Anthony R. Vortherms

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 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.

Synthesis, Characterization, and In Vitro Assay of Folic Acid Conjugates of 3′-Azido-3′-Deoxythymidine (AZT): Toward Targeted AZT Based Anticancer Therapeutics

Conjugates of three components namely folic acid, poly(ethyleneglycol) and 3 ′-azido-3 ′-deoxythymidine (AZT) are presented. Folate-PEG units were coupled to AZT to facilitate delivery of the nucleoside into the cell. A convenient separation of the polydisperse PEGylated-folic acid regioisomers produced upon conjugation is described. This is to select for the active γ-regioisomer over the inactive α-regioisomer. In vitro cytotoxicity assays were conducted against an ovarian cell line (A2780/AD) that overexpresses the folate receptor (FR) and compared to a FR free control cell line. Compared to AZT a ∼20-fold greater potency against the resistant ovarian line was observed for the conjugates.

Expanding Monomeric Pyrophosphate Complexes beyond Platinum

Four new monomeric pyrophosphate complexes, namely [Co(phen)(2)(H(2)P(2)O(7))] x 4 H(2)O (1 x 4 H(2)O), [Ni(phen)(2)(H(2)P(2)O(7))] x 8 H(2)O (2 x 8 H(2)O), [Cu(phen)(H(2)O)(H(2)P(2)O(7))] (3) and {[Cu(phen)(H(2)O)(P(2)O(7))][Na(2)(H(2)O)(8)]} x 6 H(2)O (4 x 14 H(2)O) have been isolated and structurally characterized. The impact of pH and stoichiometry in obtaining 1-4 is described. These complexes have been tested against the adriamycin-resistant ovarian cancer cell line A2780/AD, revealing highly significant (nM) IC(50) values, compared to microM IC(50) values for cisplatin controls.

Targeting Gallium to Cancer Cells through the Folate Receptor

The development of gallium(III) compounds as anti-cancer agents for both treatment and diagnosis is a rapidly developing field of research. Problems remain in exploring the full potential of gallium(III) as a safe and successful therapeutic agent or as an imaging agent. One of the major issues is that gallium(III) compounds have little tropism for cancer cells. We have combined the targeting properties of folic acid (FA) with long chain liquid polymer poly(ethylene glycol) (PEG) spacers’. This FA-PEG unit has been coupled to the gallium coordination complex of 1,4,7,10-tetraazacyclo-dodecane-N, N′, N′, N′′-tetraacetic acid (DOTA) through amide linkages for delivery into target cells overexpressing the folate receptor (FR). In vitro cytotoxicity assays were conducted against a multi-drug resistant ovarian cell line (A2780/AD) that overexpresses the FR and contrasted against a FR free Chinese hamster ovary (CHO) cell line. Results are rationalized taking into account stability studies conducted in RPMI 1640 media and HEPES buffer at pH 7.4.

Anticancer conjugates and cocktails based on methotrexate and nucleoside synergism

Conjugates of methotrexate (MTX) and the nucleoside analogs 3-azidodeoxythymidine (AZT), iododeoxyuridine (IUdR) and dideoxycytidine (ddC) linked using poly(ethyleneglycol) are presented. In vitro cytotoxicity assays of the conjugates against drug resistant ovarian cell line A2780/AD are preformed and comparisons made to such assays performed for unconjugated (cocktail) systems. All systems tested were inactive, or had low activity, at 24 h. After 72 hr incubation however, the cocktails of MTX and AZT, IUdR or ddC showed high cytotoxicity in the low nanomolar range. The conjugates were only very moderately active with IC50 values in the [0.1 to 1.0 mM] range. Conjugation of the antifolate to the nucleoside analogs has it seems reduced the activity significantly when compared to a cocktail of the components, indicating a conjugate approach is unlikely to translate into success in vivo. The positive note comes from the observation that by combining two of the new conjugates, namely those based on MTX with IUdR or AZT, an IC50 at 24 hours of~ [180 μM] was produced.