Enhanced radioimmunotargeting of 125I-labeled L6-biotin monoclonal antibody (MAb) by combining preload of cold L6 MAb and subsequent immunoadsorption in rats.

The present study investigates whether tumor:normal tissue uptake ratios of radiolabeled monoclonal antibodies can be further improved by a combination of extracorporeal immunoadsorption (ECIA) and preload with unlabeled idiotypic monoclonal antibody. Athymic rats, heterotransplanted with human lung carcinoma under the kidney capsule (SR tumor) and i.m. (IM tumor), were divided into four study groups: controls, ECIA, preload, and combined preload+ECIA. The preload+ECIA procedure reduced the whole-body and plasma activity by 48 and 89%, respectively. After such combined procedure, the uptake of 125I-labeled L6-biotin in SR tumors was unchanged, while the uptake in normal tissues was considerably reduced. Tumor (T):bone marrow ratio was then increased by 17.5 times (after ECIA) and by 4.5 times (24 h after ECIA). Similar enhancements were achieved for T:liver and T:kidney ratios. For the IM tumors, the ratios were not as high as for SR tumors. The effects on T:normal ratios of preload+ECIA in combination were synergistic. The combined procedure resulted both in an increased uptake and prolonged persistence of 125I-labeled L6-biotin in the SR tumors and also in a reduction of corresponding uptake values in organs critical for radiation.

Antitumor effect of radioactive cisplatin (191Pt) on nude mice.

PURPOSE: To investigate the effect of (191)Pt-cisplatin in vivo in terms of the antitumor effect and general toxicity on tumor-bearing nude mice. METHODS AND MATERIALS: Tumor-bearing (human squamous cell carcinoma, AB) nude mice were divided into four groups and given, i.p., physiological saline (controls), cisplatin, (191)Pt-cisplatin (80 MBq/mg), or (191)Pt-cisplatin (160 MBq/mg), respectively. Mortality and weight were used as parameters for monitoring general toxic effect, while specific growth delay (SGD) and the area under the logarithm of the relative tumor size curve (AUC-log[RTS]) were used to evaluate the antitumor effect of the treatments. RESULTS: Both SGD and AUC-log(RTS) values showed that (191)Pt-cisplatin was significantly (P < 0.05) more effective in retarding tumor growth than nonradioactive cisplatin. No differences in mortality between the different groups could be observed and no significant differences in weight change between the mice treated with cisplatin or (191)Pt-cisplatin could be seen. CONCLUSION: (191)Pt-cisplatin is a more effective drug than nonradioactive cisplatin in retarding tumor growth on nude mice without adding systemic toxic effects. We believe that radioactive cisplatin may prove to be an alternative to conventional cisplatin; however, the possible toxic effects on organs at risk have to be thoroughly investigated.

Biokinetics of radiolabeled monoclonal antibodies in heterotransplanted nude rats: evaluation of corrected specific tissue uptake.

A tumor model is presented to study the biokinetics and localization of radiolabeled monoclonal antibodies (MAb) in the nude rat (Rowett RNu/RNu) heterotransplanted with human melanoma metastases. The nude rat is larger, less sensitive, and lives longer than the nude mouse. It is, therefore, well suited for in vivo studies of tumor localization with radiolabeled monoclonal antibodies. The tumor-to-host weight ratio was closer to the human situation for the nude rat than for the mouse, and quantitative imaging could be performed with a parallel hole collimator. We followed the antibody biokinetics for as long as 8 days, with repeated blood sampling and imaging. Specific uptake of MAb was higher in tumor tissue than in all other tissues except blood. Initial high uptake was also recorded in the bone marrow. The lymph glands showed a slow uptake of specific and control antibody. A simple in vitro correction procedure is described to calculate the corrected specific tissue uptake (STUcorr) that takes the blood activity into account. Thus it was shown that 80% of the tissue uptake in the dissected liver at 30 hr was due to labeled antibodies circulating in the blood. The specific tissue uptake ratio of antibodies 96.5 and OKT3 (nonspecific control) was unity for all other organs except for tumor tissue, where the ratio was greater than two and even higher when correction for blood content of labeled antibody was made.

