Robert P. Doyle

Traveling the Vitamin B12 Pathway: Oral Delivery of Protein and Peptide Drugs

Oral routes of administration for therapeutic peptides and proteins face two major barriers: proteolytic degradation in the stomach and an inadequate absorption mechanism for polypeptides within the intestinal lumen. As a result, peptide-based therapeutics are administered by injection, a painful process associated with lower patient compliance. The development of a means of overcoming these two major obstacles and enabling the successful delivery of peptide therapeutics by the oral route of administration has therefore been the target of extensive scientific endeavor. This Minireview focuses on oral peptide/protein delivery by the dietary uptake pathway for vitamin B(12). Recent progress in this field includes the delivery of erythropoietin, granulocyte-colony-stimulating factor, luteinizing-hormone-releasing hormone, and insulin.

Vitamin B12 in drug delivery: breaking through the barriers to a B12 bioconjugate pharmaceutical

Importance of the field: Vitamin B12 (B12) is a rare and vital micronutrient for which mammals have developed a complex and highly efficient dietary uptake system. This uptake pathway consists of a series of proteins and receptors, and has been utilized to deliver several bioactive and/or imaging molecules from 99mTc to insulin. Areas covered in this review: The current field of B12-based drug delivery is reviewed, including recent highlights surrounding the very pathway itself. What the reader will gain: Despite over 30 years of work, no B12-based drug delivery conjugate has reached the market-place, hampered by issues such as limited uptake capacity, gastrointestinal degradation of the conjugate or high background uptake by healthy tissues. Variability in dose response among individuals, especially across ageing populations and slow oral uptake (several hours), has also slowed development and interest. Take home message: This review is intended to stress again the great potential, as yet not fully realized, for B12-based therapeutics, tumor imaging and oral drug delivery. This review discusses recent reports that demonstrate that the issues noted above can be overcome and need not be seen as negating the great potential of B12 in the drug delivery field.

Targeting the Cubilin Receptor through the Vitamin B12 Uptake Pathway: Cytotoxicity and Mechanistic Insight through Fluorescent Re(I) Delivery

The intrinsic factor (IF) vitamin B(12) ileum anchored receptor, cubilin, mediates endocytotic uptake of the IF complex of vitamin B(12) to the blood serum. This receptor was targeted for the selective delivery and accumulation of a new bioprobe, a B(12) conjugate of rhenium 2, in the cubilin expressing placental choriocarcinoma BeWo cell line. Competitive uptake and cytotoxicity assays of 2 were investigated and interactions with nuclear DNA explored. In addition, the mechanism of internalization of 2 was confirmed to proceed in an IF-cubilin mediated fashion via siRNA transfection experiments. These studies show the great potential of cubilin as a new target for the delivery of B(12) based conjugates for cancer diagnostics and/or treatment.

Targeting the Folate Receptor (FR): Imaging and Cytotoxicity of ReI Conjugates in FR‐Overexpressing Cancer Cells

The synthesis, characterization, in vitro imaging, and cytotoxic properties of a new folate conjugate of rhenium(I) are reported. The conjugate [FA‐PEG‐BQAV‐Re(CO)3]+ (γ‐4) was screened against an adriamycin‐ and cisplatin‐resistant human ovarian cancer cell line (A2780/AD) that overexpresses the folate receptor (FR). Compound γ‐4 was internalized by a folate‐receptor‐mediated endocytotic pathway, which results in internal accumulation of γ‐4. This was contrasted with a FR‐negative Chinese hamster ovary cell line in which no internalization of γ‐4 was observed. γ‐4 was found to be cytotoxic with IC50 values of 189 and 78 μM at 6 and 24 h, respectively, toward the FR‐positive cell line. This is in contrast to the IC50 value of 502 μM at 6 h and 84 μM at 24 h for cisplatin in the same cell line, with a significantly greater toxicity at the earlier time point. The cytotoxicity of γ‐4 as explained by interactions that occur between the rhenium(I) complex moiety and DNA is described.

Vitamin B12 as a carrier for the oral delivery of insulin.

The noninvasive delivery of insulin continues to be a major goal for the treatment of diabetes mellitus. Oral-enteric administration would make insulin delivery easier and more effective, as higher patient compliance and improved glycemic control are likely; yet the oral-enteric pathway has been unfeasible owing to insulins susceptibility to proteolytic degradation and inefficient enteric uptake. Herein we show that a noninvasive oral delivery route for insulin is possible through the vitamin B12 uptake pathway. In diabetic rat models, insulin-B12 conjugates can significantly lower blood glucose levels when administered orally.

