Anatomical study of serotonergic innervation and 5-HT(1A) receptor in the human spinal cord.

Serotonergic innervation of the spinal cord in mammals has multiple roles in the control of motor, sensory and visceral functions. In rats, functional consequences of spinal cord injury at thoracic level can be improved by a substitutive transplantation of serotonin (5-HT) neurons or regeneration under the trophic influence of grafted stem cells. Translation to either pharmacological and/or cellular therapies in humans requires the mapping of the spinal cord 5-HT innervation and its receptors to determine their involvement in specific functions. Here, we have performed a preliminary mapping of serotonergic processes and serotonin-lA (5-HT(1A)) receptors in thoracic and lumbar segments of the human spinal cord. As in rodents and non-human primates, 5-HT profiles in human spinal cord are present in the ventral horn, surrounding motoneurons, and also contact their presumptive dendrites at lumbar level. 5-HT(1A) receptors are present in the same area, but are more densely expressed at lumbar level. 5-HT profiles are also present in the intermediolateral region, where 5-HT(1A) receptors are absent. Finally, we observed numerous serotonergic profiles in the superficial part (equivalent of Rexed lamina II) of the dorsal horn, which also displayed high levels of 5-HT(1A) receptors. These findings pave the way for local specific therapies involving cellular and/or pharmacological tools targeting the serotonergic system.

Potential adverse effects of cyclosporin A on kidneys after spinal cord injury.

STUDY DESIGN: Cell transplantation strategies are gaining increasing interest for spinal cord injury (SCI) with the objective of promoting spinal cord repair. To avoid allogenic graft rejection, an adequate immune suppression is required, and one of the most potent and commonly used immunosuppressives is cyclosporin A (CsA). In SCI, permanent sensory motor loss is combined with modifications of drug absorption, distribution and elimination. OBJECTIVES: The objectives of this study were to thoroughly explore histological and functional outcomes of CsA treatment in a rat model of spinal cord compression. SETTING: Experiments were carried out at the Institute for Neurosciences of Montpellier (France), the Integrative Biology of Neurodegeneration Laboratory (Spain) and in the Novartis Institutes for BioMedical Research (Switzerland) for CsA blood concentration determination. METHODS: We first evaluated histological outcomes of CsA treatment on kidneys and spinal cord after SCI. We then investigated whether SCI modified CsA blood concentration. Finally, using behavioral analysis, we assessed the potential CsA impact on functional recovery. RESULTS: When spinal-cord-injured rats were treated with a CsA dose of 10 mg kg(-1) per day, we observed deleterious effects on kidneys, associated with modifications of CsA blood concentration. Adding an antibiotic treatment reduced kidney alteration without modifying CsA blood concentration. Finally, we showed that CsA treatment per se modified neither functional recovery nor lesion extension. CONCLUSION: This study pinpoints the absolute requirement of careful CsA monitoring in the clinical setting for patients with SCI to minimize potential unexpected effects and avoid therapeutic failure.

Dosimetry-based therapy in metastatic breast cancer patients using 90Y monoclonal antibody 170H.82 with autologous stem cell support and cyclosporin A.

