Comparison of microendoscopic discectomy and open discectomy for single-segment lumbar disc herniation.

Background: Lumbar disc herniation is a common disease. Endoscopic treatment may have more advantages than traditional surgery.

Aim: To compare the clinical efficacy and safety of microendoscopic discectomy (MED) and open discectomy with lamina nucleus enucleation in the treatment of single-segment lumbar intervertebral disc herniation.

Detection of Klebsiella. Pneumoniae Infection with an Antisense Oligomer Against its Ribosomal RNA.

Purpose: Previously, we demonstrated specific accumulation into bacteria of a 12-mer phosphorodiamidate morpholino (MORF) oligomer complementary to a ribosomal RNA (rRNA) segment found in all bacteria using the universal probe called Eub338 (Eub). Here, two MORF oligomers Eco and Kpn with sequences specific to the rRNA of Escherichia coli (Eco) and Klebsiella pneumoniae (Kpn) were investigated along with Eub and control (nonEub).

Procedures: To determine bacterial rRNA binding, oligomers were tagged with Alexa Fluor 633 (AF633) for fluorescence in situ hybridization (FISH) and fluorescence microscopy, and radiolabeled with technetium-99m (Tc-99m) for biodistribution and SPECT imaging in infected mice.

Novel DNA Polymer for Amplification Pretargeting.

In this Letter, different from conventional pretargeting, an additional novel DNA polymer with multiple copies of a target was first designed to be administrated between the antitumor antibody, and the labeled effector served as an amplification pretargeting strategy. Two phosphorothioate DNA strands, a bridging and a target strand, were hybridized to form a polymer. Polymer size, as a function of molar ratios, was then monitored by size exclusion HPLC and electrophoretic mobility shift assay.

Quantitative Correlation of in Vivo Properties with in Vitro Assay Results: The in Vitro Binding of a Biotin-DNA Analogue Modifier with Streptavidin Predicts the in Vivo Avidin-Induced Clearability of the Analogue-Modified Antibody.

Quantitative prediction of in vivo behavior using an in vitro assay would dramatically accelerate pharmaceutical development. However, studies quantitatively correlating in vivo properties with in vitro assay results are rare because of the difficulty in quantitatively understanding the in vivo behavior of an agent. We now demonstrate such a correlation as a case study based on our quantitative understanding of the in vivo chemistry.

A feasible approach to evaluate the relative reactivity of NHS-ester activated group with primary amine-derivatized DNA analogue and non-derivatized impurity.

Synthetic DNA analogues with improved stability are widely used in life science. The 3'and/or 5' equivalent terminuses are often derivatized by attaching an active group for further modification, but a certain amount of non-derivatized impurity often remains. It is important to know to what extent the impurity would influence further modification.

Differentiation between temporary and real non-clearability of biotinylated IgG antibody by avidin in mice.

Although an increasing number of antibody conjugates are being used in the clinic, there remain many unmet needs in antibody targeting. Normal tissue background is one of the key issues that limits the therapeutic efficacy and the detection sensitivity. Background reduction coupled with dose increase may provide the required target accumulation of the label or toxin at an acceptable normal tissue background.

Comparison between two labeled agents in mice using a coinjection-ratio approach in contrast to a conventional group approach.

Introduction: The differences between two agents often need to be accurately defined in vivo. Usually they are injected respectively into two groups of subjects. However, if the two agents do not interact with each other in vivo, a coinjection would serve the same purpose.

(99m)Tc-MORF oligomers specific for bacterial ribosomal RNA as potential specific infection imaging agents.

Purpose: Radiolabeled oligomers complementary to the 16S rRNA in bacteria were investigated as bacterial infection imaging agents.

Methods And Results: Identical sequences with backbones phosphorodiamidate morpholino (MORF), peptide nucleic acid (PNA), and phosphorothioate DNA (PS-DNA) were (99m)Tc-labeled and evaluated for binding to bacterial RNA. MORF binding to RNA from Escherichia coli strains SM101 and K12 was 4- and 150-fold higher compared to PNA and PS-DNA, respectively.

Intraperitoneal injection is not always a suitable alternative to intravenous injection for radiotherapy.

Abstract Intraperitoneal (IP) injection is frequently reported to be as effective as intravenous (IV) injection. Because it allows administering a larger volume with more radioactivity, we have investigated this route and the possibility of using it to circumvent the volume constraint we earlier experienced with pretargeting radiotherapy. Using (99m)Tc as the label, the pharmacokinetics (PK) of the cMORF effector (a DNA analogue) was evaluated after IP or IV injection in normal mice by necropsy and SPECT/CT imaging.

