Retention In Normal Tissues Research Articles

Abstract 3303: Pre-clinical characterization of the novel FAP targeting ligand PNT6555 for imaging and therapy of cancer

Abstract Background: Fibroblast Activation Protein-α (FAP) is a transmembrane glycoprotein highly expressed on activated fibroblasts. It is a constitutively active 170 kDa serine protease and a member of the dipeptide peptidase (DPP) family, sharing ~50% homology with DPPIV. FAP expression is only rarely expressed in normal adult tissues and is overexpressed in many epithelial cancers through upregulation on cancer-associated fibroblasts present in the stroma of various types of tumor. POINT BioPharma is developing PNT6555, which comprises a DOTA chelator linked to a FAP-targeting moiety, for imaging and therapeutic applications. Methods: PNT6555 and its radiometal chelates were evaluated for potency, selectivity, biodistribution and efficacy using biochemical and cellular assays as well as imaging, biodistribution and efficacy studies in tumor bearing mice. Results: PNT6555 and its gallium (natGa-PNT6555) and lutetium (natLu-PNT6555) chelates showed potent activity in FAP inhibition assays using human, mouse, and rat sources of FAP. PNT6555, natLu-PNT6555 and natGa-PNT6555 also showed significantly reduced potency when tested against PREP and DPPIV, two closely related homologous proteins. In vivo time-course biodistribution studies (by PET-imaging) with 68Ga-PNT6555 showed rapid clearance of 68Ga-PNT6555 from blood through the kidneys and urinary tract, with rising 68Ga-PNT6555 activity observed in the tumor through 60 minutes. At 60 minutes, the tumor was the only site of significant retained activity (>10 %ID/g). In vivo biodistribution studies (by SPECT imaging and direct organ assay) with 177Lu-PNT6555 showed rapid renal clearance into the bladder. After 24 hours, the tumor was the only tissue with significant activity retention. Direct organ assay showed little 177Lu-PNT6555 accumulation and retention in normal tissues with a high level of tumor retention observed out to 168h (>10 %ID/g). Therapeutic studies, using a single dose of 177Lu-PNT6555 or 225Ac-PNT6555, were completed in pre-clinical mouse models of cancer. In the HEK-mFAP model, significant dose responsive efficacy was observed in mice treated with either 177Lu-PNT6555 or 225Ac-PNT6555, with no apparent weight loss observed at all tested dose levels. Several mice experienced long-term survival >100 days at multiple of the tested dose levels. Conclusions: PNT6555, and its radiometal chelates, are potent and specific inhibitors of FAP. 68Ga/177Lu-PNT6555 showed rapid and prolonged uptake into FAP expressing tumors with limited uptake or retention observed in normal tissues. 177Lu/225Ac-PNT6555 showed compelling efficacy in pre-clinical tumor models that expressed FAP. Clinical studies with imaging and therapeutic chelates of PNT6555 are warranted. Citation Format: Robin M. Hallett, Sarah E. Poplawski, Mark H. Dornan, Shin Hye Ahn, Shuang Pan, Wu Wengen, Liu Yuxin, David G. Sanford, Valerie S. Hergott, Quang-De Nguyen, Anthony P. Belanger, Jack H. Lai, William Bachovchin, Joe A. McCann. Pre-clinical characterization of the novel FAP targeting ligand PNT6555 for imaging and therapy of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3303.

Alpha radioimmunotherapy using 225Ac-proteus-DOTA for solid tumors - safety at curative doses.

