[(68)Ga-labeled peptides for clinical trials - production according to the German Drug Act: the Göttingen experience]. / (68)Ga-markierte Peptide für klinische Studien. Herstellung gemäß Arzneimittelgesetz: Göttinger Erfahrungen.

The AMG implies far-reaching implications for the synthesis of new radiopharmaceuticals for clinical trials. AIM, METHODS: As a part of the DFG-funded Clinical Research Group (KFO 179) a project designated "Immuno-PET for assessment of early response to radiochemotherapy of advanced rectal cancer" was initiated. This trial is focused on a trivalent bispecific humanized monoclonal antibody, and a 68Ga-labeled peptide. Following the new regulatory framework we established a GMP-compliant cleanroom laboratory and applied for a manufacturing permission. RESULTS: During the project constructural, personnel and organizational conditions for a successful application were established, including a quality management system. A GMP-conform cleanroom laboratory class C was constructed, equipped with a two-chamber lock. The actual manufacturing is performed in a closed system with subsequent sterile filtration. The manufacturing processes have been automatised and validated as well as the necessary quality controls. The manufacturing permission was granted after an official inspection. CONCLUSIONS: The new German Drug Act is considered as a break in the production practice of nuclear medicine. The early involvement and communication with the authorities avoids time-consuming and costly planning errors. It is much to be hoped that the new legal situation in Germany will not cause serious impairments in the realization of clinical trials in German nuclear medicine.

[Clinical evaluation of a new thyroglobulin immunoradiometric assay in the follow-up of differentiated thyroid carcinoma]. / Klinischer Stellenwert eines neuen Thyreoglobulin-immunoradiometrischen Assays in der Nachsorge des differenzierten Schilddrüsenkarzinoms.

AIM: Formal and clinical comparison of a new 3 (rd) -generation-Tg-IRMA (3-G-IRMA; Dynotest Tg-plus) with a conventional Tg-IRMA (3-G-IRMA; SELco Tg-assay) for patients with differentiated thyroid carcinoma. In addition we evaluated, if thyroglobulin (Tg) levels above a specific threshold concentration indicate the need for further investigations for residual disease. PATIENTS, METHODS: Tg concentration of 105 sera of 93 consecutive patients with a differentiated thyroid cancer was determined with both assays and compared at different cut-off values (Dynotest Tg-plus: 0.2, 1, 2 ng/ml; SELco Tg-assay: 0.5, 1, 2 ng/ml) with the clinical results in respect to the corresponding TSH concentration. RESULTS: Tg concentration did not show any significant difference (SELco Tg-assay 0.5 ng/ml, Dynotest Tg-plus 0.2 ng/ml). The Tg-values of both assays correlated with 97%. However, correlation of recovery in both assays was small (40%). The sensitivities and specificities of both assays at different cut-offs and TSH values did not reveal significant differences. In patients with TSH concentration > 30 micro U/ml the functional assay sensitivity was superior to arbitrary cut-offs in the decision to start further evaluations. CONCLUSIONS: In our study neither formal nor clinical significant differences between two Tg-assays were found. In a hypothyroid patient (TSH > 30 micro U/ml, Tg concentration exceeding the functional assay sensitivity) further investigations for residual disease are warranted. Higher thresholds are of limited value, due to an unacceptably high rate of false negative results.

(99m)Tc-E-selectin binding peptide for imaging acute osteomyelitis in a novel rat model.