A general, extracorporeal immunoadsorption method to increase the tumor-to-normal tissue ratio in radioimmunoimaging and radioimmunotherapy.

The aim of this study was to investigate a new extracorporeal immunoadsorption method to improve tumor-to-normal tissue ratios in radioimmunoimaging (RII) and radioimmunotherapy (RIT). We have developed and investigated a general method using biotinylated antibodies and an agarose-avidin column for extracorporeal immunoadsorption. The studies were made in an animal model and extracorporeal immunoadsorption (ECIA) was performed 24 or 48 hr after the injection of 125I-labeled biotinylated antibodies. In athymic rats, heterotransplanted with human malignant melanoma, 90%-95% of the circulating activity was removed with ECIA. The tumor-to-normal tissue ratios at 24 hr was increased 4 times (from 1.2 to 5.1) in the liver, 2.5 times (0.7 to 1.8) in the lung, 4 times (1 to 4) in the kidneys and 4 times (1.4 to 5) in the bone marrow. Whole body activity was reduced by 40%-50%. Tumor-to-organ ratios at 48 hr were increased 3.5 times (from 1.5 to 5.2) in the liver, 2 times (0.9 to 1.7) in the lung, 3 times (1.3 to 3.8) in the kidneys and 4 times (1.4 to 5.5) in the bone marrow. Whole body activity was reduced by 35% when ECIA was performed 48 hr after injection. This study proves that an important reduction in background activity, and thereby an improvement in the tumor-to-background ratio, can be achieved by using this generally applicable, biotin-avidin ECIA method. For RII, the improved ratio increases the possibilities of detecting tumors and metastases in blood-rich organs. For RIT, the procedure may lead to a decreased absorbed dose to bone marrow and other critical organs.

Heterogeneous distribution of radiolabelled monoclonal antibody. Autoradiographic evaluation in the nude rat model.

Iodinated monoclonal antibody 96.5 was injected intravenously in the nude rat model transplanted with human melanoma. The activity distribution was evaluated by: 1) direct application of dissected tissue on autoradiographic film, 2) autoradiography of whole-body sections, and 3) beta-camera imaging of fresh frozen tissue. Method (1) is non-quantitative and has a poor resolution. It can only be recommended for simple screening. The whole-body method (2), although complicated, gives more accurate digital information of tissue uptake in individual pixels, or in larger regions of interest (ROIs). The beta camera technique (3) is a rapid method but its accuracy is less than the whole-body method. All three methods showed that the activity distribution in the tumours was more heterogenous than in other tissues. An overlap of activity uptake in tumours and other tissues was often seen. Mean uptake ratios in the whole body autoradiograms correlated well with in vivo uptake ratios from measurements in dissected tissues. At present, whole body autoradiography appears to be the method of choice for imaging the uptake of radiolabelled monoclonal antibodies in experimental animals.

Performance of simultaneous emission-transmission systems for attenuation-corrected SPEct: a method for validation applied to two camera systems.

Several commercially available systems for attenuation correction in single photon emission computed tomography (SPECT) based on a transmission scan have been introduced that vary in performance. A test procedure for attenuation correction in SPECT is described and applied to two principally different gamma camera systems (the Siemens Multispect 3 triple-headed system [3HS] and the ADAC Genesys Vertex double-headed system [2HS]). The test procedure was based on geometrically well-defined phantoms. A torso phantom was used to illustrate the attenuation correction methods. The test procedure can be used without detailed knowledge of or access to the algorithms used for attenuation correction. The influence on the transmission measurement of radioactivity in a phantom was higher for the 2HS than for the 3HS. The 3HS produced satisfactory attenuation maps and corrected emission count rates to a constant value independent of phantom density and size. With the 2HS, there was a progressive decrease in the correction of emission count rates with increasing phantom density, and about 30% lower corrected count rates in the large compared with the small phantom. A decrease in measured attenuation coefficients in the vicinity of an emission source was demonstrated in large but not small phantoms. A likely explanation is erroneous correction of downscatter into the transmission energy window. This study demonstrates the need for independent evaluation of systems for attenuation correction in SPECT.

Radio-immunotherapy dosimetry with special emphasis on SPECT quantification and extracorporeal immuno-adsorption.

