Cure of Burkitt's lymphoma in severe combined immunodeficiency mice by T cells, tetravalent CD3 x CD19 tandem diabody, and CD28 costimulation.

To increase the valency, stability, and therapeutic potential of bispecific antibodies, we have constructed a tetravalent tandem diabody (Tandab) that is specific to both human CD3 (T-cell antigen) and CD19 (B-cell marker; S. M. Kipriyanov et al., J. Mol. Biol., 293: 41-56, 1999). It was generated by the functional dimerization of a single chain molecule that contained four antibody variable domains (V(H) and V(L)) in an orientation that prevented intramolecular pairing. Compared with a previously constructed heterodimeric CD3 x CD19 diabody, the Tandab exhibited a higher apparent affinity to both CD3+ and CD19+ cells and longer blood retention when injected into mice. Biodistribution studies in mice bearing Burkitt's lymphoma xenografts demonstrated specific accumulation of the radioiodinated Tandab in a tumor site with tumor-to-blood ratios of 1.5, 8.1, and 13.3 at 3, 18, and 24 h, respectively. Treatment of severe combined immunodeficiency mice bearing established Burkitt's lymphoma (5 mm in diameter) with human peripheral blood lymphocytes, Tandab, and anti-CD28 MAbs resulted in the complete elimination of tumors in all of the animals within 10 days. In contrast, mice receiving human peripheral blood lymphocytes in combination with either the diabody alone or the diabody plus anti-CD28 MAbs showed only partial tumor regression. These data demonstrate that the CD3 x CD19 Tandab may be a promising tool for the immunotherapy of human B-cell leukemias and lymphomas.

Immunoscintigraphy of human mammary carcinoma xenografts using monoclonal antibodies 12H12 and BM-2 labeled with 99mTc and radioiodine.

For immunoscintigraphic localization of human breast cancer two monoclonal antibodies (mabs) 12H12 (immunoglobulin G1) and BM-2 (immunoglobulin G3) were developed. The mabs, directed against two different epitopes on the mucin glycoprotein TAG-12, showed reactivity with 96% of all primary mammary carcinomas. The antibodies were labeled with either 125I or 131I. In addition, 12H12 was directly labeled with 99mTc according to the method of Schwarz and Steinsträsser (A. Schwarz and A. Steinsträsser, J. Nucl. Med., 28:721, 1987). Biodistribution was measured in female nude mice bearing the human mammary carcinoma SF-15. Both radioiodinated mabs showed similar biodistribution with fast tumor uptake (8.5% injected dose/g at 6 h postinjection), which increased to 10-11% injected dose/g at 24 h and subsequently remained constant up to 120 h. 99mTc-Labeling of the mab 12H12 led to an enhanced tumor uptake of 10.5 and 14% injected dose/g at 6 and 24 h postinjection, respectively, and to significantly accelerated blood clearance of radioactivity. Similar results were obtained with a second mammary tumor (AR-1), while an endometrial tumor (EK-3) showed a 3-fold lower accumulation of radioactivity and no difference in uptake of radio-iodinated and 99mTc-labeled 12H12. Scintigraphic imaging of tumor-bearing nude mice with the 99mTc 12H12 at 24 h postinjection clearly demonstrated a diagnostic potential of the new mab for tumor localization and staging.

Immunoscintigraphy with positron emission tomography: gallium-68 chelate imaging of breast cancer pretargeted with bispecific anti-MUC1/anti-Ga chelate antibodies.

