First-in-human phase 0 study of 111In-CHX-A"-DTPA trastuzumab for HER2 tumor imaging.

INTRODUCTION: Tumors over-expressing the human epithelial receptor 2 (HER2) or exhibiting amplification or mutation of its proto-oncogene have a poorer prognosis. Using trastuzumab and/or other HER2 targeted therapies can increase overall survival in patients with HER2(+) tumors making it critical to accurately identify patients who may benefit. We report on a Phase 0 study of the imaging agent, 111In-CHX-A"-DTPA trastuzumab, in patients with known HER2 status to evaluate its safety and biodistribution and to obtain preliminary data regarding its ability to provide an accurate, whole-body, non-invasive means to determine HER2 status. METHODS: 111In-CHX-A"-DTPA trastuzumab was radiolabeled on-site and slowly infused into 11 patients who underwent single (n=5) or multiple (n=6) ɣ-camera (n=6) and/or SPECT (n=8) imaging sessions. RESULTS: No safety issues were identified. Visual and semi-quantitative imaging data were concordant with tissue HER2 expression profiling in all but 1 patient. The biodistribution showed intense peak liver activity at the initial imaging timepoint (3.3h) and a single-phase clearance fit of the average time-activity curve (TAC) estimated t1/2=46.9h (R2=0.97; 95%CI 41.8 to 53h). This was followed by high gastrointestinal (GI) tract activity peaking by 52h. Linear regression predicted GI clearance by 201.2h (R2 =0.96; 95%CI 188.5 to 216.9h). Blood pool had lower activity with its maximum on the initial images. Non-linear regression fit projected a t1/2=34.2h (R2 =0.96; 95%CI 25.3 to 46.3h). Assuming linear whole-body clearance, linear regression projected complete elimination (x-intercept) at 256.5hr (R2=0.96; 95%CI 186.1 to 489.2h). CONCLUSION: 111In-CHX-A"-DTPA trastuzumab can be safely imaged in humans. The biodistribution allowed for visual and semiquantitative analysis with results concordant with tissue expression profiling in 10 of 11 patients. Advances in Knowledge and Implications for Patient Care Using readily available components and on-site radiolabeling 111In-CHX-A"-DTPA trastuzumab SPECT imaging may provide an economical, non-invasive means to detect HER2 over-expression.

Association of obesity, serum glucose and lipids with the risk of advanced colorectal adenoma and cancer: a case-control study in Korea.

BACKGROUND: Previous studies on colorectal cancer risk suggest that obesity, serum lipids and glucose might be related to colorectal carcinogenesis. This case-control study was conducted to investigate the association between obesity, serum lipids and glucose, and the risk of advanced colorectal adenoma and cancer. METHODS: Patients with histologically confirmed colorectal cancers (n=105), same number of patients with advanced colorectal adenomas matched by age and sex, and the same number of controls matched by age and sex were selected in Hanyang University Guri Hospital between January 2002 and June 2004. RESULTS: Adenoma and cancer group showed significantly higher levels of mean body mass index and serum glucose. Cancer group also showed significantly lower mean serum lipids levels than controls. We used an unordered polytomous logistic model to calculate multivariate odds ratios for advanced adenoma and cancer relative to controls. Higher serum glucose level was more strongly associated with increased risk of cancer relative to controls (odds ratio, 3.0; 95% confidence interval, 0.9-9.8) than with increased risk of advanced adenoma (odds ratio, 2.1; 95% confidence interval, 0.9-5.4). Higher body mass index was strongly associated with increased risk of advanced adenoma (odds ratio, 10.8; 95% confidence interval, 4.6-25.3), but associated with attenuated risk of cancer (odds ratio, 2.3; 95% confidence interval, 0.9-5.8). Serum triglycerides and cholesterol levels were strongly associated with reduced risk of cancer (odds ratio, 0.3; 95% confidence interval, 0.1-0.8 and odds ratio, 0.2; 95% confidence interval, 0.1-0.6, respectively). CONCLUSIONS: Obesity and hyperglycaemia are positively related to advanced colorectal adenoma formation. Furthermore, hyperglycaemia plays an important role in progression to cancer. Findings on an inverse relationship between serum triglyceride and cholesterol levels and the risk of colorectal cancer may be the secondary results from metabolic or nutritional changes in advanced colorectal cancer patients and should be clarified in further studies.

Huge gastric diospyrobezoars successfully treated by oral intake and endoscopic injection of Coca-Cola.

A diospyrobezoar is a type of phytobezoar that is considered to be harder than any other types of phytobezoars. Here, we describe a new treatment modality, which effectively and easily disrupted huge gastric diospyrobezoars. A 41-year-old man with a history of diabetes mellitus was admitted with lower abdominal pain and vomiting. Upper gastrointestinal endoscopy revealed three huge, round diospyrobezoars in the stomach. He was made to drink two cans of Coca-Cola every 6 h. At endoscopy the next day, the bezoars were partially dissolved and turned to be softened. We performed direct endoscopic injection of Coca-Cola into each bezoar. At repeated endoscopy the next day, the bezoars were completely dissolved.