This is the initial report of an α-based pre-targeted radioimmunotherapy (PRIT) using 225Ac and its theranostic pair, 111In. We call our novel tumor-targeting DOTA-hapten PRIT system “proteus-DOTA” or “Pr.” Herein we report the first results of radiochemistry development, radiopharmacology, and stoichiometry of tumor antigen binding, including the role of specific activity, anti-tumor efficacy, and normal tissue toxicity with the Pr-PRIT approach (as α-DOTA-PRIT). A series of α-DOTA-PRIT therapy studies were performed in three solid human cancer xenograft models of colorectal cancer (GPA33), breast cancer (HER2), and neuroblastoma (GD2), including evaluation of chronic toxicity at ~20 weeks of select survivors.Methods: Preliminary biodistribution experiments in SW1222 tumor-bearing mice revealed that 225Ac could not be efficiently pretargeted with current DOTA-Bn hapten utilized for 177Lu or 90Y, leading to poor tumor uptake in vivo. Therefore, we synthesized Pr consisting of an empty DOTA-chelate for 225Ac, tethered via a short polyethylene glycol linker to a lutetium-complexed DOTA for picomolar anti-DOTA chelate single-chain variable fragment (scFv) binding. Pr was radiolabeled with 225Ac and its imaging surrogate, 111In. In vitro studies verified anti-DOTA scFv recognition of [225Ac]Pr, and in vivo biodistribution and clearance studies were performed to evaluate hapten suitability and in vivo targeting efficiency.Results: Intravenously (i.v.) administered 225Ac- or 111In-radiolabeled Pr in mice showed rapid renal clearance and minimal normal tissue retention. In vivo pretargeting studies show high tumor accumulation of Pr (16.71 ± 5.11 %IA/g or 13.19 ± 3.88 %IA/g at 24 h p.i. for [225Ac]Pr and [111In]Pr, respectively) and relatively low uptake in normal tissues (all average ≤ 1.4 %IA/g at 24 h p.i.). Maximum tolerated dose (MTD) was not reached for either [225Ac]Pr alone or pretargeted [225Ac]Pr at administered activities up to 296 kBq/mouse. Single-cycle treatment consisting of α-DOTA-PRIT with either huA33-C825 bispecific anti-tumor/anti-DOTA-hapten antibody (BsAb), anti-HER2-C825 BsAb, or hu3F8-C825 BsAb for targeting GPA33, HER2, or GD2, respectively, was highly effective. In the GPA33 model, no complete responses (CRs) were observed but prolonged overall survival of treated animals was 42 d for α-DOTA-PRIT vs. 25 d for [225Ac]Pr only (P < 0.0001); for GD2, CRs (7/7, 100%) and histologic cures (4/7, 57%); and for HER2, CRs (7/19, 37%) and histologic cures (10/19, 56%) with no acute or chronic toxicity.Conclusions: [225Ac]Pr and its imaging biomarker [111In]Pr demonstrate optimal radiopharmacologic behavior for theranostic applications of α-DOTA-PRIT. For this initial evaluation of efficacy and toxicity, single-cycle treatment regimens were performed in all three systems. Histologic toxicity was not observed, so MTD was not observed. Prolonged overall survival, CRs, and histologic cures were observed in treated animals. In comparison to RIT with anti-tumor IgG antibodies, [225Ac]Pr has a much improved safety profile. Ultimately, these data will be used to guide clinical development of toxicity and efficacy studies of [225Ac]Pr, with the goal of delivering massive lethal doses of radiation to achieve a high probability of cure without toxicity.

Renal-clearable hyaluronic acid functionalized NaGdF4 nanodots with enhanced tumor accumulation.

Integration of high tumor-targeting capacity, controlling in vivo transport and low normal tissue retention into one engineered nanoparticle is a critical issue for future clinically translatable anti-cancer nanomedicines. Herein, hyaluronic acid functionalized 3.8 nm NaGdF4 nanodots (named NaGdF4 ND@HAs) have been prepared through conjugation of tryptone capped NaGdF4 nanodots (NaGdF4 ND@tryptone) with hyaluronic acid (HA, a naturally occurring glycosaminoglycan), which can recognize the overexpressed CD44 on cancer cell membranes. The as-prepared NaGdF4 ND@HAs have good paramagnetic properties (longitudinal relaxivity (r1) = 7.57 × 10−3 M S−1) and low cytotoxicity. The in vivo experimental results demonstrate that the NaGdF4 ND@HAs can not only efficiently accumulate in mouse-bearing MDA-MB-231 tumors (ca. 5.3% injection dosage (ID) g−1 at 2 h post-injection), but also have an excellent renal clearance efficiency (ca. 75% injection dosage (ID) at 24 h post-injection). The as-prepared NaGdF4 ND@HAs have good paramagnetic properties with enhanced tumor-targeting capacity, which provides a useful strategy for the preparation of renal clearable magnetic resonance imaging (MRI) contrast agents for tumors.