Pretargeting techniques that are based on the sequential administrations of bispecific antitumor/antimetal chelate antibodies (BS-MAbs), a blocker to saturate the anti-chelate binding sites of the BS-MAb still present in the circulation, and the radiolabeled chelate are suitable to increase tumor-to-normal tissue contrasts and enable positron emission tomography (PET) as an imaging method. As demonstrated in the nude mouse model, a combination of pretargeted immunoscintigraphy and PET markedly improved the detection of tumor xenografts. With the presented preliminary clinical trial, we attempted to assess the efficacy of pretargeting and PET for breast cancer localization in patients. The BS-MAb used for pretargeting was synthesized from the F(ab')(2) fragments of the anti-MUC1 MAb 12H12, which reacts with the vast majority of breast tumors, and the F(ab') fragments of an anti-gallium (Ga) chelate MAb via a mixed functional chemical linker. For labeling of the Ga-chelate, we used the short-lived positron emitter Ga-68 (t(1/2), 68 min; beta(+), 88%). The dose and time schedule of pretargeting was deduced from previous animal experiments. Ten patients with biopsy-proven, primary breast carcinoma were infused with 10 mg of the BS-MAB: Eighteen h later, they received i.v. injections of 10.7 mg of a blocker and, 15 min later, 9.6 microg of the Ga chelate labeled with 230-300 MBq of (68)GA: PET imaging was started 60-90 min after injection of the (68)Ga chelate. Average tumor-to-blood and tumor:normal breast tissue ratios were 0.9 and 3.0 at 1 h postinjection. Tumor uptake amounted to approximately 0.003% iD/g corresponding to a standard uptake value of approximately 2. Blood clearance of the (68)Ga chelate showed a t(1/2) beta of approximately 100 min. Fourteen of 17 known lesions, averaging 25 +/- 16 mm in size, were clearly visualized as foci of increased activity with PET. No false-positive but three false-negative readings were obtained. An enhanced, bilateral activity uptake in the whole breast parenchyma, found in 4 of the 10 patients, compromised the recognition of these tumor sites. Although the shedding of the MUC1 antigen and the comparatively low tumor affinity of the BS-MAb, common to all anti-mucin MAbs, proved not to be optimal for increasing tumor:tissue ratios with a pretargeting technique, PET imaging offered better sensitivity for the detection of breast cancer at low tumor contrasts than conventional immunoscintigraphy. This could be demonstrated by the clear visualization of tumor sites 10 mm in size, which contrasted only by a factor of 2 from surrounding normal breast tissue.

Multistep tumor targeting in nude mice using bispecific antibodies and a gallium chelate suitable for immunoscintigraphy with positron emission tomography.

To improve tumor:tissue ratios in immunoscintigraphy, a three-step targeting method has been developed. The reagents used were (a) a radioactive, low molecular weight chelate prepared from ionic gallium and a phenolic polyaminocarboxylic acid, which can be labeled either with the single-photon emitter 67Ga or with the short-lived positron emitter 68Ga (t1/2 = 68 min); (b) a bispecific monoclonal antibody (bs-mAb) synthesized from the F(ab)2 fragment of the 1.1ASML antibody specific for the glycoprotein CD44v associated with a rat pancreas carcinoma cell line and the F(ab') fragment of an antibody specific for the gallium chelate; and (c) the nonradioactive gallium chelate covalently coupled to transferrin, which served as a high molecular weight blocker to prevent binding of the radioactive gallium chelate to bs-mAbs in the circulation. Targeting experiments in tumor-bearing nude mice with different doses of bs-mAbs, blocker, and 67Ga chelate were adjusted to maximize tumor to tissue contrasts and tumor uptake. Compared with the biodistribution of the 131I-labeled, native 1.1ASML antibody 24 h postinjection, a schedule using 100 pmol bs-mab 24 h later 100 pmol blocker, 15 min later 16 pmol 67Ga chelate, 1 h later examination, increased tumor:blood and tumor: liver ratios by a factor of 5 while keeping the localization of radioactivity in the tumor constant (10.1% injected dose/g). High-contrast images using either 67Ga or 68Ga were obtained within 1 h. The targeting method described enables the use of the short-lived positron emitter 68Ga and thus allows the combination of an improved immunoscintigraphy and positron emission tomography.

Bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics.

To increase the valency, stability and therapeutic potential of bispecific antibodies, we designed a novel recombinant molecule that is bispecific and tetravalent. It was constructed by linking four antibody variable domains (VHand VL) with specificities for human CD3 (T cell antigen) or CD19 (B cell marker) into a single chain construct. After expression in Escherichia coli, intramolecularly folded bivalent bispecific antibodies with a mass of 57 kDa (single chain diabodies) and tetravalent bispecific dimers with a molecular mass of 114 kDa (tandem diabodies) could be isolated from the soluble periplasmic extracts. The relative amount of tandem diabodies proved to be dependent on the length of the linker in the middle of the chain and bacterial growth conditions. Compared to a previously constructed heterodimeric CD3xCD19 diabody, the tandem diabodies exhibited a higher apparent affinity and slower dissociation from both CD3(+)and CD19(+)cells. They were also more effective than diabodies in inducing T cell proliferation in the presence of tumor cells and in inducing the lysis of CD19(+)cells in the presence of activated human PBL. Incubated in human serum at 37 degrees C, the tandem diabody retained 90 % of its antigen binding activity after 24 hours and 40 % after one week. In vivo experiments indicated a higher stability and longer blood retention of tandem diabodies compared to single chain Fv fragments and diabodies, properties that are particularly important for potential clinical applications.

