A kit of human polyclonal IgG for the diagnosis of infectious processes

Journal of Radioanalytical and Nuclear Chemistry, Vol. 240, No. 2 (1999) 481-487

A kit of human polyclonal IgG for the diagnosis of infectious processes

A. Perera, 1. C. Perez, 2 M. B. Torres, 2 A. Henandez, 1 F. C. Heres, 2 T. Moreira, 3 A. Gutierrez, 3 A. Gigato, 31. Hernandez, 4 L. Alberti, 4 O. Marrero, 2 L. Martinez, 5 J. M. Sanfiz, 5 E. Sanehez, 5

J. Rodriguez, s L. O. Marrero, 6 G. Parra 4

1 Center for Clinical Research, 2 Institute for Cardiology and Cardiovascular Surgery,

3 National Center for Scientific Researches, 4 Isotopes Center,

5 National Institute for Oncology and Radiobiology, 6 Orthopedic Hospital "Frank Pais", Havana, Cuba

(Received April 16, 1998)

The aim of this work was to obtain a freeze-dried kit for direct 99mTc-labeling of human polyclonal IgG. The labeling procedure was carried out by Schwarz's method. The best yields of 99mTc-IgG were obtained by using sodium pyrophosphate decahydrate as a weak chelating agent. Performed tests showed the stability of the radiopharmaceutical up to 24 hours. Plasma clearance in rats was fitted to a biexponential curve with Tl/2u = (0.1+ 0.9) h and Ti/2f ~ = (10-Z-_3) h. The organs with higher uptake of radiopharmaceutical were lung, kidneys and blood. In a rabbit model the abscess target/background ratio was 3-6 according to time of the scintigraphic images. Thirty patients with musculoskeletal infection were studied. Twenty-one lesions were detected and confirmed by culture/biopsy.

Introduction

Infections and inflammatory processes have a high incidence in population world-wide. In Cuba, for example, 7500 persons died by infectious diseases in the 1990 to 1995 period. It represented 1.7% of all deaths. Early diagnosis and determination of extension of the infection are important for the prognosis of this disease and the selection of the therapy to employ.

Different radiopharmaceuticals have been used in Nuclear Medicine for scintigraphic detection of inflammations. 1 Polyclonal human immunoglobulin, labeled with either indium-1 l 1 or technetium-99m, is a well-established radiotracer for the detection of infectious and inflammatory areas. It has some advantages over other employed radiopharmaceuticals, such as: (a) gallium-67 citrate: 2,3 low radiation burden; (b) labeled leukocytes: 4-6 no time-consuming procedure, (c) labeled monoclonal antibodies: 7-9 no formation of human anti-mouse antibodies (HAMA) response in patients, and (d) small chemotactic peptides: 1~ no weakening of the host defence.

Nowadays, the exact mechanism of localization of labeled-IgG at sites of infection or inflammation is not yet well understood. Some explanations have been proposed: increased vascular permeability, IgG binding to the Fc-receptors of tissue leukocytes at site of infection, IgG molecule binding to the Fc-receptors of blood circulating white cells and binding directly to microorganism, depending on the amount of protein A on its surface. 1,11,12

The main aim of the present work was to obtain a freeze-dried kit for direct one-step labeling of human polyclonal non-specific IgG with technetium-99m.

Experimental

Labeling procedure

It was started from Intacglobin (50 mg/ml of human polyclonal IgG and 50mg/ml of dextrose, sterile and pyrogen free solution), supplied by Center for Sera and Hemoderivatives, Havana, Cuba. The labeling procedure was performed according to S C H W A R Z ' s m e t h o d . 13,14