Comparative cellular catabolism and retention of astatine-, bismuth-, and lead-radiolabeled internalizing monoclonal antibody.

UNLABELLED: Monoclonal antibodies (mAbs) labeled with alpha-emitting radionuclides such as (211)At, (212)Bi, (213)Bi, and (212)Pb (which decays by beta-emission to its alpha-emitting daughter, (212)Bi) are being evaluated for their potential applications for cancer therapy. The fate of these radionuclides after cells are targeted with mAbs is important in terms of dosimetry and tumor detection. METHODS: In this study, we attached various radionuclides that result in alpha-emissions to T101, a rapidly internalizing anti-CD5 mAb. We then evaluated the catabolism and cellular retention and compared them with those of (125)I- and (111)In-labeled T101. T101 was labeled with (211)At, (125)I, (205,6)Bi, (111)In, and (203)Pb. CD5 antigen-positive cells, peripheral blood mononuclear cells (PBMNC), and MOLT-4 leukemia cells were used. The labeled T101 was incubated with the cells for 1 h at 4 degrees C for surface labeling. Unbound activity was removed and 1 mL medium added. The cells were then incubated at 37 degrees C for 0, 1, 2, 4, 8, and 24 h. The activity on the cell surface that internalized and the activity on the cell surface remaining in the supernatant were determined. The protein in the supernatant was further precipitated by methanol for determining protein-bound and non-protein-bound radioactivity. Sites of internal cellular localization of radioactivity were determined by Percoll gradient centrifugation. RESULTS: All radiolabeled antibodies bound to the cells were internalized rapidly. After internalization, (205,6)Bi, (203)Pb, and (111)In radiolabels were retained in the cell, with little decrease of cell-associated radioactivity. However, (211)At and (125)I were released from cells rapidly ((211)At < (125)I) and most of the radioactivity in the supernatant was in a non-protein-bound form. Intracellular distribution of radioactivity revealed a transit of the radiolabel from the cell surface to the lysosome. The catabolism patterns of MOLT-4 cells and PBMNC were similar. CONCLUSION: (211)At catabolism and release from cells were somewhat similar to that of (125)I, whereas (205,6)Bi and (203)Pb showed prolonged cell retention similar to that of (111)In. These catabolism differences may be important in the selection of alpha-radionuclides for radioimmunotherapy.

In vitro and in vivo characterization of 67Ga(3+) complexes with cis,cis-1,3,5-triamino-cyclohexane-N,N',N"-triacetic acid derivatives.

The aim of this study was to investigate the in vitro and in vivo performance of a 67Ga complex with cis,cis-1,3,5-triaminocyclohexane-N,N',N"-triacetic acid (tachta) as a potential ligand for use as a Ga(III) radiopharmaceutical for PET imaging. The radiolabeling procedure, electrophoretic properties, lipophilicity, acid stability, human serum stability and biodistribution in mice of 67Ga(tachta) were investigated. The 67Ga(tachta) complex forms at 10(-3) M tachta concentration at 40 degrees C in 100% yield; it is neutral, non-lipophilic, 90% stable at pH = 4 and 5 and 100% stable at pH = 6, for at least 8 d. Serum stability experiments demonstrated that at 5 hr 67Ga(tachta) exists in serum as a free complex. At 24 hr, 30% of 67Ga(tachta) is reversibly bound to transferrin-albumin fraction of serum, and that this percentage remains unchanged for a period of 4 d. Biodistribution in mice showed that 67Ga(tachta) rapidly clears via the kidneys from the body with less than 10% of injected activity left in the body at 3 hours and only 6% remaining after 24 hr. The complex also cleared rapidly from all of the major organs, with bone showing some slightly increased (1.15% ID/g) 24 hr accumulation, in comparison with the 3 hr time point. Based upon these data, 67Ga(tachta) may be considered as a candidate for developing new Ga(III) radiopharmaceuticals for PET.

Differences of biodistribution, pharmacokinetics, and tumor targeting between interleukins 2 and 15.