INTRODUCTION: In the present study, (99m)Tc-radiolabelled E-selectin binding peptide ((99m)Tc-IMP-178) was investigated for its potential to image acute pyogenic osteomyelitis in a new animal model. Intraindividual comparisons were performed using an irrelevant peptide ((99m)Tc-IMP-100) to demonstrate specificity. METHODS: An acute pyogenic osteomyelitis was induced by injecting 0.05 ml of 5% sodium morrhuate and 5x10(8) CFU of Staphylococcus aureus into the medullary cavity of the right tibia in 16 rats. Sixteen additional rats served as untreated controls. Whole-body imaging of pyogenic (n=4) and untreated (n=4) animals was performed continuously during the first 8 h (12 MBq i.v. of (99m)Tc-IMP-178 and (99m)Tc-IMP-100 for control), and one further single image was acquired after 16 h p.i. Tissue biodistribution studies were performed in 12 rats with an acute pyogenic osteomyelitis and in 12 untreated rats 1, 4 and 24 h after injection. Data of the histological/radiological and haematological investigations were obtained in all animals. RESULTS: Histopathologically, 15 of 16 treated rats (93%) developed an acute pyogenic osteomyelitis showing a major infiltration of the bone marrow by polymorphonuclear leukocytes as well as the formation of sequestra. Haematologically, the number of leukocytes increased by 100%, the lymphocytes by 11% and the granulocytes decreased by 39%. After i.v. injection, (99m)Tc-IMP-178 rapidly cleared from the body resulting in good scintigraphic target-to-background (T/B) ratios. The highest uptake of the tracer in the pyogenic bone was observed at 60 min p.i. (0.43+/-0.02% ID.g-1 for (99m)Tc-IMP-178 and 0.30+/-0.02% ID.g-1 for (99m)Tc-IMP-100), resulting in a higher osteomyelitis-to-healthy collateral ratio with T/B of 2.40+/-0.65 ((99m)Tc-IMP-178) compared with 1.85+/-0.48 ((99m)Tc-IMP-100). No adverse reactions were seen after injection of (99m)Tc-IMP-178. CONCLUSIONS: (99m)Tc-IMP-178 allows imaging of an acute osteomyelitic lesions, presumably by interaction of (99m)Tc-IMP-178 with activated upregulated vascular endothelium.

Biodistribution, blood half-life, and receptor binding of a somatostatin-dextran conjugate.

Derivatives of somatostatin (sms) are attracting increasing interest as part of the treatment of several cancer diseases expressing sms receptors (srs). Radiolabeled sms analogs can additionally be used for systemic radiotherapy and for diagnostic investigations. Glycosylated sms-14 (sms-dextran70) was characterized regarding in vitro srs binding, biodistribution, and blood half-life in mice. Rat brain cortex membranes (expressing srs 2) were used for the srs binding study. Tyr3-Octreotide was used as positive control. The binding data were analyzed by competition curve analysis. In the biodistribution study, the Bolton-Hunter reagent was used for the radioiodination of sms-dextran70. Organs and blood were collected at different time-points and the percentage of the injected dose per gram of tissue (%ID/g) was calculated. The conjugate was administered subcutaneously (sc). The sms-dextran70 showed high srs binding affinity (i.e., in the same nanomolar range as the reference ligand Tyr3-octreotide (IC50 approximately 2.5 nM). The blood half-life was approx 27 h after reaching maximum blood concentration 24 h postinjection. Because of the molecular weight of the conjugate (i.e., approx 75,000) being above the kidney threshold for dextran (i.e., 50,000), the digestion and excretion is assumed to be mainly through the hepatobiliary system. Increased uptake was seen in the adrenals, which are receptor-positive organs. Some accumulation was seen in the stomach, indicating certain deiodination of the conjugate label. The sms-dextran70 showed promising properties and its clinical relevance is currently being evaluated in clinical phase I-II studies.

Therapeutic advantages of Auger electron- over beta-emitting radiometals or radioiodine when conjugated to internalizing antibodies.

