Biomedical subjects

Tad H Koch

Publications and source records attributed to Tad H Koch.

11 recordsLinked to original sources

PubMed2006

Design, synthesis, and preliminary evaluation of doxazolidine carbamates as prodrugs activated by carboxylesterases.

The synthesis and tumor cell growth inhibition by doxazolidine carbamate prodrugs are reported. The carbamates were designed for selective hydrolysis by one or more human carboxylesterases to release doxazolidine (Doxaz), the formaldehyde-oxazolidine of doxorubicin that cross-links DNA to trigger cell death. Simple butyl and pentyl, but not ethyl, carbamate prodrugs inhibited the growth of cancer cells that overexpress carboxylesterase CES1 (hCE1) and CES2 (hiCE). Relative CES1 and CES2 expression levels were determined by reverse transcription of the respective mRNAs, followed by polymerase chain reaction amplification. More complex structures with a p-aminobenzyl alcohol (PABA) self-eliminating spacer showed better growth inhibition (IC50=50 nM for Hep G2 liver cancer cells) while exhibiting reduced toxicity toward rat cardiomyocytes, relative to the parent drug doxorubicin. Pentyl 4-(N-doxazolidinylcarbonyloxymethyl)phenylcarbamate, the lead compound for further investigation, appears to be activated in Hep G2 cells that express both CES1 and CES2.

Animals↗

PubMed2005

Doxazolidine, a proposed active metabolite of doxorubicin that cross-links DNA.

A crystal structure establishes doxoform as a dimeric formaldehyde conjugate of the oxazolidine of doxorubicin. Doxoform is a prodrug of doxazolidine, a monomeric doxorubicin formaldehyde-oxazolidine. Both doxoform and doxazolidine inhibit the growth of cancer cells at 1-4 orders of magnitude lower concentration than doxorubicin. They also inhibit the growth of cancer cells better than doxsaliform, a prodrug for an acyclic doxorubicin-formaldehyde conjugate. Doxoform rapidly hydrolyzes to doxazolidine, which then hydrolyzes to doxorubicin with a half-life of 3 min in human serum at 37 degrees C. Both doxoform and doxazolidine are taken up by multidrug-resistant MCF-7/Adr cells 3- to 4-fold better than doxorubicin. A molecular model suggests that doxazolidine can cross-link DNA by direct reaction with a G-base in a tautomeric form with synchronous ring opening of the oxazolidine. These results point to doxoform being a prodrug for doxazolidine that is the reactive species that directly cross-links DNA.

Antibiotics, Antineoplastic↗

PubMed2004

Antiestrogen binding site and estrogen receptor mediate uptake and distribution of 4-hydroxytamoxifen-targeted doxorubicin-formaldehyde conjugate in breast cancer cells.

The anthracycline antitumor drug, doxorubicin (DOX), has long been used as a broad spectrum chemotherapeutic. The literature now documents the role of formaldehyde in the cytotoxic mechanism, and anthracycline-formaldehyde conjugates possess substantially enhanced activity in vitro and in vivo. We have recently reported the design, synthesis, and preliminary evaluation of a doxorubicin-formaldehyde conjugate targeted, via 4-hydroxytamoxifen, to the estrogen receptor (ER) and antiestrogen binding site (AEBS), which are commonly present in breast cancer cells. The lead targeted doxorubicin-formaldehyde conjugate, called DOX-TEG-TAM, was found to possess superior cell growth inhibition characteristics relative to clinical doxorubicin and an untargeted control conjugate, especially in ER-negative, multidrug resistant MCF-7/Adr cells. The enhanced activity in the absence of estrogen receptor raised the possibility that targeting was also mediated via AEBS. Fluorescence microscopy of an ER-negative, AEBS-positive cell line as a function of time showed initial DOX-TEG-TAM localization in cytosol, in contrast to initial DOX and untargeted doxorubicin-formaldehyde conjugate localization in the nucleus. DOX-TEG-TAM was taken up by four AEBS-positive cell lines to a greater extent than doxorubicin and an untargeted doxorubicin-formaldehyde conjugate. Of the four cell lines, three were ER negative. DOX-TEG-TAM uptake was inhibited in a dose-dependent manner by the presence of a competing AEBS ligand. DOX-TEG-TAM retains 60% of the affinity of 4-hydroxytamoxifen for AEBS. DOX-TEG-TAM was also taken up by the AEBS-negative, ER-positive cancer cell line Rtx-6; with these cells uptake was inhibited in a dose-dependent manner by the ER ligand, estradiol. The data support the hypothesis that uptake of 4-hydroxytamoxifen targeted doxorubicin-formaldehyde conjugate is mediated by both the antiestrogen binding site and estrogen receptor.

