Down-regulation of c-MYC antigen expression in lymphocytes of Emu-c-myc transgenic mice treated with anti-c-myc DNA methylphosphonates.

In transgenic mice bearing a murine immunoglobulin enhancer/c-myc fusion transgene (Emu-myc), it was found that antisense DNA methylphosphonates targeted against c-myc mRNA inhibited production of c-MYC protein in peripheral lymphocytes. The decrease in protein was measured 3-4 h after i.v. administration of a 300-nmol dose. c-MYC was detected by immunofluorescence of fixed cells stained with an anti-c-MYC antiserum. In addition, DNA methylphosphonates did not induce acute toxicity following i.v. administration of a 300-nmol dose. An identically administered scrambled sequence oligomer did not decrease c-MYC protein or induce toxicity. Finally, recovery of DNA methylphosphonates from the blood plasma of treated mice indicated that the oligomers remained intact up to 3 h, while their concentrations decreased rapidly for the first h, then slowly decreased over the next 2 h. This is the first demonstration of sequence-specific antisense DNA methylphosphonate inhibition of gene expression in the bloodstream of an animal model.     

PET imaging of oncogene overexpression using 64Cu-vasoactive intestinal peptide (VIP) analog: comparison with 99mTc-VIP analog.

The purpose of this study was to assess the feasibility of PET imaging of oncogene VPAC1 receptors overexpressed in human breast cancer cells. METHODS: Vasoactive intestinal peptide (VIP) analog (TP3982) was synthesized to harbor a carboxy-terminus lysine (Lys) residue separated from VIP-asparagine (Asn(28)) by 4-aminobutyric acid (Aba) as a spacer. Lys was derivatized with diaminopropionic acid coupled to a pair of dibenzoylthioglycolic acid residues as protecting groups. The analog was labeled with (64)Cu at pH 9 ((64)Cu-TP3982) and (99m)Tc at pH 12 ((99m)Tc-TP3982). (99m)Tc-TP3982 and VIP derivatized with Aba-GAGG and labeled with (99m)Tc ((99m)Tc-TP3654) were used as reference agents. Smooth muscle relaxivity assays performed with each derivative and compared with unaltered VIP(28) demonstrated functional integrity. In vitro stability of (64)Cu-TP3982 was determined by challenging the complex with 100-mol excess of diethylenetriaminepentaacetic acid (DTPA), human serum albumin (HSA), and cysteine. In vivo stability was determined in urine and serum for up to 24 h. The mass of the Cu-TP3982 complex was determined by mass spectrometry. Human T47D breast tumor xenografts were grown in athymic nude mice. Planar scintigraphic imaging was performed at 4 and 24 h after the intravenous administration of (99m)Tc-TP3982 and (99m)Tc-TP3654 and PET imaging was performed using a small animal MOSAIC PET scanner, also at 4 and 24 h after injection of (64)Cu-TP3982. Tissue-distribution studies were also performed. In a separate experiment, receptors were blocked by intravenous injection of authentic VIP(28) 30 min before the administration of (64)Cu-TP3982 and tissue distribution was examined. RESULTS: (64)Cu-TP3982 labeling yields were 98% +/- 1.2% and those for (99m)Tc-TP3982 and (99m)Tc-TP3654 were 98.2% +/- 1.1% and 97% +/- 1.6%, respectively. The biologic activity of both VIP analogs was uncompromised. When (64)Cu-TP3982 was challenged with 100-mol excess of DTPA, HSA, or cysteine, >98% radioactivity remained as (64)Cu-TP3982. In vivo, >98% of (64)Cu circulating in plasma remained as (64)Cu-TP3982. Of the (64)Cu excreted in urine 4, 20, and 24 h after injection, >98%, 89.9% +/- 0.9%, and 85% +/- 3%, respectively, were bound to TP3982. The mass of Cu-TP3982 as determined by surface-enhanced laser desorption/ionization time of flight (SELDI-TOF) was 4,049.7 Da. Four hours after receptor blocking with VIP(28), there was a significant reduction in uptake of all tissues except in the liver. With (64)Cu-TP3982, the 4-h postinjection tumor uptake was 10.8 +/- 2.1 %ID/g versus 0.5 +/- 0.02 %ID/g and 0.24 +/- 0.08 %ID/g for (99m)Tc-TP3982 and (99m)Tc-TP3654, respectively. Twenty-four hours after injection, the corresponding numbers were 17 +/- 0.7 %ID/g, 0.77 +/- 0.1 %ID/g, and 0.23 +/- 0.1 %ID/g. The severalfold greater uptake (21.2-74) of (64)Cu-TP3982 is attributable to the in vivo stability of the agent. CONCLUSION: The results suggest that the uncompromised biologic activity and the significantly greater tumor uptake of (64)Cu-TP3982, combined with the high sensitivity and enhanced resolution of PET imaging, make (64)Cu-TP3982 highly desirable for further studies in PET imaging of oncogene receptors overexpressed in breast and other types of cancers.     