Liver and kidney imaging with Ga-68-labeled dihydroxyanthraquinones.

This paper describes the preparation of alizarin (1,2-dihydroxyanthraquinone) and alizarin red S (sodium 1,2-dihydroxyanthraquinone-3-sulfonate) labeled with Ga-68, which is obtained from a new high-yield Ge-68 leads to Ga-68 generator. The uptake of Ga-68 alizarin by liver and spleen RES was studied in rats, dogs, and human, and amounted to 80-85% of the administered dose within 5 min after i.v. injection. Gallium-68 alizarin red S was preferentially accumulated in the renal parenchyma to an extent of 70% within 2 hr after i.v. administration. Both substances combine simple and fast preparation with the potential advantages of positron scintigraphy. Complete labeling of 1 mCi Ga-68 was achieved by 100 micrograms of each compound, amounts that are without any known measurable harm to humans (LD50 alizarin red S for i.v. injected mice = 70 mg/kg (8); LD50 alizarin for i.p. injected mice > 120 mg/kg (18)).

Establishment and characterization of monoclonal antibodies against an octahedral gallium chelate suitable for immunoscintigraphy with PET.

As a prerequisite for preparing bispecific antibody conjugates containing anti-tumor and anti-metal chelate binding sites that can be used for pretargeted immunoscintigraphy, monoclonal antibodies (Mabs) have been raised against an octahedral metal chelate synthetized from gallium (Ga) and the hexadentate ligand N,N'bis[2-hydroxy 5-(ethylene beta carboxy) benzyl] ethylenediamine N,N' diacetic acid (Ga-HBED-CC). With use of the Farr assay, binding studies with the 67Ga-labeled chelate and three clones of anti-chelate Mabs showed that none of the Mabs were able to precipitate more than 50% of the Ga-chelate, suggesting an enatiomerism of the Ga-chelate and a sensitivity of the Mabs to either one or the other chelate enantiomer. This could be confirmed by comparing the circular dichroism spectra of the Ga-chelate fractions that passed affinity columns containing the Mabs immobilized on sepharose without retention. With use of a Ga-HBED-CC enantiomer, whole-body retention in mice, preinjected with the corresponding anti-metal chelate Mab of ca. 70% ID, was measured compared to 2.1% retention in mice not preinjected with the Mab. Due to the high affinity of chelate-to-Mab binding in vivo, bispecific antibody conjugates prepared from the fragments of the anti-Ga-chelate Mab might be suitable for pretargeted immunoscintigraphy with the short-lived positron-emitter 68Ga.

Uptake of indium-111 in the liver of mice following administration of indium-111-DTPA-labeled monoclonal antibodies: influence of labeling parameters, physiologic parameters, and antibody dose.

Liver uptake of indium-111 (111In) in mice was investigated following administration of 111In-DTPA murine monoclonal antibodies (111In-DTPA-MAbs) labeled by the cyclic anhydride method. Biodistribution of HPLC-purified 111In-DTPA-MAb preparations was checked with a low (0.2 micrograms) and a high (8.0 micrograms) MAb dose. Using Bio Gel P-30 for desalting the MAb-conjugates, 111In uptake in the liver amounted to 8%-9% of the injected dose (ID) and was independent from the MAb dose, the DTPA-to-MAb molar ratio, tumor growth and biologic variability (different MAbs and different strains of mice). Using Sephadex G-25 for desalting, 0.2 micrograms doses from 7 out of 26 preparations showed increased liver accumulation of 111In in non-tumor mice ranging from 15%-25% of ID. Corresponding high doses led to a "normal" value of 8%-9%. Increased liver uptake of the low dose could not be reduced by coadministration of the unconjugated MAb, but was normal after reinjection of "in vivo filtered" material. An inverse intracellular distribution of 111In activity between sediment and supernatant of liver homogenates, following the administration of the low and the high MAb dose, indicated an artifact of the labeling procedure rather than an inherent biological property of labeled MAbs.