Briefly, 10 mg/ml protein solution was reduced with 2- mercaptoethanol (2-ME) and purified through a PD-10 column (Pharmacia, UK). One milliliter fractions were collected and absorbance at 280 nm measured. Fractions with higher concentration of immunoglobulin were pooled. Aliquots of 1 mg were labeled with 99mTc. In order to assess the influence of different reaction parameters on labeling efficiency, a saturated factorial design 27-4 with 4 repetitions at center of the experimental matrix was performed. Table 1 shows studied factors and employed experimental conditions. Radiolabeling yield of IgG was considered as response variable. The formulation was optimized taking into account the reaction parameters, which influenced the radiochemical purity of 99mTc-IgG higher than 10%. The influence of the following weak chelating agents was also studied: sodium potassium tartrate, sodium pyrophosphate decahydrate, sodium d-glucarate and sodium l-hydroxyethylidene- 1, l-diphosphonate.

* E-mail: [email protected]

0236-5731/99/USD 1 Z O0 �9 1999 Akad~miai Kiadd, Budapest All rights reserved

Elsevier Science B. V., Amsterdam Akadgmiai Kiad6, Budapest

A. PERERA et al.: A KIT OF HUMAN POLYCLONAL IgG FOR THE DIAGNOSIS OF INFECTIOUS PROCESSES

Table 1. Studied reaction parameters

No. Parameter Higher Lower level Center of level matrix

1 Molar ratio IgG:2ME(X1) 1:1400 1 : t 000 1 : 1200 2 Reduction time (X2) 45 min 30 min 37 min 3 Concentration of dextrose (X3) 50 mg/ml 35 mg/ml 35 mg/ml 4 Volume of chelating agent solution (X4) 100/al 50 gl 75 I11 5 Concentration of chelating agent (X5) 50 gmol/ml 30 lamol/ml 40 gmol/ml 6 Concentration of SnC12.2H20 (X6) 2.5 I.tmol/ml 1.5 I.tmol/ml 2.0 lamol/ml 7 Incubation time (X7) 60 min 30 min 45 rain

Table 2. Results of 99mTc-labeling of each experimental formulation

Experimentel Percent of each obtained product po in t s 99mTc-IgG 99mTc-Tartrate 99mTc-Dextrose 99mTcO4Na 99mTcO2

1 83.9 2.0 10.4 3.7 0.0 2 85.2 8.4 5.1 0.9 0.4 3 92.9 1.1 4.3 1.3 0.4 4 82.6 2.8 13.1 1.5 0.0 5 72.4 2.7 21.1 1.6 2.2 6 73.6 3.0 22.5 0.9 0.0 7 79.4 3.5 16.5 0.0 0.6 8 57.2 19.2 22.5 0.6 0.5 9 76.2 1.3 19.1 2.5 0.9

10 84.8 4.7 5.0 0.0 5.5 11 76.6 10.8 10.3 1.7 0.6 12 72.7 7.4 18.9 0.7 0.3 13 87.3 0.0 11.3 0.0 1.4 14 88.8 5.6 5.1 0.0 0.5 15 86.6 1.2 7.3 2.9 2.0 16 91.6 0.0 6.8 0.0 1.6 01" 66.5 6.0 25.1 1.6 0.8 02* 46.3 12.9 37.8 3.0 0.0 03* 43.2 21.4 29.3 4.0 2.1 04* 49.6 17.8 30.6 1.6 0.4

* Points at center of experimental matrix.

The formulation with best 99mTc-labeling efficiency was lyophilized. The stability of the label was monitored up to 24hours in 0.1M PBS (pH 7.2-7.4), plasma, serum and L-cysteine (10 gM, 100gM, 6 4 0 g M and 64 mM). Bearing of fragments and molecular aggregates was assessed by SDS-PAGE and HPLC using a TSK- 3000 size exclusion column.

Normal animal pharmacokinetics and biodistribution

Pharmacokinetic and biodistribution studies were performed in male Wistar rats (180-200 g weight). They were administrated 2 0 g g of 99mTc-IgG (740kBq)

through tail vein. Rats were killed and specimens taken at 0, 1, 3, 6, 12, 24 hours. Blood samples were collected also at 30 minutes, 2 and 8 hours. The following organs were studied: liver, spleen, kidneys, lung, heart, muscle, large intestine, small intestine, bone (femur) and

articulation (knee). Plasma and blood clearances were fitted to a bicompartmental model.