Recent studies suggest a higher anti-tumour efficacy of internalizing monoclonal antibodies (MAbs) when labelled with Auger electron emitters, as compared with beta-emitters. The aim of this study was to compare the anti-tumour efficacy and toxicity of the internalizing MAb, CO17-1A, labelled with Auger electron emitters (125I, (111)In) versus conventional beta(-)-emitters (131I, 90Y) in a colon cancer model, and to assess whether the residualizing radiometals may have therapeutic advantages over the conventionally iodinated conjugates. Biodistribution studies of 125I-, (111)In- or 88Y-labelled CO17-1A were performed in nude mice bearing subcutaneous human colon cancer xenografts. For therapy, the mice were injected with either unlabelled or 125I-, 131I-, (111)In- or 90Y-labelled CO17-1A IgG2a, whereas control groups were left untreated or were given a radiolabelled isotype-matched irrelevant antibody. The influence of internalization was assessed by comparing the results with those obtained with an anti-carcinoembryonic antigen (CEA) antibody which does not internalize to a relevant extent. The maximum tolerated activities (MTA) and doses (MTD) of each agent were determined. Myelotoxicity and potential second-organ toxicities, as well as tumour growth, were monitored. Bone marrow transplantation (BMT) was performed in order to enable dose intensification. Radiometals showed significantly better tumour-to-blood ratios than the respective iodinated conjugates. The MTAs of 131I- and 125I-CO17-1A without artificial support were 11.1 MBq (300 microCi) and 111 MBq (3 mCi), respectively; the MTA of the metals was reached at 4 MBq (100 microCi) for 90Y-, and at 85 MBq (2.3 mCi) for (111)In-CO17-1A. Myelotoxicity was dose limiting in all cases. BMT enabled an increase in the MTA to 15 MBq (400 microCi) of 131I-labelled CO17-1A, to 4.4 MBq (120 microCi) of 90Y-labelled CO17-1A, and to 118 MBq (3.2 mCi) of (111)In-labelled CO17-1A, while the MTA of 125I-CO17-1A had not been reached at 185 MBq (5 mCi) with BMT. Whereas no significant therapeutic effects were seen with unlabelled CO17-1A, tumour growth was retarded significantly with its radiolabelled forms. The therapeutic results were significantly (P<0.01) better with both Auger electron emitters (125I and (111)In) than with the beta-emitters, and, in accordance with the biodistribution data, a trend towards better therapeutic results was found with radiometals (more complete remissions) as compared with radioiodine. In contrast, at equitoxic doses, no significant difference was observed in the therapeutic efficacy of 131I- versus 125I-labelled non-internalizing anti-CEA antibody, F023C5. These data suggest that, at equitoxic doses, the therapeutic efficacy of internalizing MAbs labelled with Auger electron emitters, such as 125I or (111)In, is superior to that of internalizing MAbs labelled with conventional beta-emitters. The lower toxicity of Auger electron emitters may be due to the short path length of their low-energy electrons, which can reach the nuclear DNA only if the antibody is internalized (as is the case in antigen-expressing tumour tissue, but not in the stem cells of the red marrow).

Studies on the red marrow dosimetry in radioimmunotherapy: an experimental investigation of factors influencing the radiation-induced myelotoxicity in therapy with beta-, Auger/conversion electron-, or alpha-emitters.