Results from therapeutic trials with radiolabelled monoclonal antibodies are difficult to compare, because of lack of accurate macroscopic and microscopic dosimetry for both tumours and normal tissues. Requirements for such a dosimetry are covered in the paper. Accurate in vivo dosimetric measurement techniques for verification of calculated absorbed doses are also needed to verify treatment planning. In the review, important topics related to dosimetry in therapeutic trials in RIT are covered, such as, absorbed-dose calculations and activity-quantification techniques for planar imaging and SPECT. The latter is particularly discussed, including a summary of different correction techniques. Absorbed-dose calculations and treatment-planning techniques are also discussed. Possible ways of enhancing the therapeutic ratio are reviewed, especially the novel technique with extracorporeal immuno-adsorption. The review could form the basis of the development of future treatment-planning protocols and for dosimetry calculations in radio-immunotherapy, considering some of the most important parameters for approaching an accurate in vivo dosimetry.

Liposuction reduces arm lymphedema without significantly altering the already impaired lymph transport.

In a prospective study, 20 patients with arm lymphedema after breast cancer treatment underwent liposuction combined with Controlled Compression Therapy (CCT) or CCT alone. Indirect lymphoscintigraphy (ILS) was used to study lymph kinetics before and after intervention. Lymphoscintigrams from the contralateral, non-edematous arm were characterized by prompt transit of the radiotracer (99mTc-albumin nanocolloid) to the axillary nodes, whereas tracer accumulation as dermal backflow characterized tracer transport in the lymphedematous arm. Neither liposuction with CCT nor CCT alone, changed this ILS profile. Liposuction combined with CCT reduced arm edema volume by (median) 115% (range 92-179%), whereas CCT alone decreased arm edema volume by only 54% (range 7-81%) (p = 0.008). Because liposuction in conjunction with CCT was not associated with further impairment to an already restricted lymph transport, we recommend this therapy (liposuction with external compression) for chronic arm lymphedema, as it reduces edema volume safely, rapidly, and more efficiently than external compression alone. Moreover, it does not worsen an already impaired lymph transport in the lymphedematous upper extremity.

Absorbed doses to patients from 191Pt-, 193mPt- and 195mPt-cisplatin.

Cisplatin, a chemotherapeutic drug, can be synthesized using radioactive platinum and then used for pharmacokinetic studies and tumor imaging. We have calculated the absorbed doses to various organs and tissues as well as the effective doses from 191Pt-, 193mPt- and 195mPt-cisplatin after administration to humans for diagnostic purposes. Liver was the organ that received the highest absorbed dose. The effective dose from 191Pt-, 193mPt- and 195mPt-cisplatin was 0.10 +/- 0.02, 0.17 +/- 0.04 and 0.23 +/- 0.05 mSv/MBq respectively.

Some applications of the indium-113m-transferrin technique for gamma-radiation detection of microvascular effects of serotonin and ischemia.

The distribution of systematically injected In-113m (t1/2 = 100 min) in organs of the rat was analyzed, and the use of the isotope for in vivo and in vitro gamma-radiation detection studies of blood plasma protein extravasation was demonstrated in skin, muscle, and tumor. In-113m was slowly excreted from rats. One to 6 h after injection the blood held 3% and 2%, respectively, of injected radioactivity/g tissue wet weight; skin and muscle held 0.1%-0.2%/g; liver, colon, and spleen held approximately 1%/g; lungs 1.5%-1.3%/g and kidneys 2.8%-3.3%/g. Scintillation camera technique revealed 40%-80% extraaccumulation of In-113m in a control extremity upon local administration of serotonin and 20%-40% in an extremity with a transplanted tumor, thus indicating a lower effect of serotonin in tumor microvascular circulation than in muscle and skin. In vitro detection of In-113m radiation by a well-counter in dissected tissues showed no effects of serotonin in the tumor and a four- to five-fold increase of radioactivity in muscle and skin, thus confirming blood protein extravasation upon serotonin treatment in these tissues. External analyses of In-113m in the vascular system using one miniaturized probe directed toward an area of interest showed that the method was too sensitive to movements of the animal, and second probe directed toward a control area is needed.

