experienced a loss of weight from 90 to 86 kg two months after the first infusion. He remained at this weight until the end of the study. Another patient in group 3 who already had, prior to entry into the study, a mild eosinophilia of 6 percent before the first infusion, showed an increase of this value to 13 and 14 percent one

June 24, 1988

i

3;

i

4h

i

24h

i

2d

i

4d

7'd

and two months later, respectively. Afterwards, the percentage of eosinophils returned to the moderately elevated value observed at the start. Signs of allergy were not reported. Otherwise, variations in hematology and clinical chemistry were not of clinical significance. None of those adverse reactions had any influence on the schedule of administration of A1PI. It was not necessary to reduce the dose or the rate of administration. In the evaluation of safety, special attention was devoted to the liver status and hepatitis serology. Elevated serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, or alkaline phosphatase occurred sometimes individually, but never reached a level

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SYMPOSIUM ON ALPHA-1-ANTITRYPSIN DEFICIENCY--SCHMIDT ET AL

1.bO0]

1.4001

oOoOoI ' 800-4 bOO-

I

400÷

2007

x

0 B

'

TABLE IV

Patient Number

'

'

2;h

'

'

Group 2 1 2 3 4 5 •6 7 8

Vacc + Vacc + Vacc + Vacc + 0 Vacc + 0 Vacc +

IgG IgG IgG IgG IgG IgG

Protection Assured Until

Month 3 Month 3 Month 3 End of 6 months Month 5 End of 6 months End of 6 months Month 3

Vacc = vaccination. "Patients received vaccination against hepatitis B virus and hyperimmune globulins against hepatitis B virus if they were anti-hepatitis B surface antigen negative on entry. Patient 5 had been vaccinated one year before. Patient 7 was seropositive at the entry. All patients but three became anti-hepatitis B surface antigen negative during this trial and, therefore, partially or totally unprotected against hepatitis B virus, Patients of group 3 were not at all treated prophylactically with active or passive immunization against hepatitis B virus.

of more than 2.5 times the upper limit (this limit being considered as the minimal value for a possible transfusional non-A, non-B hepatitis--if it occurs on two or more consecutive occasions two weeks apart--by the International Committee on Thrombosis and Haemostasis). Clinical symptoms suggestive of hepatitis were not observed. Of eight patients in group 2, only three remained serologically protected against hepatitis B virus until the end of the sixth month (Table IV). The three patients in group 3 who entered this study much later did not receive any immunization at all since it had been .decided to abandon this

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;d

prophylactic measure. Those who were not hepatitis carriers did not convert to a hepatitis B surface antigen positive status.

Serologic Protection against Hepatitis B Virus in Function of Time* Prophylaxis before First Infusion

'

Figure 4. Concentration of free inhibitor against trypsin (mU/ml) in patients of group 1 ( x ) and group 2 (+). Moan values with SD. ' = minutes, h = hours, d = days.

COMMENTS

This was a feasibility and safety study performed in 17 adult patients with a congenital deficiency in A1PI (of the P~Z phenotype) accompanied by a chronic obstructive lung disease. Safety alone was investigated individually in three further patients. The conclusions of this study are fourfold: • It is possible to maintain a level above 35 percent (the level in healthy people of the P~M phenotype) with a single weekly infusion of A1PI concentrate of about 60 mg/kg. • The compound remains, at least partly, in its active form after it is infused. • This compound, heat treated in solution, appears to be safe with respect to hepatitis in all 20 patients. • Side effects were minimal and never interfered with the scheduled treatment. They consisted of hematoma-like spots that disappeared spontaneously without trace and did not recur later; a further but temporary elevation of the eosinophils in a patient already eosinophilic on enrollment; and a weight reduction from 90 to 86 kg in an obese patient. Following this six-month study, all 17 patients of groups 1 and 2 decided to continue their substitutive treatment as part of an ongoing study. Certain questions remain open that are worth recording: • How can the collective or individual efficacy of a substitution therapy be evaluated? Two major problems must be taken into account if attempts to answer this question

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are envisaged. A double-blind controlled study would need approximately 500 patients studied over three years [7]. The effort involved and expense of such an undertaking would be disproportionate to the value obtained, considering the small number of P~Z patients with chronic obstructive lung disease in existence. The individual efficacy of a substitution therapy could be appreciated if there was a reliable marker for lung degradation by granulocyte elastase. Such a marker is being investigated in some laboratories [8] but is not currently available. • The efficacy of long-term substitution being accepted, what is the optimal time for its initiation? Considering that the spontaneous evolution of A1PI-deficient patients is extremely variable [7], it seems reasonable to initiate a substitutive treatment in people still having 65

percent of their forced expiratory volume in one second capacity but having lost 40 ml of their total lung capacity in the preceding 12 months [9]. • Finally, consideration should be given to whether the recommended dosage, deduced from epidemiologic studies, is adequate in all cases. It is not excluded that some patients with a considerable infiltration of leukocytes in the lungs could require a higher dose of A1 PI. These considerations notwithstanding, the results of the present study combined with the well-documented hypothesis regarding the role of protease-antiprotease imbalance in the pathogenesis of emphysema in defective P~Z patients [10] suggest that long-term substitution therapy has a high probability of therapeutic success for all PiZ patients presenting with rapid degradation of their respiratory capacities.

REFERENCES

1. CarrelRW, Jeppsson J-O, Laurell CB, et al: Structure and variation of human alpha-l-antitrypsin. Nature 1982; 298: 329334. 2. Packaginginsert of the test "NOR-Partigen" (276) of the Behringwerke AG, Marburg, West Germany (issue of June 1985). 3. Packaginginsert of the Beckman alpha-l-antitrypsin (AAT) reagent to be used in conjunction with Beckman's ICS Calibrator II for the quantificationof alpha-l-antitrypsin in human serum by rate nephelometry.Beckman Instruments, Inc., Brea, California 92621 (issue of 1983). 4. GadekJE, HunninghakeGA, FianncemainRL, et al: Evaluation of the protease-antiproteasetheory of human destructivelung disease. Bull Eur Physiopathol Respir 1980; 16: 27. 5. Fritz H, et al: Proteasen-inhibitoren. In: Bergmeyer HU, ed. Methoden der enzymatischenAnalyse, Volume 1. Weinheim. Federal Republic of Germany: Verlag Chemie, 1970; 1021-

June 24, 1988

1038. 6. GadekJE, Crystal RG: Experiencewith replacementtherapy in the destructive lung disease associated with severe alpha-1antitrypsin deficiency. Am Rev Respir Dis 1983; 127: $45$46. 7. Burrows B: A clinical trial of efficacy of antiproteolytic therapy: can it be done? Am Rev Respir Dis 1983; 127: $42$43. 8. Kucich U, et al: Immunologic measurement of elastin-derived peptides in human serum. Am Rev Respir Dis 1983; 127: $28-$30. 9. GadekJE (ed.): Alpha-l-proteinase inhibitor symposium. Am J Med 1988; 84 (suppl 6A): 1-2. 10. GadekJE, Crystal RG: Alpha-l-antitrypsin deficiency. In: Stanbury JB, Wyngaarden JB, Fredrickson DS, Goldstein JL, Brown MS. eds. The metabolicbasis of inherited disease, 5th ed. New York: McGraw-Hill, 1983; 1450-1467.

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