Effect of a radiolabel biochemical nature on tumor-targeting properties of EpCAM-binding engineered scaffold protein DARPin Ec1

Radionuclide-based imaging of molecular therapeutic targets might facilitate stratifying patients for specific biotherapeutics. New type of imaging probes, based on designed ankyrin repeat proteins (DARPins), have demonstrated excellent contrast of imaging of human epidermal growth factor type 2 (HER2) expression in preclinical models. We hypothesized that labeling approaches, which result in lipophilic radiometabolites (non-residualizing labels), would provide the best imaging contrast for DARPins that internalize slowly after binding to cancer cells. The hypothesis was tested using DARPin Ec1 that binds to epithelial cell adhesion molecule (EpCAM). EpCAM is a promising therapeutic target. Ec1 was labeled with 125I using two methods to obtain the non-residualizing labels, while residualizing labels were obtained by labeling it with 99mTc. All labeled Ec1 variants preserved target specificity and picomolar binding affinity to EpCAM-expressing pancreatic adenocarcinoma BxPC-3 cells. In murine models, all the variants provided similar tumor uptake. However, 125I-PIB-H6-Ec1 had noticeably lower retention in normal tissues, which provided appreciably higher tumor-to-organ ratios. Furthermore, 125I-PIB-H6-Ec1 demonstrated the highest imaging contrast in preclinical models than any other EpCAM-imaging agent tested so far. In conclusion, DARPin Ec1 in combination with a non-residualizing label is a promising probe for imaging EpCAM expression a few hours after injection.

Integrin α6-Targeted Magnetic Resonance Imaging of Hepatocellular Carcinoma in Mice.

Magnetic resonance imaging (MRI) has a high spatial resolution for detecting hepatocellular carcinoma (HCC). Integrin α6 has emerged as a diagnostic and prognostic biomarker of HCC. Here, we developed the MR contrast agent RWY-dL-(Gd-DOTA)4 based on the integrin α6-targeted RWY peptide that we previously identified to detect HCC. Contrast-enhanced MRI was carried out to evaluate the use of RWY-dL-(Gd-DOTA)4 to detect HCC lesions in subcutaneous and diethylnitrosamine (DEN)-induced HCC mouse models. Enhancement MR signals were observed in HCC-LM3 subcutaneous liver tumors in the first 5min post-injection of RWY-dL-(Gd-DOTA)4 at a low dose of 0.03mmol Gd/kg. Moreover, RWY-dL-(Gd-DOTA)4 generated superior contrast enhancement for liver tumors in chemical-induced HCC mice. Importantly, RWY-dL-(Gd-DOTA)4 provided complementary enhancement MR signals to the clinical available hepatobiliary MR contrast agent gadoxetate disodium Gd-EOB-DTPA. Additionally, RWY-dL-(Gd-DOTA)4 showed minimal gadolinium retention in normal tissues and organs at 48h post-injection. These findings potentiate the use of RWY-dL-(Gd-DOTA)4 for the MRI of HCC to improve the diagnosis of HCC.