Radioimmunoconjugates of 170H.82 (m170), a panadenocarcinoma monoclonal antibody, are effective for imaging primary and metastatic breast cancer. To evaluate m170 as a targeting agent for therapy, we developed (111)In- and 90Y-2-iminothiolane-2-[p-(bromoacetamido)benzyl]-1,4,7,10 tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid-m170 immunoconjugates with 99% purity by molecular sieving and immunoreactivity comparable to unmodified antibody. (111)In-m170 pharmacokinetic studies were performed prior to each therapy to determine the maximum dose of 90Y-m170 that could be administered without exceeding a limit of 800 rad to the liver, lungs, or kidneys or 250 rad to the whole body or bone marrow for each of three cycles of treatment. Peripheral blood stem cells (PBSCs) were harvested and cyclosporin A (5 mg/kg twice daily) was administered as strategies to ameliorate myelosuppression and prevent the development of HAMA, respectively. An (111)In imaging/pharmacokinetic study was performed, and the 90Y dose was calculated and administered. The liver was the 90Y dose-limiting organ. The mean and range of calculated doses (in rad/mCi) for the five patients evaluated were as follows: whole body, 2.3 (2.1-2.4); liver, 17.8 (12.7-22.2); lung, 6.4 (4.8-7.2); kidney, 6.9 (6.3-11.5); marrow, 3.6 (1.9-4.4); and tumors (n = 25), 71.5 (14.1-141.5). Of the three patients treated, with doses of 37, 54, and 57 mCi of 90Y, one had a partial response, one had measurable tumor reduction but less than a partial response, and one had stable disease for more than 1 month. PBSCs prevented prolonged myelosuppression. The therapeutic responses, coupled with an absence, thus far, of significant adverse sequelae, suggest that this dosimetry-based approach combined with PBSCs may lead to effective therapy when higher 90Y doses are reached.

The effects of circulating antigen on the pharmacokinetics and radioimmunoscintigraphic properties of 99m Tc labelled monoclonal antibodies in cancer patients.

UNLABELLED: PURPOSE. This article reports the pharmacokinetics, radiation dosimetry and radioimmunoscintigraphy (RIS) of two (99m)Tc-labelled monoclonal antibodies (MAb) used to detect cancer. METHODS: The effects of circulating antigen in female cancer patients are explored and their effects on the ability of these MAbs to effectively perform as RIS agents noted. To illustrate the effects of circulating antigen, data using MAb B43.13 (OVAREX, AltaRex Corp., Waltham, MA, USA) from a Pilot study in ovarian cancer patients are presented. The results from a Phase II study of MAb 170H.82 (Tru-Scint AD, BIOMIRA INC., Edmonton, Alberta, Canada) in patients with primary and locally recurrent breast cancer were used to portray the biodistribution patterns when no circulating antigen is present. Data from planar gamma camera images were obtained for both groups and used for pharmacokinetic and radiation dosimetry analyses. RESULTS: A pharmacokinetic analysis indicated a shorter residence time and higher clearance of (99m)Tc-MAb-B43.13 that was ascribed in part to the circulating CA 125 antigen in this group of ovarian cancer patients. CONCLUSION: These clearance patterns resulted in acceptable, though higher radiation doses to the spleen and urinary bladder wall for these patients when compared to the MAb-170H.82 group. Both MAbs were found to produce acceptable radioimmunoscintigraphic images

Pre-operative assessment of axillary lymph node status in patients with breast adenocarcinoma using intravenous 99mtechnetium mAb-170H.82 (Tru-Scint AD).

Immunoscintigraphy of the axilla has potential utility for the diagnostic and prognostic assessment of patients with breast adenocarcinoma. mAb-170H.82 is a murine monoclonal antibody (mAb) derived against synthetic Thomsen-Friedenreich (TF) antigen. Tru-Scint AD, a 99mTc-mAb-170H.82 immunoconjugate, has previously been shown to localize in various human adenocarcinomas. The purpose of this study was to evaluate the accuracy of this immunoconjugate in the pre-operative assessment of axillary lymph nodes in patients with known breast adenocarcinoma. Sixteen patients with documented primary breast cancer were injected intravenously with 1 mg of immunoconjugate (radioactivity 1.8 GBq) and imaged 22-24 hrs post-injection. Both planar and single photon emission computed tomographic (SPECT) images were obtained and reviewed in a blinded fashion. Imaging results were compared with surgical and pathological findings. Seven of 16 patients were found to have histologically positive axillary nodes: 5 of these sites were detected by immunoscintigraphy (sensitivity = 71%). Nine patients had pathologically disease-free axillary nodes: only 1 of these was misidentified as positive by immunoscintigraphy (specificity = 89%). These results suggest that immunoscintigraphy with 99mTc-mAb-170H.82 has promise in the detection of axillary lymph node involvement in patients with breast cancer. Further studies are warranted to define the role of immunoscintigraphy in axillary staging.