Usually, the red marrow (RM) is the first dose-limiting organ in radioimmunotherapy. However, several studies have obtained only poor correlations between the marrow doses and the resulting toxicities. Furthermore, RM doses are mostly not determined directly but are derived from blood doses by assuming a ratio that is, over time for the respective conjugates, more or less constant between blood and marrow activities. The aim of this study was to determine, in a mouse model, this RM:blood activity ratio for various immunoconjugates, to investigate whether there may be differences between complete IgG and its fragments with various labels ((125/131)I versus (111)In, (88/90)Y, or 213Bi), and to analyze, in more detail, factors other than just total dose, such as dose rate or relative biological effectiveness factors, that may influence the resulting myelotoxicity. The maximum tolerated activities (MTAs) and doses (MTDs) of several murine, chimeric, and humanized immunoconjugates as complete IgG or fragments (F(ab)2 and Fab), labeled with beta(-)-emitters (such as 131I or 90Y), Auger electron-emitters (such as 125I or (111)In), or alpha-emitters (such as 213Bi) were determined in nude mice. Blood counts were monitored at weekly intervals; bone marrow transplantation was performed to support the assumption of the RM as dose-limiting. The radiation dosimetry was derived from biodistribution data of the various conjugates, accounting for cross-organ radiation; besides the major organs, the activities in the blood and bone marrow (and bone) were determined over time. Whereas no significant differences were found for the RM:blood ratios between various IgG subtypes, different radiolabels or various time points, differences were found between IgG and bi- or monovalent fragments: typically, the RM:blood ratios were approximately 0.4 for IgG, 0.8 for F(ab')2, and 1.0 for Fab'. Nevertheless, at the respective MTAs, the RM doses differed significantly between the three conjugates: e.g., with 131I-labeled conjugates, the maximum tolerated activities were 260 microCi for IgG, 1200 microCi for F(ab)2, and 3 mCi for Fab, corresponding to blood doses of 17, 9, and 4 Gy, respectively. However, initial dose rates were 10 times higher with Fab as compared to IgG, and still 3 times higher as compared to F(ab)2; interestingly, all three deliver approximately 4 Gy within the first 24 h. The MTDs of all three conjugates were increased by BMT by approximately 30%. Similar observations were made for 90Y-conjugates. Higher RM doses were tolerated with Auger-emitters than with conventional beta(-)-emitters, whereas the MTDs were similar between alpha- and beta(-)-emitters. In accordance to dose rates never exceeding those occurring at the single injection MTA, two subsequent injections of two doses of 80% of the single shot MTA of 131I- or 90Y-labeled Fab' and two doses of 100% of the single shot MTA of 213Bi-labeled Fab' were tolerated without increased lethality, if administered 24-48 h apart. In contrast, reinjection of bivalent conjugates was not possible within 6 weeks. These data suggest that the RM:blood activity ratios differ between IgG and fragments, although there is no anatomical or physiological explanation for this phenomenon at this point. In contrast to the current opinion, indication for a strong influence of the dose rate (or dose per unit time), not only total dose, on the resulting toxicity is provided, whereas the influence of high-linear energy transfer (alpha and Auger/conversion electrons) versus low-linear energy transfer (beta and gamma) type radiation seems to be much lower than expected from previous in vitro data. The lower myelotoxicity of Auger-emitters is probably due to the short path length of their low-energy electrons, which cannot reach the nuclear DNA if the antibody is not internalized into the stem cells of the RM.

Cholecystokinin-B/gastrin receptor binding peptides: preclinical development and evaluation of their diagnostic and therapeutic potential.