Interleukin-2 (IL-2) and interleukin-15 (IL-15) are T-cell tropic factors that share beta and gammac subunits of their receptors on T/NK-cells. Although these two cytokines share receptor components, the IL-15Ralpha molecule is expressed constitutively by various tissue cells, whereas the IL-2Ralpha expression is mostly restricted to activated mononuclear cells. Consequently, we postulated that the biodistribution of IL-15 might be different from that of IL-2 and that individual alpha chains play an important role in this respect. This study investigated the differences between IL-2 and IL-15 in pharmacokinetics, biodistribution, and their tumor-targeting abilities. It found that only IL-2 showed specific binding to a protein, alpha2-macroglobulin, which may be the reason that IL-2 displays longer blood clearance than IL-15. Upon injection of these cytokines into mice, we observed that IL-15 accumulated significantly more than IL-2 in kidney, spleen, and bone. These are all tissues that express IL-15 receptor alpha but not IL-2 receptor alpha. To evaluate the tumor-targeting ability of each cytokine, we used nude mice xenografted with three A431 tumors, parental and cells transfected with alpha subunit of the receptor for either IL-2 or IL-15. When examined using radioiodinated IL-2 or IL-15, each cytokine accumulated on the target cells, expressing its respective alpha chain, suggesting that the expression of the alpha chains is sufficient to define specific biodistribution of IL-2 and IL-15, although these cytokines share the beta and yc molecules of their receptors. IL-15 displayed better target-specific accumulation and more rapid clearance from the circulation than did IL-2, and thus it can be considered to be a novel and unique therapeutic agent.

DNA cleavage by 111In-labeled oligodeoxyribonucleotides.

UNLABELLED: We studied the fine structure of DNA damage produced by the decay of 111In incorporated into duplex and triplex DNA strands to evaluate the usefulness of this radionuclide for sequence-specific DNA cleavage. METHODS: Oligodeoxyribonucleotides (ODNs) were prepared with 111In attached by diethylenetriaminepentaacetic acid (DTPA) at the 5' end or 3' end through a long chemical linker or to an internal nucleotide position through a short linker. Subsequent formation of DNA duplexes and triplexes was confirmed by gel electrophoresis. The 111In-induced breaks were assayed in denaturing polyacrylamide gel electrophoresis with a single-nucleotide resolution. RESULTS: 111In-labeled oligonucleotides of high specific activity (740-1554 TBq/mmol) were synthesized. The presence of the bulky 111In-DTPA group did not impede duplex or triplex formation. Localized DNA breaks were observed in all duplexes and triplexes formed. The majority of DNA breaks in duplex formations were located within +/- 10 nucleotides from the site of attachment of the 111In-bearing linker. The yield of DNA breaks per decay was 0.38 in a duplex with internally modified ODNs. This is nearly 2 times less than the yield of DNA breaks in the same duplex with 1251 attached through the same linker. The yield of DNA breaks in the pyrimidine and purine strands of DNA triplexes with 111In attached to the triplex-forming ODNs through the linkers of different length varied from 0.05 to 0.10. The distribution of DNA breaks was wider in comparison with the duplex experiment. The lower yields of breaks per 111In decay compared with 125I may be not only the result of lower deposited energy but also of the ionic repulsion of the negatively charged 111In-DTPA group from the DNA strands. CONCLUSION: We have shown that decay of 111In produces highly localized DNA breaks. 111In introduced into triplex- and duplex-forming ODNs through hydrocarbon linkers produces sequence-specific DNA strand breaks with an efficiency nearly comparable with that of 1251. These findings are supportive of our proposed use of 111In-ODNs for gene-specific radiotherapy.

Pharmacokinetics of 111In- and 125I-labeled antiTac single-chain Fv recombinant immunotoxin.

UNLABELLED: The use of immunotoxins for cancer therapy is an attractive strategy that exploits the targeting specificity of monoclonal antibodies and their fragments as well as the exquisite toxicity of the toxins. However, few studies of immunotoxins have evaluated their biodistribution in vivo. Previous studies have used 125I for tracing immunotoxin biodistribution in mice. Because the immunotoxin works only when it is internalized and because of known problems with quick dehalogenation after internalization of antibodies, we decided to use 111In, which has greater intracellular retention than iodine. METHODS: To trace the in vivo pharmacokinetics of the immunotoxin in mice, we labeled the antiTac(Fv)-PE38 with 111ln and compared it with 125I-labeled antiTac(Fv)-PE38. We successfully labeled antiTac(Fv)-PE38 with 111In at up to 2.96 GBq/mg. A 3- to 4-fold decrease in cytotoxicity was observed for both radiolabeled preparations. We evaluated the internalization of 111In- and 125I-labeled antiTac(Fv)PE38 into ATAC4 cells (Tac-positive) as well as their biodistribution and pharmacokinetics in vivo in mice. In addition, some mice receiving these reagents were co-infused with 30 mg L-lysine to inhibit renal accumulation. RESULTS: Significantly more 111In- than 125I-labeled antiTac(Fv)-PE38 accumulated in the ATAC4 cells (20% versus 5% of initial surface-bound radioactivity; P < 0.001). In vivo, significantly more 111In- than 125I-labeled antiTac(Fv)-PE38 accumulated in the kidney (119 versus 31 percentage injected dose per gram [%ID/g]; P < 0.001). The tumor accumulation of 111In-labeled antiTac(Fv)-PE38 at 96 h was 13-fold greater than that of 125I-labeled antiTac(Fv)-PE38 (1.4 versus 0.1 %ID/g; P < 0.001). No antiTac(Fv)-PE38 was excreted into the urine in its intact form unless lysine was co-infused. Co-injected lysine reduced the renal accumulation of 111In-labeled antiTac(Fv)-PE38 by 62%. CONCLUSION: We evaluated the biodistribution, pharmacokinetics, and catabolism of 111In-labeled antiTac(Fv)-PE38 and found that it differed from 125I-labeled antiTac(Fv)PE38. These studies suggest that 111In-labeled antiTac(Fv)-PE38 can be used to trace the fate of antiTac(Fv)-PE38 in humans.