Radioimmunoscintigraphy in patients with breast adenocarcinoma using technetium-99m labelled monoclonal antibody 170H.82: report of a phase II study.

Fifty-three women with clinical evidence of adenocarcinoma of the breast were studied with technetium-99m labelled monoclonal antibody (MAb) 170H.82 at protein doses of 1, 2 and 4 mg. An overall per lesion efficacy of 83.5% sensitivity and 97.7% positive predictive value was obtained. Efficacy appears higher in lesions restricted to the breast and local regional disease than systemic metastases. For the 2 mg dose the breast/local regional disease efficacy was 90% sensitivity and 90.2% positive predictive value. The biodistribution of this MAb was best represented by a two-compartment model with a distribution-phase half-life of 4.0+/-1.4 h, followed by an elimination-phase half-life of 39.6+/-6.6 h. In all six patients studied, the critical organ was the kidney, with a mean radiation absorbed dose of 37+/-6.9 mGy/GBq. The accuracy of this imaging technique allows the development of diagnostic strategies for the routine use of the compound in patients with breast cancer.

Radioimmunoscintigraphy with Tc-99m labeled monoclonal antibody 170H.82 in suspected primary, recurrent, or metastatic breast cancer.

Radioimmunoscintigraphy (RIS) with Tc-99m labeled 170H.82, an intact murine monoclonal antibody (MAb) was performed in 27 patients with suspected primary, recurrent, or metastatic breast cancer. For radioimmunoimaging the authors used a double-headed, high-resolution gamma camera. The lesions detected by RIS were evaluated by histopathology, and for metastatic disease by CT, MRI, and bone scan. If the evaluation was made lesion by lesion, RIS with Tc-99m MAb 170H.82 showed 23 true-positive results, 3 false-negative results, 9 true-negative results, 3 false-positive results for primary and recurrent disease; sensitivity was 88% and specificity was 75%. In patients with distant metastases, detection rate was 67% (12 true-positive results and 6 false-negative results). All patients had normal baseline values for human anti-mouse antibodies (HAMA) and 6 weeks after RIS with 2 mg MAb, only 2 of 23 patients had developed a HAMA response (9%). No adverse effects were observed. The authors' clinical experience shows that RIS with MAb 170H.82 is a valuable additional method for detecting breast cancer, especially in patients with uncertain primaries or scar tissue after breast conserving treatment in which differential diagnosis is very difficult.

Improvement of radiation treatment planning in squamous-cell head and neck cancer by immuno-SPECT.

UNLABELLED: Previous studies have shown high accuracy for immunoscintigraphy with 99mTc-MAb-174 in patients with squamous-cell carcinoma of the head and neck region compared to CT and MRI. We conducted a prospective study to determine if immunoscintigraphy provides additional diagnostic information for radiation treatment planning. METHODS: Radioimmunoscintigraphy (RIS) was performed on 40 patients (planar, whole-body, SPECT) with histologically confirmed squamous-cell carcinoma (30 primary tumors, 10 recurrences) after injection of the 99mTc (1.1 GBq) labeled monoclonal anti-squamous-cell cancer antibody 174H0.64 (murine IgG1). Results were combined with information obtained by clinical examination, sonography, panendoscopy and x-ray CT. The strategy for radiation treatment and the required treatment volumes were defined with and without immunoscintigraphical findings. RESULTS: Additional diagnostically relevant information from RIS was obtained from 10 patients (25%) with advanced tumors or recurrences. In three patients (7.5%), the treatment volume had to be extended. The therapeutic strategy for seven patients (17.5%) had to be changed due to the detection of metastatic disease beyond the head and neck region. RIS of patients with squamous-cell cancers of the head and neck region with 99mTcMAb-174H0.64 enabled the detection of tumors that were not depicted by other conventional diagnostic imaging procedures. CONCLUSION: The use of RIS in radiation treatment planning of advanced tumors of the head and neck region appears to yield important diagnostic information that may alter patient management.