Abstract 851: Therapeutic combination of radiolabeled CLR1404 with external beam radiation in head and neck cancer murine xenograft models

Abstract Radiation therapy is a central treatment modality for head and neck cancer (HNC). Although significant technical advances have been made in delivering highly conformal radiation, normal tissue toxicity remains dose limiting. CLR1404 is a radiolabeled alkylphophocholine analog with theranostic potential as a PET imaging agent (CLR 124, labeled with 124I) and as a radiotherapy agent (CLR 131, labeled with 131I). CLR1404 exhibits preferential uptake in human cancers and provides tumor-selective internal delivery of radiation to complement external beam radiation (XRT) in the treatment of cancer. In this study, we investigated the antitumor effect of CLR 131 in combination with external beam radiation (XRT) in HNC. Using the fluorescently labeled CLR1404 analog, CLR 1501, in conjuntion with fluorescence microscopy and flow cytometry, we first confirmed CLR1404 uptake in 20 HNC cell lines in vitro. Thereafter, we examined CLR 124 uptake and retention in 12 HNC xenograft and patient-derived xenograft (PDX) mouse models via in vivo PET/CT imaging. Our results showed significant tumor uptake and retention of CLR 124 and limited retention in normal tissues. These results were corroborated by ex vivo gamma counting of excised tissues from tumor-bearing mice injected with CLR 131. We further assessed tumor response following single-dose administration of CLR 131 combined with fractionated XRT in 6 HNC xenograft models. Mice treated with non-radiolabeled CLR1404 displayed unchecked tumor progression. Mice treated with either CLR 131 or XRT showed partial inhibiton on tumor growth. Mice treated with CLR 131 combined with XRT exhibited enhanced inhibitory effect on tumor growth compared with either single treatment, confirming augmentation of XRT response by CLR 131 treatment. Targeted radionuclide therapy (TRT) is an attractive approach that employs radiolabeled molecules to specifically deliver radiation to primary and metastatic tumors. We hypothesized that TRT combined with reduced-dose XRT could reduce the normal tissue toxicity profile compared with high-dose XRT alone. In this study, we demonstrated uptake of CLR 131 across multiple HNC cell lines and xenograft models with enhanced antitumor effects when CLR 131 is combined with XRT. These results suggest the potential value of TRT via CLR 131 combined with reduced dose XRT in HNC patients, which will be further tested in a phase I clinical trial. Citation Format: Chunrong Li, Jenna M. Mylin, Jamey P. Weichert, Justin J. Jeffery, Ashley M. Weichmann, Kwang P. Nickel, Lindsey J. Abel, Reinier Hernandez, Joseph J. Grudzinski, Ian R. Marsh, Bryan P. Bednarz, Shari M. Piaskowski, Paul M. Harari. Therapeutic combination of radiolabeled CLR1404 with external beam radiation in head and neck cancer murine xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 851.

Evaluation of a metalloporphyrin (THPPMnCl) for necrosis-affinity in rat models of necrosis

The combination of an 13II-labeled necrosis-targeting agent (NTA) with a vascular disrupting agent is a novel and potentially powerful technique for tumor necrosis treatment (TNT). The purpose of this study was to evaluate a NTA candidate, THPPMnCl, using 131I isotope for tracing its biodistribution and necrosis affinity. 131I-THPPMnCl was intravenously injected in rat models with liver, muscle, and tumor necrosis and myocardial infarction (MI), followed by investigations with macroscopic autoradiography, triphenyltetrazolium chloride (TTC) histochemical staining, fluorescence microscopy and H&E stained histology for up to 9 days. 131I-THPPMnCl displayed a long-term affinity for all types of necrosis and accumulation in the mononuclear phagocytic system especially in the liver. Autoradiograms and TTC staining showed a good targetability of 131I-THPPMnCl for MI. These findings indicate the potential of THPPMnCl for non-invasive imaging assessment of necrosis, such as in MI. However, 13II-THPPMnCl is unlikely suitable for TNT due to its long-term retention in normal tissues.

Indium 111-labeled J591 anti-PSMA antibody for vascular targeted imaging in progressive solid tumors.