Detection of Aspergillus fumigatus pulmonary fungal infections in mice with (99m)Tc-labeled MORF oligomers targeting ribosomal RNA.

Purpose: Invasive aspergillosis is a major cause of infectious morbidity and mortality in immunocompromised patients. The fungus Aspergillus fumigatus (A. fumigatus) is the primary causative agent of invasive aspergillosis.

Pretargeting vs. direct targeting of human betalox5 islet cells subcutaneously implanted in mice using an anti-human islet cell antibody.

Introduction: We previously demonstrated MORF/cMORF pretargeting of human islets and betalox 5 cells (a human beta cell line) transplanted subcutaneously in mice with the anti-human islet antibody, HPi1. We now compare pretargeting with direct targeting in the beta cell transplant model to evaluate the degree to which target/non-target (T/NT) ratios may be improved by pretargeting.

Methods: Specific binding of an anti-human islet antibody HPi1 to the beta cells transplanted subcutaneously in mice was examined against a negative control antibody.

90Y labeled phosphorodiamidate morpholino oligomer for pretargeting radiotherapy.

While (188)Re has been used successfully in mice for tumor radiotherapy by MORF/cMORF pretargeting, previous radiolabeling of the amine-derivatized cMORF with (90)Y, a longer physical half-life nuclide, was not very successful. After developing a method involving a prepurification heating step during conjugation that increases labeling efficiency and label stability, the biodistribution of (90)Y-DOTA-Bn-SCN-cMORF ((90)Y-DOTA-cMORF) was measured in normal mice and in MORF-CC49 pretargeted mice that bear LS174T tumors. Absorbed radiation doses were then estimated and compared to those estimated for (188)Re.

Comparing the intracellular fate of components within a noncovalent streptavidin nanoparticle with covalent conjugation.

Introduction: Auger radiotherapy requires adequate tumor delivery and high nuclear accumulation and retention. We hypothesize that the noncovalent nature of a streptavidin/biotin three-component nanoparticle possessing these qualities may be required for dissociation of the radiolabeled oligomer and its accumulation into the cell nucleus.

Methods: As a test of our hypothesis, the intracellular fate of an antisense oligomer when incubated as the nanoparticle and when incubated while covalently conjugated to the antibody was compared.

Comparing two TAG-72 binding peptides previously identified by phage display as potential imaging agents.

Aim: To evaluate the targeting property in vitro and in vivo of two tumor-associated glycoprotein 72 (TAG-72) binding peptides, previously identified in this laboratory by phage selection using different elution conditions.

Materials And Methods: The peptides GGVSCMQTSPVCENNL (A2-6) and NPGTCKDKWEICLLNGG (A3-10) were radiolabeled with technetium-99m ((99m)Tc) using N-hydroxysuccinimidyl-S-acetyl-mercaptoacetyltriglycine (NHS-MAG(3)) as a chelator or were biotinylated. The specificity of the two peptides for the TAG-72 positive LS-174T cancer cells was demonstrated in vitro both by flow cytometry analysis using the biotinylated peptides and by competitive binding using the (99m)Tc-labeled peptides.

Improving the quantitation accuracy in noninvasive small animal single photon emission computed tomography imaging.

Introduction: Noninvasive imaging of small animals to measure biodistributions and pharmacokinetics of radiolabeled agents is increasingly seen as an effective alternative to external counting of tissues obtained by sacrifice and dissection. However, we have observed important disagreements in measuring the accumulation of (111)In-labeled antibodies in organs such as liver and kidneys when comparing imaging to ex vivo counting in the same animals. This study was conducted to establish whether this discrepancy could be minimized by selecting the region of interest (ROI) in images at the appropriate color threshold and by correcting for the estimated radioactivity within the blood pool of these organs during imaging.

Human islet cell MORF/cMORF pretargeting in a xenogeneic murine transplant model.

Noninvasive measurement of human islet cell mass in pancreas or following islet transplantation by nuclear imaging has yet to be achieved. It has been shown using mouse tumor models that pretargeting imaging strategies are sensitive and can greatly increase target to nontarget signal ratios. The objective now is to demonstrate the specific pretargeting of human islet cells in mice.

Unexpected side products in the conjugation of an amine-derivatized morpholino oligomer with p-isothiocyanate benzyl DTPA and their removal.