Technetium-99 m labelling of DD-3B6/22 antifibrin monoclonal antibody fragment Fab' for thrombus imaging.

The antifibrin DD-3B6/22 monoclonal antibody Fab' fragment, a murine immunoglobulin, IgG3, has been labelled with technetium-99 m (99mTc) via a transchelation reaction, to specific activity in excess of 30 mCi/mg protein. The radiolabelling of Fab' was dependent on time, temperature, pH, antibody concentrations and nature of intermediary transchelation complex used. The resultant radioconjugate was stable in vitro and in vivo. Blood clearance of 99mTc-Fab' in rat followed two compartment kinetics with the half time of the fast phase being 0.5 h. The main route of excretion was via the kidneys with little uptake indicated by other tissues. The results suggest that the inherent specificity of the antibody, small molecular size, rapid plasma clearance, high specific radioactivity, together with the physical properties of the 99mTc label, combine to make this labelled monoclonal antibody (MoAb), potentially suitable as a radiopharmaceutical for the scintigraphic detection of thrombi in humans.

A novel technetium-99m labeled monoclonal antibody (174H.64) for staging head and neck cancer by immuno-SPECT.

A novel murine monoclonal antibody (MAb 174H.64) was labeled with 99mTc by a direct method. MAb 174H.64 detects a cytokeratin-associated antigen which is expressed by over 90% of all squamous cell carcinomas. Panendoscopy, sonography and computerized tomography scan were performed in all cases as well as magnetic resonance imaging (in selected patients). Pre-operative immunoscintigraphy was performed in 21 patients with histologically proven primary carcinomas (18 with remaining primary tumors and 3 with lymph node recurrences). Scintigraphic images were obtained 4-6 h after injection of 1.1 GBq of the 99mTc-labeled antibody (2 mg). Late images were acquired 18 to 24 h after injection. Single-Photon-Emission-Computed Tomography (SPECT) of the head and thorax was performed in all patients. The primary tumors were immunoscintigraphically visualized in all 18 patients with remaining primary tumor. Fifteen of 18 loco-regional lymph node metastases were visualized by immunoscintigraphy (the smallest lesions had a diameter of < 1 cm), in one patient lymph node metastases were detected by immunoscan only. Two metastatically involved lymph nodes were identified by histology only (micrometastases). Distant metastases were present in 3 patients, of which two were identified by immunoscintigraphy. Immuno-SPECT according to this method was a sensitive and specific imaging modality for preoperative staging of patients with squamous cell carcinoma of the head and neck and detected lymph node metastases with higher accuracy than conventional clinical and imaging modalities.

An improved method for labeling monoclonal antibodies with samarium-153: use of the bifunctional chelate 2-(p-isothiocyanatobenzyl)-6- methyldiethylenetriaminepentaacetic acid.

Samarium-153 (153Sm) radioimmunoconjugates of the monoclonal antibody K-1-21 were produced using the bifunctional chelate 2-(p-isothiocyanatobenzyl)-6- methyldiethylenetriaminepentaacetic acid (Mx-DTPA). The specific activity (up to 150 MBq mg-1) and percent retained immunoreactivity (greater than 75%) were similar to that of 153Sm-K-1-21 conjugates formed with cyclic DTPA anhydride (cDTPAa). In vivo biodistribution studies showed specific localization of 153Sm-Mx-DTPA-K-1-21 to target antigen implants and higher blood pool and lower uptake in liver, spleen, kidney, and bone when compared to 153Sm-cDTPAa-K-1-21. The improved in vivo distribution of 153Sm-Mx-DTPA-K-1-21 should result in lower radiotoxicity to nontarget tissues when used for radioimmunotherapy purposes.

Evaluation of thrombus detection in a rabbit model using a technetium-99m-labelled anti-fibrin monoclonal antibody.