Improving radioimmunotargeting of tumors: the impact of preloading unlabeled L6 monoclonal antibody on the biodistribution of 125I-L6 in rats.

In the radioimmunotherapy of malignancies the uptake of monoclonal antibodies (MoAb) is commonly low in tumors compared with normal tissue. Several methods have been suggested to increase the tumor-to-normal tissue (T/N) ratio. In this study we have investigated the biodistribution of different amounts of 125I-L6-biotin MoAb in combination with a preload of unlabeled L6 MoAb. Nude rats were injected with 50 micrograms or 250 micrograms of unlabeled L6 24 hours prior to the injection of 10 micrograms, 50 micrograms or 250 micrograms of 125I-L6, antipancarcinoma MoAb. Dissections were performed 24 hours after the injection of radiolabeled MoAb. The maximal enhancement of tumor uptake with simultaneously decreased uptake in normal tissues was with 250 micrograms of 125I-L6 preceded by a preload of 50 micrograms unlabeled L6. Mean T/N ratios were improved by a factor of 2.9 for bone marrow, 3.4 for liver, 3.7 for lungs and 2.3 for kidneys as compared with the corresponding controls. This study demonstrated that preinjection of optimal amounts of unlabeled L6 MoAb may increase the uptake of 125I-L6 by tumor and improve the T/N ratios. Based on present data, preloading with unlabeled MoAb should be considered in future clinical studies with immunoconjugates to improve the radioimmunotargeting of tumors. It is essential to titrate an appropriate amount of the preload, thus avoiding possible tumor antigen saturation of unlabeled MoAbs but simultaneously decreasing the uptake of subsequently injected radiolabeled MoAb in normal tissues.

A 14-year prospective study of autonomic nerve function in Type 1 diabetic patients: association with nephropathy.

AIMS: Prospective studies of autonomic nerve function are rare. We have followed the progression of autonomic dysfunction in relation to nephropathy over 14 years in Type 1 diabetic patients. METHODS: Autonomic nerve function was assessed by heart-rate responses to deep breathing (E/I ratio) and tilting (acceleration and brake indices) and by the postural blood pressure reaction in 58 patients, 43 of whom were reassessed after 14 years. Nephropathy was evaluated by the degree of albuminuria (albuminuria > 20 micro g/min or > 0.03 g/24 h) and glomerular filtration rate ((51)Cr-EDTA plasma clearance). The acceleration index had deteriorated after 7 years (P = 0.0155), whereas the E/I ratio (P = 0.0070) and the diastolic postural blood pressure reaction (P = 0.0054) had deteriorated 14 years after the baseline examination (age-corrected values). All those with albuminuria at the third examination showed signs of autonomic neuropathy at baseline (10 of 10) compared with only nine of 22 without (P = 0.0016). Multiple regression analysis showed that the association between autonomic dysfunction and future albuminuria was due to the E/I ratio. In addition, individuals with an abnormal postural diastolic blood pressure fall (n = 7) at baseline showed a greater fall in glomerular filtration rate more than others 7-14 years later [29 (16.5) ml/min/1.72 m(2) vs. 11 (9) ml/min/1.72 m(2); P = 0.0074]. CONCLUSION: Autonomic nerve function had deteriorated after 14 years. Autonomic neuropathy and abnormal postural diastolic blood pressure falls at baseline were associated with future renal complications.

Time course of the inflammatory response to histamine and allergen skin prick test in guinea-pigs.

Plasma exudation and vasodilatation are key microvascular features of acute inflammation. Exudation and vasodilatation responses in the weal area after skin prick testing with histamine are essentially completed within 30 min. There is evidence to suggest that vasodilatation lasts considerably longer after provocation with allergen, but there is no information on the duration of plasma exudation. The purpose of this study was to measure the time course of the microvascular inflammatory response in the skin after histamine and allergen provocation. Skin prick tests were performed with histamine, allergen (ovalbumin) or saline (control) on guinea-pigs which were shaved on their backs. Radioactive 113mIn was used to label transferrin as a plasma tracer. Radioactivity was recorded from the superficial part of the skin by external detection of conversion electrons from the decay of 113mIn. The increase in count rate, corresponding to tracer accumulation by vasodilatation and/or plasma exudation, was used as a measure of the microvascular inflammatory response to skin prick test. The microvascular response was studied immediately and up to 30 min after provocation. The largest response to histamine and allergen occurred immediately after provocation. The exudative response then gradually declined to be absent after 25-30 min. Skin prick test with saline resulted in a small response of shorter duration. We conclude that the microvascular reaction to histamine as well as allergen provocation in guinea-pig skin has a rapid onset and a duration of approximately 30 min.