Effects of insulinlike growth factor binding proteins on insulinlike growth factor-I biodistribution in tumor-bearing nude mice.

UNLABELLED: This study evaluated the biodistribution and tumor targeting ability of radiolabeled insulinlike growth factor (IGF)-I. Because IGF binding proteins (IGFBPs) play a critical role in modulating IGF activity, the binding properties of 125I-labeled IGF-I to IGFBPs were investigated in vitro and in vivo. Because a large amount of the IGF-I was catabolized in vivo, we also studied the catabolism of IGF-I by tumor cells in vitro. METHODS: 125I-labeled-IGF-I was prepared using the chloramine T method. The biodistribution of 125I-labeled-IGF-I in tumor-bearing nude mice was compared between groups injected with 125I-labeled IGF-I alone or coinjected with unlabeled peptide. In vitro and in vivo chromatography studies were performed to evaluate the binding profile to IGFBPs and the degree of catabolites in serum as well as urine. RESULTS: Data indicated that the binding of radiolabeled IGF-I to IGFBPs in vitro was dose dependent. However, there was a difference in complex formation between the serum and the heparinized plasma. In heparinized plasma, the radioactivity shifted from a 30- to 50-kDa complex to a 150-kDa complex and to a free ligand, because the binding of heparin with IGFBPs decreased its affinity for IGF-I. In plasma prepared with acid citrate dextrose a binding pattern identical to that of serum was observed. Moreover, there was a binding difference between mouse and rat. The 125I-labeled IGF-I catabolized very quickly when incubated at 37 degrees C but not at all at 4 degrees C. In tumor-bearing nude mice, the uptake of radioactivity in normal tissues decreased quickly, particularly in the kidneys. In mice coinjected with unlabeled carrier, the radioactivity in most normal tissues was lower and the tumor uptake higher than in the mice without carrier. CONCLUSION: These data confirm that 125I-labeled IGF-I is avidly bound to IGFBPs, both in vitro and in vivo. By partially saturating this binding with unlabeled peptides, a favorable biodistribution was achieved, including faster clearance from normal tissue and higher tumor uptake, which resulted in better tumor-to-nontumor ratios. Nevertheless, the rapid catabolism and release of the radiolabel from tumor tissue result in a suboptimal targeting agent.

Candidate neuroanatomic substrates of psychosis in old-aged depression.

1. The authors investigated the candidate neuroanatomic substrates underlying delusional thought disorder in old-aged depressed patients by using magnetic resonance imaging (MRI), and examined the relationship between volumes for individual brain structures and clinical correlates of particular relevance to depression: executive cognitive impairment and global severity of depression. 2. MR morphometry was performed on nineteen deluded depressed patients and 26 non-deluded depressed patients, all older than 55 years of age. Subjects were administered a neuropsychological test battery and measures of depression. 3. The absolute volume of prefrontal cortex (PFC) was smaller in the deluded depressed group than in non-deluded depressed group (131.79 +/- 37.26 ml vs. 152.65 +/- 26.13 ml, p = 0.03); a difference that was statistically significant even after adjusting for the effect of whole brain volume (p = 0.01). No group differences were observed in the volumes of the basal ganglia, the temporal lobes, the superior temporal gyri, the amygdala-hippocampal complex, the lateral ventricles, or whole brain. The relative volume of PFC correlated inversely and significantly with the index of Wisconsin Card Sorting Test (WCST) performance (r = -0.76, p < 0.01) in depressed patients. 4. PFC may be one of the candidate neuroanatomic substrates underlying delusional thought disorder in old-aged depression.

Methods to avoid adverse effect of circulating antigen on biodistribution of 125I-labeled antiTac dsFv: preinjection of intact antibody versus clearance of antigen with adivin-biotin system.