Technetium-99 m (99mTc)-labelled conjugates of an anti-fibrin monoclonal antibody, DD-3B6/22, have been assessed for their detection of vascular thrombi in a rabbit model. DD-3B6/22 binds to a D-dimer epitope present on cross-linked fibrin but absent from the fibrin monomer or fibrinogen. Injection of a 99mTc-labelled Fab' fragment of DD-3B6/22 allowed delineation of model thrombi as early as 30 min postinjection (p.i.) with optimal localization at 4-5 h. Thrombus label uptake at 4 h p.i. was 0.304 +/- 0.106% injected dose/g (% ID/g) compared with 0.022 +/- 0.001% ID/g after the injection of a control Fab' fragment. These results suggest that the 99mTc-labelled Fab' fragment of DD-3B6/22 has excellent potential for scintigraphic detection of vascular thrombi in humans.

Preclinical evaluation of 99m technetium-labeled DD-3B6/22 Fab' for thrombus detection.

Injection of 99mTc-labeled Fab' fragments of the anti-fibrin antibody DD-3B6/22, in the baboon, resulted in clear visualisation of both fresh and aged autologous thrombi by gamma scintigraphy. Whole body scintigraphy, pharmacokinetics and urine analysis showed rapid renal excretion of the conjugate with little accumulation of label in other organs. 99mTc-DD-3B6/22 Fab' appears a suitable candidate for further investigation as a radioimaging agent for thrombi in humans.

Growth and metastasis of human bladder cancer xenografts in the bladder of nude rats. A model for intravesical radioimmunotherapy.

A potentially useful therapeutic approach to the treatment of human bladder cancer is intravesical therapy with radiolabelled monoclonal antibodies (MAbs). We have established an animal model to study this approach. Inoculation of cloned 2B8 cells derived from the human bladder cancer cell line, UCRU-BL-17, into the bladder wall of nude rats pre-irradiated with 900 rads, resulted in local tumour growth in 39/40 (97.5%) animals, with invasion or metastases to distant organs in 25% of cases. Both the bladder tumours and the metastases were morphologically similar to the original biopsy sample from which the cell line, UCRU-BL-17, was established. The cells were of human origin, as shown by expression of HLA antigens, Alu probing, and cytogenetic analysis. Preliminary studies indicated that i.p. injection of anti-human bladder cancer monoclonal antibody (MAb), BLCA-38, radiolabelled with either iodine 131 or samarium 153 (153Sm), resulted in tumour localisation, with tumour-to-blood ratios of 5.04 (131I), and 4.3 and 3.1 (153Sm) respectively. We now aim to examine the efficacy of the intravesical route for radioimmunotherapy in the nude rat model. This model will also serve for preclinical studies on the efficacy of systemically injected radioimmunoconjugates for control of metastatic growth.

Detection of experimental thrombi in rabbits with an 131I-labelled fibrin-specific monoclonal antibody.

The detection of thrombi in rabbits has been investigated with 131I-labelled DD-3B6/22, a monoclonal antibody (Mab) reactive at high affinity (Kd = 2.68 x 10(-10) M) with human D Dimer (DD). DD-3B6/22 bound well to both "fresh" and "aged" human clots in an in vitro assay but showed poor binding to rabbit clots. However, reactivity was restored to rabbit blood if it was seeded, before clotting, with human DD covalently coupled to Sepharose beads. Thus, a rabbit model was developed in which blood was allowed to clot around DD-Sepharose beads introduced into the jugular vein. Gamma camera imaging showed that intact 131I-labelled DD-3B6/22 localised to these clots within 24 h. Uptake at this time was 0.202 +/- 0.012% injected dose per gram (%ID/g) compared with 0.086 +/- 0.018%ID/g after injection of control antibody. 131I-labelled F(ab')2 fragments of DD-3B6/22 allowed earlier scintigraphic detection of the clot which was evident 4 h after injection. Uptake in the clot at 24 h was 0.154 +/- 0.038 %ID/g compared with 0.109 +/- 0.027 %ID/g for a control F(ab')2. As antigen levels in the clot are estimated to be less than 300 micrograms DD, thus representing a very small human clot, the DD-3B6/22 Mab would appear to have a good potential for the sensitive detection of thrombi in a clinical setting.