Radio-iodinated and internally labelled (35S) IgM monoclonal antibodies in a syngenic rat model.

To simulate the human situation concerning human monoclonal antibodies (MAbs), we have introduced a new syngenic rat model with an implanted rat colon carcinoma. Rat IgM MAbs (10B12), labelled by the chloramine-T method with 125I or internally with 35S, were injected intravenously into the rats and the biodistribution was studied for 8 days. The radioactivity uptake in the tumours of the 35S label was higher than that of the 125I label and the retention of 35S in the tumours gave tumour/blood ratios 8 times higher than those of 125I at 48 and 96 h after injection. In this model we have shown that dehalogenation of iodinated IgM MAbs is a serious problem. We therefore suggest that internally labelled MAbs should be used and that further investigations should be carried out in a syngenic rat model, since this probably reflects the clinical situation better than the nude mouse model.

Serum cystatin C advantageous compared with serum creatinine in the detection of mild but not severe diabetic nephropathy.

OBJECTIVE: To determine whether serum cystatin C is more accurate than serum creatinine in the detection of diabetic nephropathy, also after adjustment for age. METHODS: Forty-one patients with type 1 and 82 patients with type 2 diabetes were evaluated with serum creatinine, serum cystatin C, and (51)Cr-EDTA clearance (reference method). Cystatin C was measured by a particle-enhanced turbidimetric method and creatinine by an enzymatic method. Statistical estimations were performed both without and with age adjustment created by z-scores for (51)Cr-EDTA clearance, creatinine, and cystatin C. The cut-off levels for glomerular filtration rate (GFR) ((51)Cr-EDTA clearance) were 60 and 80 mL min(-1) 1.73 m(-2), respectively, in absolute values and 80, 90 and 95% CIs, respectively, in age-adjusted values (z-scores). RESULTS: Estimations without age adjustment showed significantly (P = 0.0132) closer correlation for cystatin C (r = 0.817) versus (51)Cr-EDTA clearance as compared with creatinine (r = 0.678). However, when using age-adjusted values, the correlation for cystatin C and creatinine, respectively, versus (51)Cr-EDTA clearance did not differ. When comparing the diagnostic utilities for serum cystatin C versus serum creatinine in manifest renal impairment (GFR < 60 mL min(-1) 1.73 m(-2) or z-scores <-1.28 SD), there were no significant differences between the two markers whether age adjusted or not. However, for diagnosing mild nephropathy (GFR < 80 mL min(-1) 1.73 m(-2) or z-score -0.84 SD), serum cystatin C is significantly more useful. CONCLUSIONS: Serum cystatin C performed better compared with serum creatinine even when measured enzymatically, to detect mild diabetic nephropathy. However, serum creatinine was as efficient as serum cystatin C to detect advanced diabetic nephropathy.

Tumour uptake of monoclonal antibody after regional intraarterial injection. Biokinetics in the nude rat heterotransplanted with malignant melanoma.

Nude rats were heterotransplanted with human melanoma metastases on both thighs. Ten days later a bolus of 125I-labelled monoclonal antibody (MAb) 96.5 was injected through a catheter in the common femoral artery. The femoral vein was clamped for 15 min to obstruct the venous outflow from the injected leg. The specific tissue uptake (%/g) in the tumour on the injected side compared to the non-injected side showed initially higher uptake (ratio 7.2 at 3 h). After 24 h there were no side differences. The tumour to muscle ratio was 2.8 at 3 h when injected and control sides were compared. Intravenous or subcutaneous injection gave similar specific tissue uptake as regional arterial injection after 24 h. Tissue to plasma ratios were similar after intravenous and subcutaneous injection of monoclonal antibodies. Intraarterial injection of a bolus of labelled monoclonal antibodies and obstructing the venous outflow thus increased the tumour uptake during a short period of time during which the contrast enhancement was three-fold.