Antineoplastic Agents↗

PubMed2004

Studies of targeting and intracellular trafficking of an anti-androgen doxorubicin-formaldehyde conjugate in PC-3 prostate cancer cells bearing androgen receptor-GFP chimera.

The synthesis of a doxorubicin-formaldehyde conjugate bound to the nonsteroidal anti-androgen cyanonilutamide, via a cleavable tether, and binding of the construct to cell free androgen receptor (AR) as a function of tether design were previously reported. Cyanonilutamide bearing a linear alkyne tether bound to the AR better than other designs. Fluorescence microscopy studies of binding of the lead targeted drug, as well as various tethered cyanonilutamides, to the AR and subsequent trafficking of the resulting AR complex in live PC3 prostate cancer cells transfected with AR-green fluorescent protein (GFP) chimera are now described. Cyanonilutamide and cyanonilutamide bonded to a linear alkyne tether caused translocation of AR-GFP to the nucleus. In general, the ability of tethered cyanonilutamides to cause translocation paralleled their binding affinity for the AR. However, a noncleavable form of the lead cyanonilutamide-doxorubicin-formaldehyde conjugate bound to AR-GFP but the resulting complex did not translocate to the nucleus. Binding was apparent from the drugs inhibition of Mibolerone-induced translocation. Direct observation of anthraquinone fluorescence of targeted drug in PC3 cells showed initial cytosolic localization, independent of AR expression, with predominant nuclear localization after sufficient time for release of drug from the targeting moiety. The results indicate that doxorubicin-formaldehyde conjugate bonded to cyanonilutamide via a cleavable linear tether enters PC3 cells, resides in cytosol, binds to the AR if present, and ultimately releases doxorubicin or a doxorubicin derivative to the nucleus.

Androgen Antagonists↗

PubMed2004

Kinetic analysis of site-specific photoaptamer-protein cross-linking.

ssDNA oligonucleotides containing bromodeoxyuridine, BrdU-photoaptamers, are rapidly emerging as specific protein capture reagents in protein microarray technologies. A mathematical model for the kinetic analysis of photoaptamer-protein photocross-linking reactions is presented. The model is based on specific aptamer/protein binding followed by laser excitation that can lead to either covalent cross-linking of the photoaptamer and protein in the complex or irreversible photodamage to the aptamer. Two distinct kinetic regimes, (1) frozen and (2) rapid equilibrium, are developed analytically to model binding kinetics between laser pulses. The models are used to characterize the photocross-linking between three photoaptamers and their cognate protein targets; photoaptamers 0650 and 0615 cross-link human basic fibroblast growth factor and 0518 cross-links HIV MN envelope glycoprotein. Data for cross-linking reaction yields as a function of both laser energy dose and target protein concentration are analyzed for affinity constants and cross-link reaction rates. The binding dissociation constants derived from the cross-linking data are in good accord with independent measurements; the rapid equilibrium model appears to produce results more consistent with the experimental observations, although there is significant overlap between the two models for most conditions explored here. The rate of photodamage for 0615 and 0518 is 3.5 and 2.5 times that of the specific cross-link, giving low maximum reaction yields of approximately 20% and approximately 30%, whereas 0650 cross-links with a rate over five times higher than its photodamage rate and has a maximum reaction yield exceeding 80%. Quantum yields for the three systems are estimated from the data; photoaptamer 0650 has a reasonably high quantum yield of approximately 0.2 for protein cross-linking, while 0518 and 0615 have quantum yields of 0.07 and 0.02. The work presented here provides a useful set of metrics that allow for refinement of photoaptamer properties.

Binding Sites↗

PubMed2004

Design, synthesis, and biological evaluation of doxorubicin-formaldehyde conjugates targeted to breast cancer cells.