A Ga-68-labeled tetrabromophthalein (Ga-68 BP-IDA) for positron imaging of hepatobiliary function: concise communication.

We describe the chemical synthesis of an iminodiacetic-acid-substituted tetrabromo-o-cresolphthalein (BP-IDA), which complexes Ga-68 tightly. The liver uptake, bile excretion, and urinary excretion of the complex were examined in rats. Maximum liver uptake reached 60%, and 1-hr cumulative bile excretion was 75% of injected dose. Urinary excretion in rats with ligated common bile duct remained below 1%. Competitive action of exogenous bilirubin on hepatobiliary excretion of the Ga complex was less pronounced than that of bromosulfophthalein. The absolute activity determination of the positron emitter Ga-68, the high accumulation in the liver, the low urinary excretion, and the weak competition from exogenous bilirubin are promising features of this radiopharmaceutical for the quantitative study of hepatobiliary function.

PET imaging of somatostatin receptors using [68GA]DOTA-D-Phe1-Tyr3-octreotide: first results in patients with meningiomas.

UNLABELLED: Imaging of somatostatin receptors (SSTRs) using [111In]diethylenetriaminepentaacetic-acid-octreotide (DTPAOC) has proven to be helpful in the differentiation of meningiomas, neurinomas or neurofibromas, and metastases as well as in the follow-up of meningiomas. A drawback of the SPECT method is its limited sensitivity in detecting small meningiomas. Because of PET's increased spatial resolution and its ability to absolutely quantify biodistribution, a PET tracer for SSTR imaging would be desirable. METHODS: 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic-acid-D-Phe1-Tyr3-octreotide (DOTATOC) was labeled using the positron-emitting generator nuclide 68Ga. We acquired dynamic PET images over 120 min after intravenous injection of 175 MBq [68Ga]DOTATOC in 3 patients suffering from 8 meningiomas (WHO I degrees; 7- to 25-mm diameter). Patients' heads had been fixed using individually shaped fiber masks equipped with an external stereotactic localizer system to match PET, CT, and MRI datasets. RESULTS: [68Ga]DOTATOC was rapidly cleared from the blood (half-life alpha, 3.5 min; half-life beta, 63 min). Standardized uptake values (SUVs) of meningiomas increased immediately after injection and reached a plateau 60-120 min after injection (mean SUV, 10.6). No tracer could be found in the surrounding healthy brain tissue. All meningiomas (even the 3 smallest [7- to 8-mm diameter]) showed high tracer uptake and could be visualized clearly. Tracer boundaries showed a good correspondence with the matched CT and MRI images. PET provided valuable additional information regarding the extent of meningiomas located beneath osseous structures, especially at the base of the skull. CONCLUSION: According to our initial experiences, [68Ga]DOTATOC seems to be a very promising new PET tracer for imaging SSTRs even in small meningiomas, offering excellent imaging properties and a very high tumor-to-background ratio.

Gallium-68 chelate imaging of human colon carcinoma xenografts pretargeted with bispecific anti-CD44V6/anti-gallium chelate antibodies.