Labeling of monoclonal antibodies with samarium-153 for combined radioimmunoscintigraphy and radioimmunotherapy.

The labeling of a monoclonal antibody K-1-21 with 153Sm has been investigated using the bifunctional chelate cyclic diethylenetriaminepentaacetic acid (DTPA) anhydride. Labeling efficiencies greater than 60% were obtained using high specific activity [153Sm]chloride and a cDTPAa:MAb conjugation ratio of 20:1. The resultant labeled antibody had a s.a. greater than 150 MBq.mg-1 and a % retained immunoreactivity greater than 90%. Imaging and biodistribution studies in a rat model demonstrated that specific uptake of 153Sm-K-1-21 into s.c. implants of the target antigen could be clearly detected in scintigrams at 6 days p.i. The specific uptake (1.90 +/- 0.45% ID/g, 19.95 +/- 2.20 Implant:Blood ratio) compared favorably to 131I- and 111In-labeled K-1-21 (2.52 +/- 0.20 and 3.33 +/- 0.20% ID/g, 7.69 +/- 0.45 and 10.10 +/- 0.60 I:B, respectively). Labeling of MAbs with 153Sm for combined scintigraphy/therapy is feasible at clinically appropriate specific activities using cDTPAa, with the resultant conjugates retaining immunoreactivity and in vivo antigen localization.

Anti-fibrin monoclonal antibodies for radioimmunodetection: preliminary assessment in a rat model system.

The D Dimer (DD) site formed by linkage of D domains from adjacent fibrin (FN) molecules is unique to cross-linked FN and its degradation products and is not found in FN monomer or fibrinogen (FB). Thus monoclonal antibodies (MAb) reactive to DD should have a very suitable specificity for in vivo thrombus detection. Two anti-DD MAbs have been labelled with 131-I and assessed as scintigraphic agents in a normal rat model system. Each rat received 3 sc implants of antigen covalently coupled to Sepharose beads: 1) Human DD 2) Human FB 3) Glycine (GL) (control). Scintigraphic images taken 7 days after injection of 131-I anti DD MAb showed clear localisation of both anti-DD MAbs to the DD implant rather than to the FB or GL implants with no localisation in normal tissues. This was confirmed in biodistribution studies. Injection of anti-DD MAbs DD-3B6/22 and DD-IC3/108 resulted in DD: blood ratios of 10.4 +/- 0.6 and 4.9 +/- 0.3 respectively. These results suggest that anti-DD MAbs will have potential for thrombus radioimmunodetection.

Renal tubular receptor imaging with iodine-131-labeled peanut lectin: pharmacokinetics and renal clearance mechanism in animals.

Intravenously administered peanut lectin (PNA), iodinated with 131I ([131I]PNA), is rapidly cleared from the plasma by the kidneys in dogs (clearance [total body] = 17.52 +/- 8.74 ml/min). Dynamic gamma camera renal scintigraphy demonstrated renal accumulation and excretion phases of the [131I]PNA renogram in dogs and rabbits (% injection dose-at-peak = 21.8 +/- 3.3% and 19.6 +/- 4.3%, time-to-peak = 44.6 +/- 4.8 min and 37.2 +/- 6.9 min, respectively). Immunoperoxidase staining of kidney sections, following i.v. administered PNA, demonstrated predominant accumulation by the proximal tubules of mice, rabbits, and dogs. The basement membrane was intensely stained at early times p.i. while intracellular and luminal PNA was evident within 1 hr. Urine analysis confirmed the presence of intact [131I]PNA in the bladder contents, while protein degradation products, and a small percentage of the free iodide (less than 5%) were noted within 1 hr p.i. The relative proportion of free iodide increased at later times p.i. (greater than 6 hr). A receptor mediated excretion mechanism is proposed for the clearance of PNA and may be useful for the study of renal tubular function.