Animal model

Five New Zealand rabbits were injected in posterior thigh with approximately 109 CFU of Staphilococcus aureus (ATCC 25923) in a volume of 100 I-tl. The same volume of sterile saline solution was injected in the counter-lateral leg as a negative control. Forty-eight hours after administration, 300 gg of IgG labeled with 74 MBq of 99mTc was injected intravenously into an ear

vein. Scintigraphic images were acquired at 0, 0.5, 1, 2, 4, 6, 10 and 24 hours p.i., by using a Sophy DS7 (Sopha Medical, Canada) gamma-camera.

Pilot clinical trial

Thirteen patients (18-54 years old) suspected of having musculoskeletal infection were studied. All

patients gave their written informed consent. One milligram of IgG labeled with 1110 MBq of 99mTc was

administrated i.v. Scintigraphic images were acquired at

482

A. PEP.ERA et al.: A KIT OF HUMAN POLYCLONAL IgG FOR THE DIAGNOSIS OF INFECTIOUS PROCESSES

4 and 24 hours using format 128x128 pixel, zoom 1 and 1000 kcount by employing a gamma-camera Sophy DS7 (Sopha Medical, Canada). A week later a set of scintigraphic images was acquired 4 hours after i.v. injection of 740 MBq of 99mTc-MDP (Amersham, UK). AI1 detected lesions were confirmed by culture/biopsy.

Results

Labeling procedure

The outcome of the experimental design is shown in Table 1. The response surface of the experimental model was:

Y = 78,4-3,75X1-0,375X2-7,75X 3-1,625X4+ +0,638X5-1,85X6-1,738X 7

It is seen that the reaction parameters, which negatively influenced more than 10% on labeling yield of the protein, were: molar ratio IgG:2-ME (X1), concentration of dextrose (X3) and concentration of stannous chloride (X6). Influence of the other factors was significantly lower. Taking into account that the concentration of dextrose has the highest impact on decreasing of radiochemical purity of 99mTc-IgG, the labeling procedure was continued from lyophilized human IgG (Center for Sera and Hemodefivatives, Havana, Cuba).

Figure 1 shows the radiochemical purity of 99mTc- IgG, for formulations 3 and 16, obtained using different weak chelating agents. It is noted that formulations containing sodium tartrate and sodium pyrophosphate decahydrate have no significant differences (p>0.05). By the other hand, formulation 3 showed better values of radiolabeling yield (p<0.05). Thus, this last kit was lyophilized and then labeled with 99mTc. Labeling efficiency for the kit with sodium tartrate decreased (88+ 2%) after the freeze-drying procedure. Nevertheless, in case of the kit with sodium pyrophosphate the yield values were very similar (97+2%). This formulation was considered the best and the study continued using it.

The stability of the radiopharmaceutical in 0.1M PBS (pH 7.2-7.4), plasma, serum and L-cysteine (10ktM, 100 ~M, 640 ktM and 64 mM) up to 24 hours is showed in Figs 2 and 3, respectively. A lack of fragments and molecular aggregates by HPLC and SDS-PAGE was observed.

Normal animal pharmacokinetics and biodistribution

Plasma clearance was fitted to a biexponential curve with T 1/2ct = (1.0-!--0.9) h and T 1/213 = (10-!-_3) h. The virtual dilution volume was (5.1_+0.1)ml and plasma clearance (0.34+0.05) ml/h. The organs with higher uptake of radiopharmaceutical were lung, kidneys and blood. Figure 4 shows the percent of injected dose for these organs.