UNLABELLED: Recently, we demonstrated the feasibility of combining improved tumor-to-tissue contrasts and PET imaging for immunoscintigraphic tumor localization using a multistep targeting technique that consists of the administration of an antitumor/antihapten bispecific monoclonal antibody (BS-MAb), a blocker to saturate the antihapten binding sites of the BS-MAb that are still present in the circulation, and a low molecular weight Ga chelate, labeled with positron emitter 68Ga, serving as the hapten. Due to this technique, the biodistribution of the radiolabeled hapten is governed mainly by the binding characteristics of both the antitumor and the antihapten part of the BS-MAb. For a future clinical implementation of the method, we investigated MAb VFF18, which is reactive with the adhesion molecule CD44V6, a tumor-associated antigen, and up-regulated in colon, squamous cell and pancreas carcinoma, and two anti-Ga chelate MAbs, which are highly selective for only one of the two enantiomers (optical isomers) of the inherently racemic Ga chelate. METHODS: From the VFF18 MAb and the anti-Ga chelate MAbs, two BS-MAbs containing the same antitumor parts, but different antihapten parts, were prepared and tested for multistep targeting in human colon carcinoma-bearing nude mice. RESULTS: Despite identical biodistributions of both BS-MAbs and their very similar affinities for the corresponding Ga chelate enantiomers, tumor uptake of the two enantiomers 1 hr postinjection was significantly different [8.7 +/- 1.9% versus 5.8% +/- 1.6% of the injected dose/g (%i.d./g)], with tumor-to-blood ratios being higher for the BS-MAb showing the lower tumor uptake (7.6 +/- 1.6 versus 4.7 +/- 0.6). From data obtained with each BS-MAb, a similar initial tumor binding of approximately 15.5%i.d./g, but different in vivo half-lives of the corresponding BS-MAb-enantiomer immune complexes, could be estimated. Pretargeting with a mixture of both BS-MAbs followed by the administration of the racemic Ga chelate resulted in the lowest tumor uptake (3.9% +/- 1.5%i.d./g). PET imaging of nude mice with the enantiomeric, as well as with the racemic, 68Ga chelate demonstrated a clear delineation of tumors against blood pool background. CONCLUSION: Multistep immunoscintigraphy with BS-MAbs markedly increases tumor-to-tissue ratios in nude mice and enables PET imaging. Using a BS-MAb containing MAb VFF18, a more sensitive localization of CD44V6-positive tumors in patients should also be obtained.

Effects of disulfiram on mixed function oxidase system and trace element concentration in the liver of rats.

Disulfiram (DSF), an inhibitor of chemically induced carcinogenesis, and its metabolite diethyldithiocarbamate (DDTC) have been investigated for their influence on trace element distribution and on certain enzymes of the drug metabolizing system in the livers of phenobarbital (PB) treated rats. Both substances diminished the PB induced enzyme response in liver microsomes, DDTC being more effective (-85%) than DSF (-60%). The copper, cobalt and zinc content of the livers of DSF treated animals were increased by factors of 6, 3 and 1.5 respectively as compared to controls, while DDTC treatment had no influence on liver trace element content. A correlation between enzyme inhibition and enhanced trace element uptake of the liver after DSF administration could not be observed. The change of trace element transport into the liver during DSF treatment is discussed.

Contribution of paramagnetic trace elements to the spin-lattice relaxation time in the liver.

The relaxation times of water protons in rat liver tissue were measured with a NMR spectrometer at 20 MHz. The paramagnetic trace elements Cu, Fe, and Mn were determined by neutron activation analysis. No shortening of T1 could be observed when liver Cu or Fe concentration was increased in the microgram range. T1 was strongly correlated with the liver Mn concentration of untreated animals and animals whose liver Mn concentration was artificially increased or decreased by intravenous injection of manganous acetate or a metal chelating agent with high affinity for hepatobiliary excretion. Deviations from this Mn-T1 correlation were found in the initial phase of liver cirrhosis induced by thioacetamide (elongated T1, normal Mn concentration) and after stimulation of liver growth by phenobarbital (normal T1, decreased Mn concentration). An increased or decreased enhancement factor for Mn may have contributed to the observed deviations during phenobarbital and thioacetamide treatment.

Influence of a prolonged treatment with disulfiram and D-penicillamine on nitrosodiethylamine-induced biological and biochemical effects. II. Investigations on trace elements in the liver.

The influence of a 28-week treatment with disulfiram (DSF), D-penicillamine (PA), and nitrosodiethylamine (NDEA), as well as with a combination of DSF or PA with NDEA on the concentrations of eight essential trace elements in the whole liver tissue of rats was measured by means of neutron activation analysis. While NDEA treatment lowered the Zn content of the liver, DSF alone or in combination with NDEA enhanced the Zn and Se concentration by 50%-80%. Co, Cu, and Cd levels were increased by factors of 10, 60, and 110, respectively. The Mo concentration was decreased by 50% after DSF administration. PA reduced Cu, Co, and Zn in the liver. PA/NDEA treatment also lowered Cu, Co, and Zn content, but there was no strengthening effect of PA on the decrease in Zn observed with NDEA. The change of trace element concentrations, especially of Cu, is discussed with regard to the observed tumor induction in the liver, which tended to be increased by a combined NDEA/PA administration compared with NDEA treatment alone, whereas a protective action of DSF against NDEA induced liver tumors could not be established.

Influence of a prolonged treatment with disulfiram and D(-)penicillamine on nitrosodiethylamine-induced biological and biochemical effects in rats. I. Investigations on the drug metabolizing system.