UNLABELLED: The presence of circulating antigen may adversely affect the biodistribution of a radiolabeled antibody. The alpha subunit of the interleukin-2 receptor (IL-2Ralpha) is a cell-surface receptor that is overexpressed in various hematologic malignancies and in benign disorders. This receptor is cleaved from the cell surface and can be found in high concentrations in serum. Radiolabeled antiTac antibodies are being evaluated to target this receptor. Previous studies have shown that circulating soluble IL-2Ralpha (slL-2Ralpha) adversely affected the biodistribution of radiolabeled antiTac disulfide-stabilized (ds)Fv. In this study, we compared blocking and clearing sIL-2Ralpha to see which better minimized its interference with the biodistribution of radiolabeled antiTac dsFv. METHODS: Two models of sIL-2Ralpha were used: one consisted of mice given intravenous sIL-2Ralpha and the other consisted of mice bearing SP2/Tac tumor xenografts (IL-2Ralpha positive), which shed sIL-2Ralpha. We biotinylated humanized antiTac monoclonal antibody (bt-HuTac) and radiolabeled it with 125I. We then compared its biodistribution with that of humanized antiTac monoclonal antibody IgG (HuTac). We examined the biodistribution of an injected dose of 125I-labeled antiTac dsFv after a preinjection of HuTac to block the sIL-2Ralpha epitope and after a preinjection of bt-HuTac, followed by an avidin chase. RESULT: The 125I-labeled bt-HuTac cleared from the serum at a rate similar to that of HuTac. The avidin chase effectively cleared >92% of circulating 125I-labeled bt-HuTac within 20 min and was also effective in clearing sIL-2Ralpha. In comparison, HuTac prolonged the retention of 125I-labeled sIL-2Ralpha in the circulation, and the avidin chase decreased 125I-labeled sIL-2Ralpha to <18% of control. Although the two-step antigen-clearing system effectively cleared the antigen from the circulation and improved the biodistribution of 125I-labeled dsFv, the HuTac preinjection method had a similar but longer lasting beneficial effect on 125I-labeled dsFv biodistribution. CONCLUSION: Preinjection of either HuTac or bt-HuTac with avidin chase improved the biodistribution of subsequently administered 125I-labeled antiTac dsFv by preventing the dsFv from binding to the sIL-2Ralpha, but the HuTac blocking method is simpler and longer lasting.

Chemical modification to reduce renal uptake of disulfide-bonded variable region fragment of anti-Tac monoclonal antibody labeled with 99mTc.

The anti-Tac disulfide-bonded variable region fragment (dsFv) is a genetically engineered, 25 kDa, murine monoclonal antibody fragment that recognizes the alpha subunit of the interleukin-2 receptor (IL-2Ralpha). The dsFv radiolabeled with the tetrafluorophenyl ester (TFP) of [99mTc]mercaptoacetyltriglycine ([99mTc]MAG3-TFP) showed rapid tumor uptake and fast blood clearance in mice, resulting in high tumor-to-nontumor background ratios. However, its high renal uptake was a problem. In this study, we tested the effect of lowering the isoelectric point (pI) of dsFv to <9.3 on renal and tumor uptake. To lower the pI, dsFv was acylated simultaneously with both [99mTc]MAG3-TFP and TFP-glycolate. The acylation of dsFv decreased its pI and its immunoreactivity inversely proportional to the molar ratio of TFP-glycolate to dsFv, whereas the conjugation of [99mTc]MAG3-TFP alone did not. When biodistribution studies were performed in nude mice, the effect of the lowered pI was reflected primarily in decreased kidney uptake and whole-body retention, with its highest effect seen at the earliest time point (15 min) after injection. In tumor-bearing nude mice, glycolated [99mTc]MAG3-dsFv with a pI range of 4.9 to 6.5 accumulated selectively into IL-2 receptor-positive SP2/Tac tumor similar to that of the control [125I]dsFv labeled by the Iodo-Gen method, whereas its renal uptake was 25% of [125I]dsFv at 15 min. At 90 min, the ratios of tumor to receptor-negative SP2/0 tumor, liver, kidney, stomach, and blood had peaked at 10.9, 8.5, 0.3, 5.0, and 6.2, respectively, for the glycolated [99mTc]MAG3-dsFv. The corresponding ratios for [125I]dsFv were 3.7, 5.0, 0.1, 1.5, and 2.1, respectively.

Favorable effects of glycolate conjugation on the biodistribution of humanized antiTac Fab fragment.