BackgroundJ591 is a monoclonal antibody that targets the external domain of the prostate-specific membrane antigen (PSMA). Besides prostate cancer cells, it also targets the neovasculature of non-prostate solid tumors. We provide an analysis of the antibody mass-dose dependency of lesion uptake and normal tissue retention, together with an assessment of lesion detectability using 111In-J591 imaging, compared with conventional imaging in patients with a variety of solid tumors.MethodsTwenty patients in six cohorts received fixed amounts (5, 10, 20, 40, 60, and 100 mg) of J591 in a phase I trial. A maximum of four administrations per patient was given, with each administration separated by 3 weeks. All antibody administrations included 370 MBq (10 mCi) of 111In labeled to 2 mg of J591 via the chelating agent DOTA. Three whole body (WB) gamma camera scans with at least one SPECT scan, along with multiple WB count-rate measurements and blood samples, were obtained for all patients. The effect of escalating antibody mass on lesion uptake and normal tissue retention was evaluated using lesion, liver, serum, and WB residence times and ratios thereof for each treatment cycle. Lesion detectability using 111In-J591 imaging was compared to the standard imaging on a lesion-by-lesion basis.ResultsA total of 170 lesions in 20 patients were detected by standard or 111In-J591 imaging. 111In-J591 targeted both skeletal and soft tissue diseases in all tumor types. 111In-J591 imaging identified 74% (20/27) of skeletal lesions, 53% (18/34) of nodes, and 64% (70/109) of other soft tissue/organ lesions. There was increasing 111In-J591 uptake in lesions with increasing antibody mass-dose, coupled with decreasing retention in the liver for increments up to 20 mg, and no significant change at higher antibody mass.ConclusionsRadiolabeled J591 antibody has potential as a targeting agent for solid tumor vasculature and lesion detection. Bone and soft tissue lesions arising from tumors of diverse origin were targeted by the anti-PSMA antibody J591. For the detection of lesions in these tumors by J591 antibody scans, an antibody mass of 20 mg is adequate. The optimal time of imaging is 5 to 7 days post-injection.

Efficacy and safety of selenium nanoparticles administered intraperitoneally for the prevention of growth of cancer cells in the peritoneal cavity

Peritoneal implantation of cancer cells, particularly postoperative seeding metastasis, frequently occurs in patients with primary tumors in the stomach, colon, liver, and ovary. Peritoneal carcinomatosis is associated with poor prognosis. In this work, we evaluated the prophylactic effect of intraperitoneal administration of selenium (Se), an essential trace element and a putative chemopreventive agent, on peritoneal implantation of cancer cells. Elemental Se nanoparticles were injected into the abdominal cavity of mice, into which highly malignant H22 hepatocarcinoma cells had previously been inoculated. Se concentrations in the cancer cells and tissues, as well as the efficacy of proliferation inhibition and safety, were evaluated. Se was mainly concentrated in cancer cells compared to Se retention in normal tissues, showing at least an order of magnitude difference between the drug target cells (the H22 cells) and the well-recognized toxicity target of Se (the liver). Such a favorable selective distribution resulted in strong proliferation suppression without perceived host toxicity. The mechanism of action of the Se nanoparticle-triggered cytotoxicity was associated with Se-mediated production of reactive oxygen species, which impaired the glutathione and thioredoxin systems. Our results suggest that intraperitoneal administration of Se is a safe and effective means of preventing growth of cancer cells in the peritoneal cavity for the above-mentioned high-risk populations.

Effect of Chelators on the Pharmacokinetics of 99mTc-Labeled Imaging Agents for the Prostate-Specific Membrane Antigen (PSMA)