A general extracorporeal immunoadsorption method to increase tumor-to-tissue ratio.

The idea of applying extracorporeal immunoadsorption (ECIA) in radioimmunodiagnosis and radioimmunotherapy has been proposed previously. The authors here report on the development of new concept using a general method for ECIA based on biotinylated MoAb adsorbed on an avidin column. Athymic rats heterotransplanted with either human melanomas or human lung carcinoma were injected with iodine-125-labeled biotinylated 96.5 or L6 MoAb, respectively. At 24 or 48 hours after the injection, ECIA was performed by pumping blood through a hollow-fiber plasma filter. The separated plasma then was passed through an absorbent (avidin-agarose) column. The whole ECIA procedure lasted for 3 hours. By this ECIA method, the tumor-to-normal tissue ratios were increased in various tissues (i.e., radiosensitive and blood rich organs) by a factor of four to five.

Subcutaneous injection of monoclonal antibody 96.5. Biokinetics in the nude rat heterotransplanted with malignant melanoma.

Nude rats heterotransplanted with human melanoma metastasis were injected subcutaneously on the hind paw with 125I-labelled monoclonal antibody 96.5 and with control antibody 131I-OKT3. The elimination from the injection site followed a biexponential function. The uptake in the inguinal lymph nodes on the side of the injection was initially high, but after 90 h it equalled the control side. The uptake in the tumour was slower than after i.v. injection but higher than in other tissues except blood. More than 80% of the activity in the dissected liver represented circulating blood. The uptake ratio of 96.5/OKT3 was c.3 in the tumours but c. 1 in all other tissues including blood. The capillary filtration coefficient was proportional to the uptake in organs like liver, lungs and muscle. It is concluded that subcutaneously injected radiolabelled monoclonal antibodies are initially transported via the lymph but then mainly distributed via the blood reaching the different tissues including tumours.

Improving radioimmonotargeting of tumors. Variation in the amount of L6 MAb administered, combined with an immunoadsorption system (ECIA).

Extracorporeal immunoadsorption (ECIA) is a new method for the selective removal of circulating radiolabeled monoclonal antibodies (MAb) from plasma to increase the uptake in tumor versus normal tissues (T/N-ratio). To ascertain whether the amount of MAb affects T/N ratios immediately and 24 h after ECIA, we used a rat model with two tumor sites--one intramuscular (im) and one below the subrenal capsule (SR). Extracorporeal immunoadsorption was done with an avidin-agarose column after injection of 125I-labeled biotinylated L6 MAb. The animals received 10, 50 or 250 micrograms of L6 only (controls), or followed by ECIA. The efficacy of the procedure in removing plasma activity was 80-95%. For both tumor sites, the highest T/N-ratios were obtained with 10 micrograms L6. All T/N-ratios significantly improved for SR tumors by a factor ranging from 3.2 (lung) to 12.6 (bone marrow). The T/N-ratios were still elevated 24 h after ECIA. Injection of larger amounts of MAb, probably causing a higher degree of tumor saturation, will not necessarily improve the T/N ratio after ECIA.

Gamma camera imaging of platinum in tumours and tissues of patients after administration of 191Pt-cisplatin.

The aim of this study was to visualize non-invasively the uptake of platinum in tumours and tissues after treatment with cisplatin. 191Pt-cisplatin was synthesized from 191PtCl4 with rigorous pharmaceutical quality control. The uptake of platinum by both tumorous and healthy tissues was studied by gamma camera imaging in 14 patients, 5 of whom showed a clear uptake of platinum in regions corresponding to known tumour sites. Maximum concentrations of platinum in the tumours were on average 4.9+/-1.0 microg/g, when normalized to an administered amount of 180 mg cisplatin. In all the patients, the liver was the organ that showed the highest uptake. Platinum uptake was also seen in the spleen, gall bladder, gastrointestinal tract, bladder, kidneys, ureter, neck and mediastinum and urogenital region. By using in-house production of 191Pt-cisplatin, it was possible to monitor the uptake of platinum in tumorous tissues and healthy organs.