The influence of a prolonged treatment with disulfiram (DSF) and D(-)penicillamine (PA) on biological and biochemical effects induced by nitrosodiethylamine (NDEA) was studied in rats. The combination of NDEA and DSF led to a massive and early development of esophageal tumors, which were fatal to the animals. No liver tumors were observed in this group, whereas PA in combination with NDEA led to an increased development of liver tumors compared with NDEA alone. In the last two groups, only incidental tumors of the esophagus were observed. Nasal cavity tumors also appeared earlier in the animals treated with DSF and NDEA than in animals treated with NDEA alone or with NDEA plus PA. At a biochemical level, DSF led to a significant inhibition of hepatic anilinehydroxylase and nitroso-dimethylaminedemethylase in contrast to PA, which had no influence on these enzymes. The reduced activities of these drug-metabolizing enzymes did not appear to be related to gross cytochrome P450 content. Highly significant increases in glutathione content and glutathione-S-transferase activity (GSH/GST) were induced by DSF but not by PA. Because N-nitrosodiethylamine requires enzymatic activation to form the ultimate carcinogen, it is suggested that the observed inhibition of nitrosamine-transforming enzymes in the liver during DSF treatment leads to an increased amount of intact nitrosamines in other organs, e.g., in the esophagus, where it could be transformed to the ultimate carcinogen. DSF treatment alone or in combination with NDEA leads to an accumulation of trace elements in the liver, whereas PA eliminated copper and cobalt. The possible influence of these elements on tumor development is discussed in part II of this study.

Pretargeting of human mammary carcinoma xenografts with bispecific anti-MUC1/anti-Ga chelate antibodies and immunoscintigraphy with PET.

We recently demonstrated the feasibility of combining enhanced tumor-to-tissue contrast and PET imaging for immunoscintigraphic tumor localization in pancreas and colon carcinoma bearing nude mice. Contrast enhancement was obtained with a multistep targeting technique that consists of the sequential administration of an antitumor/antihapten bispecific antibody (BS-MAb), a blocker to saturate the antihapten binding sites of the BS-MAb that remains in circulation, and a low molecular weight Ga chelate, labeled with the positron emitter 68Ga, which serves as the hapten. To evaluate the efficacy of this pretargeting technique for breast cancer localization, we synthesized a BS-MAb from the F(ab')(2) fragments of the anti-MUC1 MAb 12H12 which reacts with the vast majority of human breast carcinomas, and the F(ab') fragment of an anti-Ga chelate MAb using a bifunctional chemical linker. The BS-MAb was tested for its affinity and its biokinetics in nude mice bearing a human mammary carcinoma. Equilibrium binding of the BS-MAb for mammary carcinoma cells was low (1.2 x 10(7) M(-1)) while the binding capacity of cells was high (8.4 x 10(6) BS-MAbs per cell). Tumor uptake of the 67Ga labeled chelate in pretargeted animals was to 5.8 +/- 0.8% iD/g resulting in a tumor-to-blood ratio of 2.6 at 1h postinjection. This compares with a ratio of 0.65 and 0.85 obtained with 125I-labeled native 12H12 at 24h and 48h postinjection. No difference in the tumor uptake of both the 68Ga and 67Ga labeled chelate was observed. PET imaging of mice, started 1h postinjection of the 68Ga chelate, clearly visualized all tumors.

Abdominal pseudocysts and ascites formation after ventriculoperitoneal shunt procedures. Report of four cases.

The authors report three patients with abdominal pseudocysts and one with cerebrospinal fluid ascites as late complications of ventriculoperitoneal shunts. The presenting signs and symptoms were those of intraabdominal abnormality, with no neurological symptoms suggestive of shunt malfunction.

Determination of the free fraction and relative free fraction of drugs strongly bound to plasma proteins.