UNLABELLED: One of the major limitations of using intact immunoglobulins for targeting tumors is poor penetration into tissues. Although Fab fragments have been used because of their improved kinetics, they have undesirable high renal accumulation. In this study we tested a new approach to block renal accumulation of Fab. METHODS: We conjugated humanized antiTac Fab fragments, which are directed against the interleukin-2 receptor, with glycolate. The biodistribution, pharmacokinetics and catabolism of glycolated Fab (glyco-Fab) were evaluated at two different levels of substitution (heavy and light) compared with nonglycolated Fab in Tac-antigen-positive (ATAC4) and -negative (A431) tumor-bearing nude mice. The mice received coinjections of 125I-labeled glyco-Fab (3 microCi/1 microg) and 131I-labeled nonglycolated Fab (5 microCi/1 microg). In addition, groups of mice receiving these reagents were also coinfused with 50 mg L-lysine. RESULTS: Significantly less glyco-Fab than nonglycolated Fab accumulated in the kidney (21 versus 189 %ID/g; P < 0.001). A higher proportion of glyco-Fab was excreted into the urine in its intact form. The glyco-Fab survived longer in circulation than nonglycolated Fab. The peak tumor accumulation of glyco-Fab was 2.3-fold greater than that of nonglycolated Fab. Furthermore, the ATAC4 tumor-to-normal tissue ratio of glyco-Fab was much higher in all organs than that of nonglycolated Fab. The heavily glyco-Fab accumulated less in the kidney than the lightly glyco-Fab. The coinjected lysine reduced the renal accumulation of both nonglycolated Fab and glyco-Fab. CONCLUSION: Glyco-Fab is a promising agent because of its lower renal accumulation, higher tumor uptake and higher tumor-to-normal tissue ratio.

Evaluation of 99mTc-mercaptoacetyltriglycine-biocytin as a new hepatobiliary imaging agent in mice coinjected with bilirubin.

We evaluated 99mTc-labeled mercaptoacetyltriglycine (99mTc-MAG3)-biocytin as a hepatobiliary imaging agent in the absence and presence of bilirubin in mice. We then compared its pharmacokinetic parameters; peak liver/heart activity ratio (rmax) and half clearance time (HCT) with those of 99mTc-labeled diisopropyl-iminodiacetic acid (99mTc-disofenin). Balb/c mice were injected intravenously with hepatobiliary agent (99mTc-MAG3-biocytin or 99mTc-disofenin) alone or in combination with bilirubin at two doses (7 and 14 mg/kg) dissolved in 5% human serum albumin. Images were acquired every 15 s for 30 min with a gamma-camera equipped with a pinhole collimator. Dynamic images showed rapid hepatic uptake of 99mTc-MAG3-biocytin, with rapid clearance from the blood and rapid excretion via the biliary system. Its hepatic uptake was not affected by bilirubin coinjection, whereas 99mTc-disofenin coinjected with bilirubin showed a higher blood background than 99mTc-disofenin alone. These qualitative findings were reflected in pharmacokinetic parameters, rmax and HCT. The rmax was obtained from plots of time versus liver/heart activity ratios obtained in equal-area regions of interest over the heart and liver. The HCT was calculated from the hepatic clearance curve from plots of time versus liver activity. 99mTc-MAG3-biocytin without bilirubin coinjection showed an rmax of 8.9+/-1.3 and an HCT of 399+/-36 s. These values did not change even when 14 mg/kg of bilirubin were coinjected. By contrast, the parameters for 99mTc-disofenin with bilirubin were significantly (p < 0.01) affected by 14 mg/kg of bilirubin coinjection: rmax was decreased from 7.9+/-2.5 to 1.4+/-0.2 and HCT was increased from 292+/-32 s to 782+/-133 s. 99mTc-MAG3-biocytin hepatobiliary scintigraphy in mice is not affected by bilirubin coinjection, and this hepatobiliary agent appears to offer promise for estimating hepatic function in patients with high bilirubin levels.

The pharmacokinetic characteristics of glycolated humanized anti-Tac Fabs are determined by their isoelectric points.

To evaluate a method for preventing the nephrotoxicity caused by the high renal accumulation of radiolabeled or toxin-conjugated small immunoproteins used for cancer therapy, we conjugated humanized anti-Tac Fab fragments with various numbers of glycolate molecules [glycolated Fab fragments (glyco-Fabs)] and separated the conjugates by means of ion-exchange columns into three fractions, depending on their isoelectric points (pIs). We evaluated the biodistribution, pharmacokinetics, and catabolism in normal nude mice of nonglycolated Fab (pI > or = 9.3) and three different preparations of glyco-Fab, including strongly anionic glyco-Fab (sa-glyco-Fab: pI < or = 4.5), mildly anionic glyco-Fab (pI = 4.5-7), and mildly cationic glyco-Fab (pI = 7-9.3). In addition, the biodistributions of 125I-labeled sa-glyco-Fab and 131I-labeled nonglycolated Fab were evaluated in normal nude mice coinjected with 50 mg of L-lysine and/or 1 microg of furosemide and in a control group without coinjection. We then evaluated the serial biodistribution of 125I-labeled sa-glyco-Fab (4 microCi/1 microg) and 131I-labeled nonglycolated Fab (5 microCi/1 microg) in Tac antigen-positive (ATAC4) and -negative (A431) tumor-bearing nude mice with s.c. tumor xenografts derived from Tac antigen-positive ATAC4 cells and receptor-negative A431 cells. These animals were coinjected with 30 mg of lysine i.v. and 30 mg of lysine i.p. 15 min after the radiolabeled Fab injection. To evaluate the biodistribution data and study scintigraphic imaging, we performed serial scintigraphy on normal and tumor-bearing mice with all four 131I-labeled preparations. 125I-labeled mildly cationic glyco-Fab and 131I-labeled nonglycolated Fab had similar distributions, except in the kidney. However, both 125I-labeled anionic glyco-Fab preparations showed significantly different distributions from both cationic Fabs in the blood, liver, lung, and spleen. Renal accumulation of all four radiolabeled Fab preparations increased significantly as the pI increased (P < 0.01). In addition, the intact fraction of Fab excreted into urine increased as pI decreased. Therefore, the glomerular filtration depended on whether the charge on the Fab was positive or negative. The proportion of Fab reabsorbed by the proximal tubules increased as pI increased. 125I-labeled sa-glyco-Fab and 125I-labeled mildly anionic glyco-Fab showed a similar distribution in the blood and all organs except the kidney. Lysine led to an additional blocking effect on proximal tubular uptake of both sa-glyco-Fab and nonglycolated Fab. Addition of furosemide yielded only a small effect when used with lysine. With lysine, the sa-glyco-Fab:nonglycolated Fab estimated integral radioactivity ratios were 4.7 and 0.7 in the ATAC4 tumor and in the kidney, respectively. The use of anionic fragments, which may be used in conjunction with lysine, represents a promising approach that may help decrease the renal toxicity of other small fragments, the molecular weights of which range from Mr 40,000 to 70,000, and, thereby, allow higher doses of radiation to the tumor.