The high sensitivity of pentagastrin stimulation in detecting primary or metastatic medullary thyroid cancer (MTC) suggests widespread expression of the corresponding receptor type on human MTC. Indeed, autoradiographic studies demonstrated cholecystokinin (CCK)-B/gastrin receptors not only in >90% of MTCs but in a high percentage of small cell lung cancers and potentially a variety of gastrointestinal adenocarcinomas. In a pilot study, we have demonstrated the feasibility of radiolabeled gastrin-I to target CCK-B receptor-expressing tissues in vivo in animals and patients (T. M. Behr et al., Eur. J. Nucl. Med., 25: 424-430, 1998). The aim of the present study was to systematically optimize, in a preclinical model, suitable radioligands for targeting CCK-B receptors in vivo. For this purpose, a variety of CCK/gastrin-related peptides, all having in common the COOH-terminal CCK-receptor binding tetrapeptide sequence Trp-Met-Asp-PheNH2 or derivatives thereof, were studied. They were radioiodinated by the Iodogen or Bolton-Hunter procedures. The peptides tested were members of the gastrin- or cholecystokinin families or possessed characteristics of both, which differ by the intramolecular position of a tyrosyl moiety (occurring in native or sulfated form). Their stability and affinity were studied in vitro and in vivo; their biodistribution and therapeutic efficacy were tested in nude mice bearing s.c. human MTC xenografts. Diethylene-triamine-pentaacetate derivatives of suitable peptides were synthesized, evaluated, and labeled with (111)In. All members of the CCK or gastrin family were stable in serum (with t(1/2)s of several hours at 37 degrees C); nevertheless, the stability of those peptides was highest that bore the NH2-terminal pGlu residues (e.g., big gastrin, gastrin-I, caerulein, and others) or D-amino acids. In accordance to their comparably low affinity, nonsulfated members of the CCK family showed fairly low uptake in the tumor and other CCK-B receptor-expressing tissues (e.g., the stomach). Sulfated CCK derivatives performed significantly better but additionally displayed a high uptake in normal, CCK-A receptor-expressing tissues (such as the liver/gallbladder, pancreas, and bowel). Best tumor uptake and tumor:nontumor ratios were obtained with members of the gastrin family, probably because of their selectivity and affinity for the CCK-B receptor subtype. Pilot therapy experiments in MTC bearing animals showed significant antitumor efficacy as compared with untreated controls. (111)In-Labeled diethylene-triamine-pentaacetate derivatives of minigastrin showed excellent targeting of CCK-B receptor-expressing tissues in animals and a normal human volunteer. These data suggest that CCK/gastrin analogues may be a useful new class of receptor binding peptides for diagnosis and therapy of CCK-B receptor-expressing tumors, such as MTC or small cell lung cancer. Nonsulfated gastrin derivatives may be preferable because of their CCK-B receptor selectivity, and hence, lower accretion in normal CCK-A receptor-expressing organs. Further preclinical as well as clinical studies are ongoing.

Radioimmunotherapy of small volume disease of colorectal cancer metastatic to the liver: preclinical evaluation in comparison to standard chemotherapy and initial results of a phase I clinical study.

At the time of surgery, occult metastases (micrometastases) are present in more than 50% of colorectal cancer patients, and the liver is the most frequent site of apparent metastatic disease. Frequently, adjuvant chemotherapy is unable to prevent tumor recurrence. Thus, novel therapeutic strategies are warranted. The aim of this study was to establish a model of human colon cancer metastatic to the liver of nude mice, to assess, in this setting, the therapeutic efficacy of radioimmunotherapy (RAIT) compared to standard chemotherapy and to evaluate, in a Phase I/II trial, the toxicity and therapeutic efficacy of RAIT in colorectal cancer patients with small volume disease metastatic to the liver. Multiple liver metastases of the human colon cancer cell line GW-39 were induced by intrasplenic injection of a 10% tumor cell suspension. Whereas controls were left untreated, therapy was initiated on day 10 or 20 after tumor inoculation with the 131I-labeled, low affinity anticarcinoembryonic antigen (anti-CEA) monoclonal antibody (MAb), F023C5 (Ka = 10(7) liters/mol), or the high-affinity anti-CEA MAb, MN-14 (Ka = 10(9) liters/mol), or chemotherapy (5-fluorouracil/leucovorin (folinic acid) versus irinotecan) at their respective maximum tolerated doses (MTDs). Twelve colorectal cancer patients with small volume disease metastatic to the liver (all lesions < or = 2.5 cm) were entered into a mCi/m2-based Phase I dose escalation study with 131I-labeled humanized version of MN-14, hMN-14. The patients were given single injections, starting at 50 mCi/m2 and escalating in 10-mCi/m2 increments. The MTD was defined as the dose level at which < or = 1 of 6 patients develop grade 4 myelotoxicity. In the mice, untreated controls died from rapidly progressing hepatic metastases at 6-8 weeks after tumor inoculation. The life span of mice treated with 5-fluorouracil/leucovorin was prolonged for only 1-3 weeks, whereas irinotecan led to a 5-8-week prolongation. In contrast, at their respective MTDs, the 131I-labeled low-affinity anti-CEA MAb, F023C5, led to a 20% permanent cure rate, and the high affinity MAb, MN-14, led to an 80% permanent cure rate, when therapy was initiated at 10 days after tumor inoculation. In the 20-day-old tumor stage, although it prolonged life, 131I-F023C5 was unable to achieve cures, whereas 131I-MN-14 was still successful in 20%. Histologically, no remaining viable tumor cells could be demonstrated in these animals surviving > 6 months. In patients, the MTD was reached at 60 mCi/m2 of hMN-14 (at 70 mCi/m2, two of three grade 4 myelotoxicities). Of 11 assessable patients, 2 had partial remissions (corresponding to an objective response rate of 18%), and 5 (45%) had minor/mixed responses or experienced stabilization of previously rapidly progressing disease. These data suggest that in small volume disease, RAIT may be superior to conventional chemotherapy. Antibodies of higher affinity seem to be clearly superior. The clinical response rates in patients with small volume disease are encouraging, being comparable to the response rates of conventional chemotherapeutic regimens but with fewer side effects. Ongoing studies will show whether treatment at the MTD will further improve therapeutic results.