Technetium-99m, the most commonly used radionuclide in nuclear medicine, can be attached to biologically important molecules through a variety of chelating agents, the choice of which depends upon the imaging application. The prostate-specific membrane antigen (PSMA) is increasingly recognized as an important target for imaging and therapy of prostate cancer (PCa). Three different (99m)Tc-labeling methods were employed to investigate the effect of the chelator on the biodistribution and PCa tumor uptake profiles of 12 new urea-based PSMA-targeted radiotracers. This series includes hydrophilic ligands for radiolabeling with the [(99m)Tc(CO)3](+) core (L8-L10), traditional NxSy-based chelating agents with varying charge and polarity for the (99m)Tc-oxo core (L11-L18), and a (99m)Tc-organohydrazine-labeled radioligand (L19). (99m)Tc(I)-Tricarbonyl-labeled [(99m)Tc]L8 produced the highest PSMA+ PC3 PIP to PSMA- PC3 flu tumor ratios and demonstrated the lowest retention in normal tissues including kidney after 2 h. These results suggest that choice of chelator is an important pharmacokinetic consideration in the development of (99m)Tc-labeled radiopharmaceuticals targeting PSMA.

Preclinical evaluation and validation of [ 18 F]HX4, a promising hypoxia marker for PET imaging

Hypoxia has been shown to be an important microenvironmental parameter influencing tumor progression and treatment efficacy. Patient guidance for hypoxia-targeted therapy requires evaluation of tumor oxygenation, preferably in a noninvasive manner. The aim of this study was to evaluate and validate the uptake of [(18)F]HX4, a novel developed hypoxia marker for PET imaging. A heterogeneous accumulation of [(18)F]HX4 was found within rat rhabdomyosarcoma tumors that was significantly (P < 0.0001) higher compared with the surrounding tissues, with temporal increasing tumor-to-blood ratios reaching a plateau of 7.638 ± 0.926 and optimal imaging properties 4 h after injection. [(18)F]HX4 retention in normal tissues was found to be short-lived, homogeneous and characterized by a fast progressive temporal clearance. Heterogeneity in [(18)F]HX4 tumor uptake was analyzed based on 16 regions within the tumor according to the different orthogonal planes at the largest diameter. Validation of heterogeneous [(18)F]HX4 tumor uptake was shown by a strong and significant relationship (r = 0.722; P < 0.0001) with the hypoxic fraction as calculated by the percentage pimonidazole-positive pixels. Furthermore, a causal relationship with tumor oxygenation was established, because combination treatment of nicotinamide and carbogen resulted in a 40% reduction (P < 0.001) in [(18)F]HX4 tumor accumulation whereas treatment with 7% oxygen breathing resulted in a 30% increased uptake (P < 0.05). [(18)F]HX4 is therefore a promising candidate for noninvasive detection and evaluation of tumor hypoxia at a macroscopic level.

Biodistribution and Clearance of Small Molecule Hapten Chelates for Pretargeted Radioimmunotherapy

The favorable pharmacokinetics and clinical safety profile of metal-chelated 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) suggests that it might be an ideal hapten for pretargeted radioimmunotherapy. In an effort to minimize hapten retention in normal tissues and determine the effect of various chemical adducts on in vivo properties, a series of DOTA-based derivatives were evaluated. Biodistribution and whole-body clearance were evaluated for (177)Lu-labeled DOTA, DOTA-biotin, a di-DOTA peptide, and DOTA-aminobenzene in normal CD1 mice. Kidney, liver, and bone marrow doses were estimated using standard Medical Internal Radiation Dose methodology. All haptens demonstrated similar low tissue and whole-body retention, with 2-4% of the injected dose remaining in mice 4h postinjection. The kidney is predicted to be dose limiting for all (177)Lu-labeled haptens tested with an estimated kidney dose of approximately 0.1mGy/MBq. We present here a group of DOTA-based haptens that exhibit rapid clearance and exceptionally low whole-body retention 4h postinjection. Aminobenzene, tyrosine-lysine, and biotin groups have minimal effects on the blood clearance and biodistribution of (177)Lu-DOTA.