Similarities and differences in 111In- and 90Y-labeled 1B4M-DTPA antiTac monoclonal antibody distribution.

UNLABELLED: Monoclonal antibodies (MoAb) labeled with 90Y are being used for radioimmunotherapy. Because 90Y is a beta emitter, quantitative information from imaging is suboptimal. With the concept of a "matched pair" of isotopes, 111In is used as a surrogate markerfor90Y. We evaluated the differences in biodistribution between 111In- and 90Y-labeled murine antiTac MoAb directed against the IL-2Ralpha receptor. METHODS: The antiTac was conjugated to the 2-(4-isothiocyanatobenzyl)-6-methyl-diethylenetriamine pentaacetic acid (1B4M-DTPA, also known as MX-DTPA). Nine patients with adult T-cell leukemia were treated. Patients received approximately 185 MBq (5 mCi) 111In-labeled antiTac for imaging and 185-555 MBq (5-15 mCi) 90Y-labeled antiTac for therapy. The immunoreactivity of 111In-labeled antiTac was 90%+/-6%, whereas for 90Y-labeled antiTac, it was 74%+/-12%. RESULTS: The differences in blood and plasma kinetics of the two isotopes were small. The area undemeath the blood radioactivity curve was 1.91 percentage+/-0.58 percentage injected dose (%ID) x h/mL for 111In and 1.86%+/-0.64 %ID x h/mL for 90Y. Urinary excretion of 90Y was significantly greater than that of 111In in the first 24 h (P = 0.001), but later, the excretion of 111In was significantly greater (P = 0.001 to P = 0.04). Core biopsies of bone marrow showed a mean of 0.0029+/-0.0012 %ID/g for 111In, whereas the 90Y concentration was 0.0049+/-0.0021 %ID/g. Analyses of activity bound to circulating cells showed concentrations of 500-30,000 molecules of antiTac per cell. When cell-bound activity was corrected for immunoreactive fraction, the ratio of 111In to 90Y in circulating cells was 1.11+/-0.17. Three biopsies of tumor-involved skin showed ratios of 111In to 90Y of 0.7, 0.9 and 1.1. CONCLUSION: This study shows that differences typically ranging from 10% to 15% exist in the biodistribution between 111In- and 90Y-labeled antiTac. Thus, it appears that 111In can be used as a surrogate marker for 90Y when labeling antiTac with the 1 B4M chelate, although underestimates of the bone marrow radiation dose should be anticipated.

Evaluation of the in vivo biodistribution of indium-111 and yttrium-88 labeled dendrimer-1B4M-DTPA and its conjugation with anti-Tac monoclonal antibody.