Radiolabeled peptides for targeting cholecystokinin-B/gastrin receptor-expressing tumors.

UNLABELLED: The high sensitivity of pentagastrin stimulation in detecting primary or metastatic medullary thyroid cancer (MTC) suggests widespread expression of the corresponding receptor type on human MTC. Indeed, autoradiographic studies have demonstrated cholecystokinin (CCK)-B/gastrin receptors not only in more than 90% of MTCs but also in a high percentage of small cell lung cancers, some ovarian cancers, astrocytomas and potentially a variety of adenocarcinomas. The aim of this study was to systematically screen and optimize, in a preclinical model and a pilot clinical study, suitable radioligands for targeting CCK-B receptors in vivo. METHODS: A variety of CCK/gastrin-related peptides, all bearing the C-terminal CCK receptor-binding tetrapeptide sequence Trp-Met-Asp-PheNH2 or derivatives thereof, were studied. They were radioiodinated by the lodogen or Bolton-Hunter procedures. The peptides were members of the gastrin or CCK families, which differ by the intramolecular position of a tyrosyl moiety. Their stability and affinity were studied in vitro and in vivo; their biodistribution and therapeutic efficacy were tested in nude mice bearing subcutaneous human MTC xenografts. Diethylenetriamine pentaacetic acid (DTPA) derivatives of suitable peptides were synthesized successfully, and their preclinical and initial clinical evaluations were performed, labeled with 111In. RESULTS: All members of the CCK or gastrin families were stable in serum (with half-lives of several hours at 37 degrees C); nevertheless, the stability of those peptides bearing N-terminal pGlu residues or D-amino acids was significantly higher. In accordance with their comparably low affinity, nonsulfated members of the CCK family showed fairly low uptake in the tumor and other CCK-B receptor-expressing tissues. Sulfated CCK derivatives performed significantly better but also displayed a comparably high uptake in normal CCK-A receptor-expressing tissues. This effect was probably due to their similar affinity for both CCK-A and CCK-B receptors. Best tumor uptake and tumor-to-nontumor ratios were obtained with members of the gastrin family because of their selectivity and affinity for the CCK-B receptor subtype. Pilot therapy experiments in MTC-bearing animals showed significant antitumor efficacy compared with untreated controls. DTPA derivatives of minigastrin were successfully developed. In a pilot clinical study, radioiodinated and 111In-labeled derivatives showed excellent targeting of physiological CCK-B receptor-expressing organs, as well as all known tumor sites. CONCLUSION: CCK/gastrin analogs may be a useful new class of receptor-binding peptides for diagnosis and therapy of CCK-B receptor-expressing tumors, such as MTC or small cell lung cancer. Nonsulfated gastrin derivatives may be preferable because of their CCK-B receptor selectivity, hence lower accretion in normal CCK-A receptor-expressing organs.