The anthracycline antitumor drug doxorubicin (DOX) has been utilized for decades as a broad-spectrum chemotherapeutic. Recent literature evidence documents the role of formaldehyde in the cytotoxic mechanism, and anthracycline-formaldehyde conjugates possess substantially enhanced activity in vitro and in vivo. Targeting a doxorubicin-formaldehyde conjugate specifically to cancer cells may provide a more efficacious chemotherapeutic. The design and 11-step synthesis of doxorubicin-formaldehyde conjugates targeted to the estrogen receptor, which is commonly overexpressed in breast cancer cells, are reported. The formaldehyde is incorporated in a masked form as an N-Mannich linkage between doxorubicin and salicylamide. The salicylamide triggering molecule, previously developed to release the doxorubicin-formaldehyde active metabolite, is tethered via derivatized ethylene glycols to an E and Z mixture of 4-hydroxytamoxifen. The targeting group, E/Z-4-hydroxytamoxifen, was selected for its ability to tightly bind the estrogen receptor and antiestrogen binding sites. The targeted doxorubicin-formaldehyde conjugates' estrogen receptor binding and in vitro growth inhibition were evaluated as a function of tether length. The lead compound, DOX-TEG-TAM, bearing a triethylene glycol tether, binds the estrogen receptor with a binding affinity of 2.5% relative to E/Z-4-hydroxytamoxifen and inhibits the growth of four breast cancer cell lines with 4-fold up to 140-fold enhanced activity relative to doxorubicin.

Antineoplastic Agents↗

PubMed2004

Doxorubicin-formaldehyde conjugates targeting alphavbeta3 integrin.

We have reported the synthesis and biological evaluation of a prodrug to a doxorubicin active metabolite. Under physiologic conditions, release of the active metabolite, a conjugate of doxorubicin with formaldehyde, occurs with a half-life of 1 hour. To direct this prodrug to tumor, we designed two conjugates of the prodrug, doxsaliform, with the alphavbeta3-targeting peptides, CDCRGDCFC (RGD-4C) and cyclic-(N-Me-VRGDf) (Cilengitide). We now report the synthesis of these doxsaliform-peptide conjugates and their evaluation using MDA-MB-435 cancer cells. A hydroxylamine ether tether was used to attach 5''-formyldoxsaliform to RGD-4C in its acyclic form via an oxime functional group. The construct acyclic-RGD-4C-doxsaliform showed good binding affinity for alphavbeta3 in the vitronection cell adhesion assay (IC50 = 10 nmol/L) and good growth inhibition of MDA-MB-435 breast cancer cells (IC50 = 50 nmol/L). In its bicyclic forms, RGD-4C showed less affinity for alphavbeta3 and significantly less water solubility. Cyclic-(N-Me-VRGDf) was modified by substitution of D-4-aminophenylalanine for D-phenylalanine to provide a novel attachment point for doxsaliform. The conjugate, cyclic-(N-Me-VRGDf-NH)-doxsaliform, maintained a high affinity for alphavbeta3 (IC50 = 5 nmol/L) in the vitronectin cell adhesion assay relative to the peptide bearing only the tether (0.5 nmol/L). The IC50 for growth inhibition of MDA-MB-435 cells was 90 nmol/L. Flow cytometry and growth inhibition experiments suggest that the complete drug construct does not penetrate through the plasma membrane, but the active metabolite does on release from the targeting group. These drug conjugates could have significantly reduced side effects and are promising candidates for in vivo evaluation in tumor-bearing mice.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2003

Rational design and synthesis of androgen receptor-targeted nonsteroidal anti-androgen ligands for the tumor-specific delivery of a doxorubicin-formaldehyde conjugate.