Novel Prodrugs for Targeting Diagnostic and Therapeutic Radionuclides to Solid Tumors

Most cancer therapeutics (chemo, radiation, antibody-based, anti-angiogenic) are at best partially and/or temporarily effective. In general, the causes for failure can be summarized as: (i) poor diffusion and/or nonuniform distribution of drug/prodrug molecules in solid tumors; (ii) high drug concentration and retention in normal tissues (leading to side effects); (iii) requirement for plasma-membrane permeability and/or internalization of drug/prodrug molecules; (iv) low uptake of drug by tumor; (v) lack of retention of drug within tumor (most have gradient-driven reversible binding); and (vi) multidrug resistance. We are developing an innovative technology that aims to surmount these problems by actively concentrating and permanently entrapping radioimaging and radiotherapeutic prodrugs specifically within solid tumors. The approach will enable noninvasive sensing (imaging) and effective therapy of solid tumors, allowing tumor detection, diagnosis, and treatment to be closely coupled (personalized medicine).

ZD9331: discovery to clinical development

Thymidylate synthase (TS) has been targeted in cancer therapy for many years. As a result of a prolonged and extensive drug development program specific TS inhibitors have come into clinical practice. Following on from the development of the polyglutamatable TS inhibitor raltitrexed (Tomudex, ZD1694), ZD9331 is a rationally designed third-generation specific inhibitor of TS that does not require polyglutamation for its activity. Its development was based on the dual rationale of increased efficacy, by overcoming the potential for resistance due to reduced expression of folylpolyglutamate synthetase (FPGS), whilst potentially reducing the toxicities associated with polyglutamation and drug retention in normal tissues. Preclinical studies have shown it to be transported by the ubiquitously expressed reduced folate carrier as well as the alpha-folate receptor which is overexpressed in some cancers, especially ovarian. In vivo studies demonstrated a broad range of activity, leading to an extensive phase I program with several administration schedules. Whilst not being targeted to any individual tumor type, a large number of phase I, II, monotherapy and combination studies have been undertaken, and overall activity has been most promising, particularly in platinum-refractory relapsed ovarian, pancreatic and gastric cancers. Its role in the treatment of these diseases may be important, especially if patients were to be selected on the basis of their folate transport and FPGS status. The true potential of the drug remains to be determined.

Synthesis and preliminary biological evaluation of new radioiodinated MMP inhibitors for imaging MMP activity in vivo

Non-invasive measurement of matrix metalloproteinase (MMP) activity in vivo is a clinical challenge in many disease processes such as inflammation, tumor metastasis and atherosclerosis. Therefore, radioiodinated analogues of the non-peptidyl broad-spectrum MMP inhibitor (MMPI) CGS 27023A 1a were synthesized for non-invasive detection of MMP activity in vivo using single photon emission computed tomography (SPECT). The compounds Br-CGS 27023A 1b and HO-CGS 27023A 1d were synthesized from the amino acid D-valine and used as precursors for radioiodinated derivatives of CGS 27023A and their non-radioactive references I-CGS 27023A 1c and HO-I-CGS 27023A 1e. Radioiodination of the precursors with [ 123I]NaI or [ 125I]NaI produced the no-carrier-added MMP inhibitors [ 123I]I-CGS 27023A 1f, [ 125I]I-CGS 27023A 1g, HO-[ 123I]I-CGS27023A 1h, and HO-[ 125I]I-CGS 27023A 1i. In vitro studies showed that the non-radioactive analogues of the MMP inhibitors exhibited affinities against gelatinase A (MMP-2) and gelatinase B (MMP-9) in the nanomolar range, comparable to the parent compound CGS 27023A. In vivo biodistribution using HO-[ 125I]I-CGS 27023A 1i in CL57 Bl6 mice showed rapid blood and plasma clearance and low retention in normal tissues. The preliminary biological evaluation warrant further studies of these radioiodinated MMP inhibitors as potential new radiotracers for imaging MMP activity in vivo.

Uptake by cells and photosensitizing effectiveness of novel pheophorbide derivatives in vitro.