We evaluated the in vivo biodistribution of indium- and yttrium-labeled second-generation polyamidoamine dendrimer (PAMAM) conjugated with 2-(p-isothiocyanatobenzyl)-6-methyl-diethylenetriaminepentaacetic acid (1B4M), a derivative of DTPA. In addition, we conjugated PAMAM-1B4M to humanized anti-Tac IgG (HuTac) and evaluated its in vitro and in vivo properties. PAMAM-1B4M was labeled with 111In at 37-48 MBq/mg (1.0-1.3 mCi/mg) or with 88Y at 3.7-4.8 MBq/mg (0.1-0. 13 mCi/mg), and an aliquot of radiolabeled conjugate was saturated with the corresponding stable yttrium or indium. Nontumor-bearing nude mice were injected intravenously with 55.5-66.6 kBq (1.5-1.8 microCi) of 88Y-labeled PAMAM-1B4M or with 185 kBq (5 microCi) of 111In-labeled PAMAM-1B4M. The mice were then sacrificed at 15 min, 90 min, 1 day, and 4 days postinjection. Then the PAMAM-1B4M was conjugated with HuTac and labeled with 111In at 111-259 MBq/mg (3-7 mCi/mg). Another preparation of 111In-labeled HuTac-PAMAM-1B4M was saturated with stable indium. Immunoreactivity of both preparations and biodistribution in normal mice 1 h after injection and in ATAC4 and A431 tumor-bearing mice 18 h after injection were evaluated and compared with those of 111In-labeled 1B4M-HuTac. We noted significantly higher accumulations (p < 0.05) of 111In-labeled and 88Y-labeled unsaturated PAMAM-1B4M than saturated preparations in the liver, kidney, spleen, and bone at most time points. The whole-body clearance times of unsaturated preparations were significantly slower than those of saturated preparations at all time points, with the exception of 168 h for 111In-labeled PAMAM-1B4M. The saturated preparation of 111In-labeled HuTac-PAMAM-1B4M showed lower hepatic uptake (27 +/- 2%ID/g) than the unsaturated (32 +/- 2%ID/g), but greater than the HuTac-1B4M control (10 +/- 0%ID/g). The splenic uptake showed 15 +/- 1, 38 +/- 5, and 8 +/- 1%ID/g for the saturated, unsaturated, and control, respectively. The biodistribution of the dendrimer conjugated HuTac in normal organs of tumor-bearing mice was similar to nontumor-bearing mice. Specific tumor (ATAC4) uptake was higher than that in nonspecific tumor (A431). In conclusion, we evaluated the biodistribution of radiolabeled PAMAM-1B4M. We noted high accumulation in the liver, kidney, and spleen, which significantly decreased when the chelates were saturated with the stable element. A similar phenomenon was observed between unsaturated and saturated 111In-labeled HuTac-PAMAM-1B4M, indicating that the PAMAM dendrimer had a detrimental effect on biodistribution.

Preparation and in vitro studies of [125I]IUDR-T101 antibody conjugate.

Idoxuridine labeled with 125I was conjugated to polylysine. This conjugate was then coupled to the carbohydrate side chains of T101 monoclonal antibody (anti-CD5). The immunoreactivity, cell retention, cytotoxicity, and intracellular localization of this conjugate was tested in CCRF-CEM cells (CD5 positive). The conjugate had 68% immunoreactivity. The retention of 125I by CCRF-CEM cells was higher for the conjugate than for T101 directly labeled with 125I and more of it localized in the nucleus than did the 125I-labeled T101. The 125I IUDR-polylysine-T101 conjugate was more cytotoxic than the 125I-labeled T101. In conclusion, the conjugation of [125I]IUDR to T101 is feasible, and preferential targeting of the 125I to the nucleus is obtained.

Use of an antibody against the soluble interleukin 2 receptor alpha subunit can modulate the stability and biodistribution of interleukin-2.

The authors have previously reported that the soluble serum form of the alpha subunit of the IL-2 receptor (sIL-2Ralpha), whose natural half-life is approximately 40 min, survived much longer in the circulation when bound by a specific antibody. In the present study, the authors evaluated the extent to which sIL-2Ralpha protected IL-2 in freshly collected serum using biochemical analyses, and a functional CTLL-2 assay. In particular, sIL-2Ralpha protected IL-2 from forming complexes with alpha(2)-macroglobulin and from inactivation in vitro. In addition, the authors demonstrated that the anti-IL-2Ralpha monoclonal antibody 7G7/B6, which does not inhibit the binding of IL-2 to its binding site on sIL-2Ralpha, protected IL-2 from degradation and inactivation in vivo in the presence of sIL-2Ralpha. Both(125)I-labelled and unlabelled IL-2 were injected into mice preinjected with humanized anti-Tac (hTac) or 7G7/B6 and sIL-2Ralpha, or sIL-2Ralpha alone. Using size-exclusion HPLC, ELISA, and CTLL-2 cell proliferation assays, we observed that the presence of 7G7/B6 led to formation of complexes with sIL-2Ralpha and increased the serum levels of IL-2 more than 3- to 40-fold those of groups receiving IL-2 alone, sIL-2Ralpha, or hTac. Taken as a whole, these results suggest that the complex of 7G7/B6 and sIL-2Ralpha not only prolongs the survival of IL-2 in vivo, but also maintains the bioactivity of IL-2. The use of antibodies against endogenous soluble receptors could increase the in vivo survival of cytokines, protect their bioactivity and thereby facilitate their clinical use in the treatment of various malignancies and AIDS.