A Modular Phase Transfer and Ligand Exchange Protocol for Quantum Dots

In this paper, we describe a quantum dot (qdot) phase transfer protocol using ligand exchange and the amino acid histidine. The phase transfer from nonpolar solvents to aqueous buffers is homogeneous, and no appreciable precipitation occurs. The molecule histidine was chosen in order to first displace the organic encapsulation and second to provide a weakly chemisorbing intermediate at the qdot ionic interface. This allows the histidine to act as an intermediate shell upon which further direct ligand exchange can occur. Since this intermediate encapsulation is easily displaced by an assortment of different molecules while in aqueous buffers, we refer to this approach as modular. Characterization via FTIR and NMR revealed the extent of ligand exchange, and provides insights into the interfacial binding mechanism. The colloidal stability and photostability of the qdots was probed via UV-vis and steady state fluorescence, which revealed promising quantum yield stability of greater than 1 year. The qdots have hydrodynamic diameters of <12 nm and surface charges dependent upon ligand type and coverage. The modularity of this approach is shown by tailoring the qdot surface charge via sequential ligand exchange using mixed monolayers of carboxylic acid and poly(ethylene glycol)-terminated thiols.

Synthesis, Characterization and Pharmacodynamics of Vitamin-B12-Conjugated Glucagon-Like Peptide-1

Clearing the way: Glucagon-like peptide-1 (GLP-1) receptor agonists are proving a potent weapon in the treatment of type II diabetes. A new vitamin B(12)-GLP-1 conjugate is investigated and shown to have insulinotropic properties similar to the unmodified peptide. These results are critical to the exploitation of the vitamin B(12) oral uptake pathway for peptide delivery.

Oral Delivery of the Appetite Suppressing Peptide hPYY(3–36) through the Vitamin B12 Uptake Pathway

hPYY(3-36) injections have shown positive effects on appetite regulations, sparking increased interest in hPYY(3-36) research. Of great interest is oral delivery of hPYY(3-36) that can achieve clinically relevant weight-loss outcomes in what would be a highly patient compliant route. Successful oral delivery of other peptides utilizing the vitamin B12 pathway has been shown but below clinically relevant levels. Herein, we present clinically relevant in vivo oral delivery of B12-hPYY(3-36) conjugates.

A water soluble vitamin B12-Re(I) fluorescent conjugate for cell uptake screens: use in the confirmation of cubilin in the lung cancer line A549

A water soluble vitamin B(12)-rhenium conjugate was synthesized and used in concert with intrinsic factor to screen for cubilin receptor-mediated uptake in lung cancer cells. Internalization of the conjugate demonstrated that it could be used to rapidly screen for the cubilin receptor in living cells, subsequently confirmed with Western blotting and RT-PCR.

Synthesis, cytotoxicity, and insight into the mode of action of Re(CO) 3 thymidine complexes

Nucleoside analogues are extensively used in the treatment of cancer and viral diseases. The antiproliferative properties of organorhenium(I) complexes, however, have been scarcely explored to date. Herein we present the syntheses, characterization, and in vitro evaluation of ReI(CO)3 core complexes of thymidine and uridine. For the binding of the ReI(CO)3 core, a tridentate dipicolylamine metal chelate was introduced at positions C5′, C2′, N3, and C5 with spacers of various lengths. The corresponding organometallic thymidine complexes were fully characterized by IR and NMR spectroscopy and mass spectrometry. Their cytotoxicity was assessed against the A549 lung carcinoma cell line. Toxicity is dependent on the site and mode of conjugation as well as on the nature and the length of the tether. Moderate toxicity was observed for conjugates carrying the rhenium moiety at position C5′ or N3 (IC50=124–160 μM). No toxicity was observed for complexes modified at C2′ or C5. Complex 53, with a dodecylene spacer at C5′, exhibits remarkable toxicity and is more potent than cisplatin, with an IC50 value of 6.0 μM. To the best of our knowledge, this is the first report of the antiproliferative properties of [M(CO)3]+1–nucleoside conjugates. In competitive inhibition experiments with A549 cell lysates and purified recombinant human thymidine kinase 1 (hTK‐1), enzyme inhibition was observed for complexes modified at either N3 or C5′, but our results suggest that the toxicity cannot be attributed solely to interaction with hTK‐1.