Pheophorbide a prepared from the algae Spirulina was derivatized at the C(7)-carboxylic group by linking amino alkyls of various lengths and terminal functional groups. The compounds were purified by thin-layer chromatography (TLC) and by high-pressure liquid chromatography (HPLC). Solubilization of compounds by serum lipoproteins, the kinetics of compound uptake into mammalian cells, and photosensitizing effectiveness when activated by 673 nm laser light have been studied. Optimal photosensitizer uptake into cells and the greatest photosensitizing activity were observed with compounds having side-chain lengths of 4-6 carbon atoms which terminated in -OH and -CH3 groups. The most effective compounds were 3 orders of magnitude more potent than Photofrin in the degree of photoinactivation of cultured EMT-6 tumor cells. HDL and LDL significantly promoted the efflux of these photosensitizing drugs from cells, suggesting that their long-term retention in normal tissues in vivo would be minimal and produce little phototoxicity.

A cross-linked monoclonal antibody fragment for improved tumor targeting.

Cross-linked F(ab')2 fragments derived from PR1A3, a murine monoclonal antibody used in radioimmunoscintigraphy of colorectal tumors, were produced using the bifunctional reagent bismaleimidohexane (BMH) as follows: Digestion of PR1A3 with pepsin gave F(ab')2 fragments which were purified by ion-exchange chromatography. Fab' was produced by reduction of F(ab')2 with cysteine. Following reaction with BMH, cross-linked F(ab')2 fragments, XL-F(ab')2, were isolated by preparative size-exclusion HPLC. Analysis by HPLC and SDS-PAGE demonstrated the presence of a molecule of approximately 100 kDa containing a nonreducible 50,000 MWt chain. Competitive and direct radioligand binding assays demonstrated that the XL-F(ab')2 had a capacity to bind to antigen similar to that of unmodified F(ab')2. The biodistribution of 125I-labeled XL-F(ab')2 and unmodified F(ab')2 was compared in a nude mouse human tumor xenograft model at 4, 24, and 48 h after injection. Differences between the two preparations were most significant after 24 or 48 h. Tumor uptake of the XL-F(ab')2 was greater and normal tissue retention less than with the unmodified fragment. Tumor to normal tissue ratios at 48 h ranged from 6.2 to 35.2 for XL-F(ab')2 while for the normal F(ab')2 they ranged from 1.5 to 14.2. These results suggest that cross-linked antibody fragments may produce better tumor targeting in clinical application.

Problems of delivery of monoclonal antibodies. Pharmaceutical and pharmacokinetic solutions.

Monoclonal antibodies to tumour-associated antigens have great theoretical potential for the specific targeting of radioactivity and anti-neoplastic agents to tumours. The clinical success of monoclonal antibody-based cancer diagnosis and therapy depends, however, on solving a number of pharmacokinetic delivery problems. These include: (i) slow elimination of monoclonal antibodies from the blood and poor vascular permeability; (ii) low and heterogeneous tumour uptake; (iii) cross-reactivity with normal tissues; (iv) metabolism of monoclonal antibody conjugates; and (v) immunogenicity of murine forms in humans. As a result of extensive pharmaceutical and pharmacokinetic research conducted over the past 10 to 15 years, several potential solutions to these delivery problems have been identified. Blood concentrations of antibody conjugates may be reduced through regional administration, the use of antibody fragments, interventional strategies and various pre-targeting techniques. Tumour uptake may be increased through administration of higher doses, or the use of agents to increase tumour vascular permeability. Tumour retention of antibody conjugates may be improved by inhibition of metabolism, by using more stable linkage chemistry. Alternatively, normal tissue retention may be decreased through the use of metabolisable chemical linkages inserted between the antibody and conjugated moiety. Very small antigen-binding fragments and peptides that exhibit improved tumour penetration and more rapid elimination from the blood and normal tissues have been prepared by genetic engineering techniques. Chimeric (mouse/human) and human monoclonal antibodies have been developed to circumvent the problem of immunogenicity. Future research will continue to be focused on improvements in the design of monoclonal antibodies for tumour targeting, with the ultimate goal of finally uncovering the 'magic bullet' envisioned by Paul Ehrlich almost a century ago.