The synthesis and preliminary evaluation of a doxorubicin-formaldehyde conjugate tethered to the nonsteroidal antiandrogen, cyanonilutamide (RU 56279), for the treatment of prostate cancer are reported. The relative ability of the targeting group to bind to the human androgen receptor was studied as a function of tether. The tether served to attach the antiandrogen to the doxorubicin-formaldehyde conjugate via an N-Mannich base of a salicylamide derivative. The salicylamide was selected to serve as a trigger release mechanism to separate the doxorubicin-formaldehyde conjugate from the targeting group after it has bound to the androgen receptor. The remaining part of the tether consisted of a linear group that spanned from the 5-position of the salicylamide to the 3'-position of cyanonilutamide. The structures explored for the linear region of the tether were derivatives of di(ethylene glycol), tri(ethylene glycol), N,N'-disubstituted-piperazine, and 2-butyne-1,4-diol. Relative binding affinity of the tethers bound to the targeting group for human androgen receptor were measured using a (3)H-Mibolerone competition assay and varied from 18% of nilutamide binding for the butynediol-based linear region to less than 1% for one of the piperazine derivatives. The complete targeted drug with the butynediol-based linear region has a relative binding affinity of 10%. This relative binding affinity is encouraging in light of the cocrystal structure of human androgen receptor ligand binding domain bound to the steroid Metribolone which predicts very limited space for a tether connecting the antiandrogen on the inside to the cytotoxin on the outside.

Androgen Antagonists↗

PubMed2003

Sensitivity and specificity of photoaptamer probes.

The potential of photoaptamers as proteomic probes was investigated. Photoaptamers are defined as aptamers that bear photocross-linking functionality, in this report, 5-bromo-2'-deoxyuridine. A key question regarding the use of photoaptamer probes is the specificity of the cross-linking reaction. The specificity of three photoaptamers was explored by comparing their reactions with target proteins and non-target proteins. The range of target/non-target specificity varies from 100- to >10(6)-fold with most values >10(4)-fold. The contributions of the initial binding step and the photocross-linking step were evaluated for each reaction. Photocross-linking never degraded specificity and significantly increased aptamer specificity in some cases. The application of photoaptamer technology to proteomics was investigated in microarray format. Immobilized anti-human immunodeficiency virus-gp120 aptamer was able to detect subnanomolar concentrations of target protein in 5% human serum. The levels of sensitivity and specificity displayed by photoaptamers, combined with other advantageous properties of aptamers, should facilitate development of protein chip technology.

Bromodeoxyuridine↗

PubMed2002

Chemical ionization mass spectrometric determination of acrolein in human breast cancer cells.

A selected ion flow tube-chemical ionization mass spectrometric method is presented for the first determination of acrolein metabolically produced in biological tissues. Acrolein in aqueous samples (2.5 ml) is preconcentrated by distillation and directly analyzed using gas-phase proton transfer from H3O+. This method provides sensitive detection of acrolein with the method detection limit of 15 nM at the 99% confidence level. Detection is linear up to the highest concentration studied (13.5 microM, R2 = 0.998). Acrolein levels are determined in doxorubicin-sensitive (MCF-7) and doxorubicin-resistant (MCF-7/Adr) human breast cancer cells in vitro. The intracellular acrolein concentrations differ insignificantly: 0.61 microM for sensitive cells and 0.54 microM for resistant cells. Treatment with a physiological concentration of doxorubicin (0.5 microM) for 24 h at 37 degrees C increased acrolein levels by factors of 2.6 and 1.9 for MCF-7 and MCF-7/Adr cells, respectively. The differential enhancement observed is consistent with the lower levels of enzymes that neutralize oxidative stress in sensitive MCF-7 cells and overexpression of an active drug efflux pump P-170 glycoprotein in resistant MCF-7/Adr cells.

Acrolein↗

PubMed2002

Evaluation of the epidoxorubicin--formaldehyde conjugate, epidoxoform, in a mouse mammary carcinoma model.

This study was conducted to evaluate the toxicity and efficacy of Epidoxoform, a prodrug of the active metabolite of epidoxorubicin, in a mouse model of mammary carcinoma. A dose escalation trial established a maximal tolerated dose of 20 mg/kg given intraperitoneally (i.p.) to 6-8 week old female C3 HeJ mice. Two days following injection of 10(6) Gollin-B mouse mammary tumor cells into the mammary fat pad, Epidoxoform was given and tumor growth compared to mice treated similarly with the maximum tolerated dose of epidoxorubicin and untreated controls. In all efficacy trials, a significant difference was found for tumor volume between Epidoxoform and epidoxorubicin treated mice and controls. In mice treated with a two-dose regimen, significantly increased efficacy was also found between Epidoxoform compared to epidoxorubicin. Epidoxoform appears to be efficacious in this model of mammary carcinoma, with improved efficacy over the parent compound epidoxorubicin. Further evaluation of this analogue appears warranted.

Animals↗