This report describes a new method for the determination of protein binding and relative protein binding (ratio of f(u) for different species) for compounds strongly bound to proteins. The method used is based on the distribution of the drug in plasma water, plasma proteins, and blood cells. Incubations were performed in diluted plasma. In diluted plasma, the erythrocyte/plasma distribution was determined with greater precision than in undiluted plasma. Formulae were derived for calculating f(u) in undiluted plasma based on the f(u) values determined in diluted plasma. These formulae are also valid in the event of more than one independent binding site in plasma. All incubations with plasma of different species were performed using rat erythrocyte suspensions, thereby making it possible for relative f(u) values in different species to be calculated without knowing the absolute free fractions. This method avoids the determination of the erythrocyte/buffer distribution in cases where it is sufficient to know relative f(u) values (e.g., exposure comparisons). Relative protein binding can also be quantified for compounds that tend to adsorb to surfaces of vials or test tubes, thus avoiding errors caused by adsorption when quantifying the drug in a protein-free aqueous solution. This method was validated by making comparisons of free fraction values obtained by the method herein described with those obtained by either ultrafiltration or equilibrium dialysis for two compounds that bind predominantly to albumin and another compound that binds to alpha(1)-acid glycoprotein. The results confirm our method produces identical free fractions in comparison with other established techniques. In addition, the range of applications of our method is much wider.

[Immunoscintigraphy of breast cancer xenografts. 99m-Tc-labeled anti-mucin monoclonal antibodies BM-7 and 12H12]. / Immunszintigraphie xenotransplantierter Mammakarzinome. 99mTc-markierte monoklonale Anti-Muzinantikörper BM-7 und 12H12.

The present study was undertaken to evaluate the correlation of the favorable in vitro characteristics of the anti-mucin Mabs 12H12 and BM-7 with high tumor accumulation in vivo. They were labeled with 99mTc; their biodistribution in nude mice bearing mammary tumor xenograft AR was examined and immunoscintigraphy was performed after 24 h. 99mTc-labeling of the Mabs 12H12 and BM-7 led to tumor uptakes of 20.7% and 8.8% ID/g, respectively, after 48 h. Tumor-to-muscle ratios were 31 (12H12) and 18 (BM-7). Tumor xenografts were clearly visualized in immunoscintigrams. Combination of Mab 12H12 and 99mTc provides high tumor-to-tissue ratios shortly after administration.

Synthetic macromolecular drug carriers: biodistribution of poly[(N-2-hydroxypropyl)methacrylamide] copolymers and their accumulation in solid rat tumors.

To optimize polymer design for tumor directed drug delivery, the fate and the total body distribution of soluble synthetic macromolecules, derived from copolymers of [(N-2-(hydroxypropyl)methacrylamide] (HPMA) were monitored scintigraphically after radiolabeling with 131I during a seven day time window. Equimolar concentrations of radioiodinated copolymers of HPMA with small amounts of methacryloyltyrosinamide (pHPMA) differing in molecular weight (23.4 kD, 27.3 kD, 30.5 kD, 44 kD, 58.4 kD, 60.1 kD) were injected intravenously into Copenhagen rats bearing Dunning prostate carcinomas (subline R3327-AT1). Scintigraphic data were validated by determining absolute amounts of [131I]pHPMA in both tumor tissue and normal organs after sacrificing the animals. Copolymers were cleared from blood circulation in a molecular-weight dependent manner, either via excretion or by extravasation into normal and neoplastic tissues. While distribution patterns for pHPMAs in normal organs were quite similar, absolute amounts of copolymer uptake differed. The higher the molecular weight, the more radioactivity was taken up by the organs. Highest amounts of radioactivity were seen in the lung, liver, and spleen. In solid tumors, kinetics of pHPMA accumulation was clearly dependent on molecular weight. pHPMAs below the renal threshold peaked at 24 hours p.i. and then remained constant. In contrast, copolymers above the renal clearance threshold displayed a continuous accumulation reaching a significantly higher tumor uptake, presumably due to the very small or non existent polymer release from tumor tissue. Absolute amounts of tumor uptake determined by dissection analysis were 0.5 +/- 0.1% of injected dose/g tissue for the 27.3 kD pHPMA and 1.2 +/- 0.1% for the 60.1 kD pHPMA, respectively. In conclusion, our results demonstrate the influence of the molecular weight of the synthetic polymer pHPMA on plasma circulation time, excretion and organ clearance. While pHPMAs are cleared from all normal tissues except the spleen quite effectively, these polymers accumulate in solid tumors in a size dependent manner, due to the well known "enhanced permeability and retention" (EPR) effect. These data are of fundamental interest for ongoing studies on the pharmacokinetics of synthetic polymers, especially when these molecules are conjugated with targeting moieties and therapeutic or diagnostic agents.