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Sodium pyrophosphate

Sodium d- glucarate

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Sodium 1- hydroxyethyl idene-l , l -d iphosphonate

Fig. 1. Labeling efficiency of human polyclonal IgG using different weak chelating agents

483

A. PERERA et al.: A KIT OF HUMAN POLYCLONAL I g G FOR THE DIAGNOSIS OF INFECTIOUS PROCESSES

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Time, hours

Fig. 3. L-Cysteine challenge assay of 99mTc-IgG

Animal model

Scintigraphic detection of abscess in posterior thigh of a New Zealand rabbit using 99mTc-IgG is shown in Fig. 5. Target/background ratio increased from 3 to 6 according to acquisition time. Good quality diagnostic images were obtained at 4-6 hour post-injection.

Pilot clinical trial

Thirteen patients suspected of having musculoskeletal infection were studied. A total of 21 lesions were identified by 99mTc-IgG scintigraphic

imaging and 20 by 99mTc-MDP scan. All detected lesions were confirmed by culture/biopsy. The sensitivity of the 99mTc-IgG scintigraphy was 100%. No adverse

reactions were observed. A comparison between 99mTc- IgG and 99mTc-MDP images of the patient with osteomyelitis of the astragalous is shown in Fig. 6.

D i s c u s s i o n

Technetium-99m labeled human polyclonal IgG has been widely employed for diagnosis of infections and inflammatory disease. 15-18

Under conditions employed in the present study, the empirical mathematical model showed that initially used concentrations of dextrose negatively influenced the labeling efficiency of the IgG. It is obvious because molar excesses were 1:600 and 1:800, respectively. Thus, when dextrose was removed from the formulation, satisfactory yields were achieved. Then, no further optimization was necessary. The variability of the radiochemical purity of 99mTc-labeled antibodies, when different weak chelating agents have been employed was reported by other authors. 14,19 In this case sodium pyrophosphate decahydrate showed the best result under the present experimental conditions. That formulation was freeze-dried and successively employed.

L-Cysteine challenge is considered a good predictor of the in vivo stability of 99mTc-labeled proteins. 2~ In this study a satisfactory correlation between cysteine challenge assay and stability in serum and plasma was detected. The percentage of 99mTc-IgG was high up to 24 hours in these media. Only in case of using a 30000 fold molar excess of cysteine, a very significant transchelation (about 80%) was observed (p<0.00001). Similar results have been reported by different authors. 21,22 It indicates an in vivo stability of the radiopharmaceutical. In fact animal distributions showed no signs of free pertechnetate or radiocolloids.

Target/background ratios were high enough up to 24 hour p.i. This coefficient depends on the labeled

484


immunoglobulin and the employed radioisotope. 21 Obtained values are in range of previously reported ones by other authors. 23,24

In general, a good correlation of 99mTc-MDP and 99mTc-IgG scintigraphic imaging was observed. Only in case of a patient with pelvic abscess the 99mTc-MDP

scan was negative meanwhile labeled immunoglobulin detected it. One patient had a septic process in a prosthesis and rheumatoid arthritis at the same time, all lesions were diagnosed by both radiopharmaceuticals. Nevertheless, the advantage of the 99mTc-IgG consisted in a better definition of the affected region.

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485


Fig. 5. Scintigraphic scan of a New Zealand rabbit with abscess in left posterior thigh 6 hours after i.v. administration of 99mTc-IgG (74 MBq)

4 8 6


Fig. 6. Patient with osteomyelitis of the astragalous. Scintigraphic planar images: 99mTc-IgG 4 hour p.i. (A), 99mTc-MDP 2 hour p.i. (B)

Conclusions

A f r e e z e - d r i e d kit fo r 9 9 m T c - l a b e l i n g o f h u m a n

p o l y c l o n a l I g G w a s o b t a i n e d . F u r t h e r c l i n i c a l t r i a l s a r e

n e c e s s a r y fo r its be t t e r e v a l u a t i o n .

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A kit of human polyclonal IgG for the diagnosis of infectious processes