Radiolabeling of Methylphosphonate and Phosphorothioate Oligonucleotides and Evaluation of Their Transport in Everted Rat Jejunum Sacs

Purpose. The therapeutic use of antisense oligonucleotides will likely involve their administration over protracted periods of time. The oral route of drug dosing offers many advantages over other possible routes when chronic drug administration is necessary. However, little is known about the potential for oligonucleotide uptake from the gastrointestinal tract. This issue is addressed in the current work. Methods. We have developed a simple procedure for radiolabeling oligonucleotides by reductive alkylation with ¹⁴C-formaldehyde. We have utilized this approach, as well as 5 addition of fluorophores, to prepare labeled methylphosphonate and phosphorothioate oligonucleotides for use in intestinal transport studies. An everted rat gut sac model was employed to compare the transport of oligonucleotides to that of model compounds whose permeation properties are better understood. Results. We demonstrate that both methylphosphonate and phosphorothioate oligonucleotides are passively transported across the intestinal epithelium, probably by a paracellular route. The rates of transport for both types of oligonucleotides were similar, and were significantly greater than that of the very high MW polymer blue dextran, but were lower than the transport rate of valproic acid, a low MW compound known to have high oral availability. Conclusions. A significant degree of permeation of oligonucleotides across the gastrointestinal epithelium does occur, but it is still unclear whether this is sufficient to permit effective oral administration of oligonucleotides as drugs.     

Targeting VPAC1 Receptors for Imaging Glioblastoma

Molecular Imaging and Biology - Scintigraphic imaging of malignant glioblastoma (MG) continues to be challenging. We hypothesized that VPAC1 cell surface receptors can be targeted for positron...     

Sequence specificity of alternating hydroyprolyl/phosphono peptide nucleic acids against zebrafish embryo mRNAs

Morpholino phosphorodiamidate (MO) DNA mimics display excellent water solubility and hybridization properties toward DNA and RNA, and have been utilized in the model vertebrate zebrafish (Danio rerio) for genome-wide, sequence-based, reverse genetic screens during embryonic development. Peptide nucleic acids (PNAs) exhibit excellent mismatch discrimination, nuclease resistance, and protease resistance, but low solubility. Negatively charged DNA mimics composed of alternating residues of trans-4-hydroxy-L-proline peptide nucleic acid monomers and phosphono peptide nucleic acid monomers (HypNA-pPNA) combine all of the positive features of both MOs and PNAs. Thus, we evaluated PNA oligomers and HypNA-pPNA oligomers as an alternative to MOs for oligonucleotide inhibition of gene expression in zebrafish embryos. We observed that HypNA-pPNA 18-mers displayed comparable potency to MO 25-mers as knockdown agents against chordin, notail and uroD, with greater mismatch stringency. Furthermore, we observed that a specific HypNA-pPNA 18-mer elicited the dharma (bozozok)(-/-) phenotype in zebrafish embryos, which MO 25-mers do not. These observations validate HypNA-pPNAs as an alternative to MO oligomers for reverse genetic studies. The stronger hybridization and greater specificity of HypNA-pPNAs enable knockdown of mRNAs unaffected by MO oligomers.     