In connection with pretargeting, an amine-derivatized morpholino phosphorodiamidate oligomer (NH(2)-cMORF) was conjugated conventionally with p-isothiocyanate benzyl-DTPA (p-SCN-Bn-DTPA). However, after (111)In radiolabeling, unexpected label instability was observed. To understand this instability, the NH(2)-cMORF and, as control, the native cMORF without the amine were conjugated in the conventional manner.

Adding a clearing agent to pretargeting does not lower the tumor accumulation of the effector as predicted.

Clearing agents are often used in pretargeting despite the potential for decreased tumor accumulation of the effector. However, according to the authors' semiempirical model, a clearing agent should not necessarily decrease tumor accumulation. In this study, the authors have added a clearing step to their model-morpholino phosphorodiamidate oligomer (MORF)/complement MORF (cMORF) pretargeting system-to confirm this prediction.

Reducing the background fluorescence in mice receiving fluorophore/inhibitor DNA duplexes.

In principle, a DNA duplex consisting of an antisense fluorophore-conjugated major strand hybridized to a shorter complementary inhibitor-conjugated minor strand should provide fluorescence only in the tumor after intravenous administration if designed to remain intact except in the presence in tumor of its mRNA target. While we have obtained impressive tumor images in mice using this approach, there remains some background fluorescence. In this study, tissue homogenates of selected mouse organs were incubated with a test duplex and the kinetics of duplex dissociation in normal tissues were measured.

A preclinical 188Re tumor therapeutic investigation using MORF/cMORF pretargeting and an antiTAG-72 antibody CC49.

The utility of MORF/cMORF pretargeting for the radiotherapy of cancer requires further validation in tumored mice before clinical trials. We now report on a therapeutic study in mice pretargeted with MORF-CC49 (an anti-TAG-72 antibody CC49 conjugated with MORF, a phosphorodiamidate morpholino oligomer) and then targeted by 188Re-cMORF (a 188Re labeled complementary MORF). Before the dose-escalating therapeutic study, a pretargeting study in LS174T tumored mice was performed at tracer levels.

Identification of a high affinity TAG-72 binding peptide by phage display selection.

Purpose: Phage display was used to select novel peptides that specifically bind the TAG-72 antigen and with properties suitable for imaging TAG-72 positive cancers.

Results: After three rounds of selection against TAG-72 and using two different elution conditions including a long elution, the consensus sequences FRERCDKHPQKCTKFL and DPRHCQKRVLPCPAWL were expressed on phages G3-15 and T3-15 respectively. ELISA, fluorescence-activated cell sorting analysis and fluorescence microscopy provided evidence that both phages specifically bound TAG-72 in vitro.

Multimodality nuclear and fluorescence tumor imaging in mice using a streptavidin nanoparticle.

Combining two or more different imaging modalities in the same agent can be of considerable value in molecular imaging. We describe the use of streptavidin nanoparticle-based complexes as multimodality imaging agents to achieve tumor detection in a mouse model by both fluorescence and nuclear imaging. Up to four biotinylated functionalities can be readily attached to these streptavidin nanoparticles without apparent influence on their properties and with reasonable pharmacokinetics and therefore may be ideally suited for multimodality imaging.

Affinity enhancement pretargeting: synthesis and testing of a 99mTc-labeled bivalent MORF.

Pretargeting with bivalent effectors capable of bridging antitumor antibodies (affinity enhancement pretargeting) has been reported to provide superior results by affinity enhancement. Phosphorodiamidate morpholinos (MORFs) and other DNA analogues used for pretargeting are ideally suited as bivalent effectors since they are easily synthesized and the distance between binding regions, a determinant of binding, may be adjusted simply by lengthening the chain. We have shown by surface plasmon resonance that bivalent MORFs will provide superior affinity enhancement provided that suitable spacing exists between the binding regions.

Preparation of (111)In-DTPA morpholino oligomer for low abdominal accumulation.

An ability to quantitate the beta cell mass by noninvasive nuclear imaging will be very useful in the prevention, diagnosis, and treatment of diabetes. However, to be successful, radioactivity from the pancreas must not be obscured by the background radioactivity in the abdomen. Pretargeting offers the promise of achieving high target organ to normal tissue ratios.

The ratio of maximum percent tumour accumulations of the pretargeting agent and the radiolabelled effector is independent of tumour size.

Our previous studies have indicated that the optimal dosage ratio of pretargeting antibody to effector is proportional to their maximum percent tumour accumulations (MPTAs). This study quantitatively describes how both MPTAs and their ratio change with tumour size, to simplify pretargeting optimisation when tumour size varies. The CC49 antibody dosages below saturation of the tumour antigen level were first examined for the LS174T tumour mouse model.