Patient preferences for care by general internists and specialists in the ambulatory setting

OBJECTIVE: To investigate patients’ preferences for care by general internists and specialists for common medical conditions. DESIGN: Telephone interview. SETTING: A convenience sample of general internal medicine practices at 10 eastern academic medical centers. PATIENT/PARTICIPANTS: A probability sample of 314 participants who had at least one visit with their primary care physician during the preceding 2 years. MEASUREMENTS AND MAIN RESULTS: Items addressed patients’ attitudes concerning continuity of care, preferences for care by general internists or specialists for common medical problems, and perceptions about the competency of general internists and specialists to manage these problems. Continuity was important to participants, with 63% reporting they preferred having one doctor. Respondents were willing to wait 3 or 4 days to see their regular doctor (85%) and wanted their doctor to see them in the emergency department (77%) and monitor their care while in the hospital (94%). A majority (>60%) preferred care from their regular doctor for a variety of new conditions. Though respondents valued continuity, 84% felt it was important to be able to seek medical care from any type of physician without a referral, and 74% responded that if they needed to see a specialist, they were willing to pay out-of-pocket to do so. Although most participants (98%) thought their regular doctor was able to take care of usual medical problems, the majority through that specialists were better able to care for allergies (79%) and better able to prescribe medications for depression (65%) and low-back pain (72%). CONCLUSIONS: Participants preferred to see their general internist despite their perceptions that specialists were more competent in caring for the conditions we examined. However, they wanted unrestricted access to specialists to supplement care provided by general internists. Presented in part at the 1996 meeting of the Society of General Internal Medicine. This work was supported in part by grant 2D28PE54004 from the Bureau of Health Professions, Health Resources and Services Administration, to the Faculty Development Program for General Internal Medicine, University of North Carolina at Chapel Hill, and by grant 52285 from the Summa Health System, Akron, Ohio.     

Variant Philadelphia translocations in chronic myeloid leukemia: correlation with cancer breakpoints, fragile sites and oncogenes

Four cases of variant Philadelphia (Ph1) translocations were found in 72 patients (5.5%) with Ph1-positive chronic myeloid leukemia (CML). One previously unreported case was a simple variant translocation, namely, 46,XY,t(11;17)(q13;p13),t(17;22)(q25;q22); 46,XY,t(1;21)(q32;q11),t(11;17)(q13;p13), t(17;22)(q25;q11). Complex variant translocations were observed in three cases, namely, 46,XY,t(5;9;22)(q31;q34;q11),46,XX,t(8;9;22) (q22;q34;q11) and 46,XX,t(9;15;22) (q34;q15;q11). The chromosomal breakpoints in the cases of variant Ph1 translocations were the following: 1q32, 5q31, 8q22, 11q13, 15q15, 17p13, 17q25 and 21q11. Eight of the eight (100%) breakpoints were located in Giemsa-negative bands. Furthermore, seven of the eight (87%) variant Ph1 breakpoints correspond to the breakpoints present in consistent cancer arrangements. Three of the eight (38%) correspond to fragile sites and four of the eight (50%) correspond to oncogenes.     

Deciphering the TLR transcriptome and downstream signaling pathway in cerebrospinal fluid in pediatric meningitis

Inflammation Research - Pediatric meningitis is characterized by a colossal inflammatory response to the pathogen in the central nervous system (CNS). This unabated inflammatory response persists...     

PET imaging of CCND1 mRNA in human MCF7 estrogen receptor positive breast cancer xenografts with oncogene-specific [64Cu]chelator-peptide nucleic acid-IGF1 analog radiohybridization probes.

Treatment of breast cancer is hampered by a large unmet need for rapid, sensitive, specific staging and stratification of palpable and nonpalpable abnormalities. Mammography and physical examination miss many early breast cancers, yet detect many benign lesions. Cyclin D1, encoded by CCND1 messenger RNA (mRNA), and insulin-like growth factor 1 receptor (IGF1R) are key regulators of cell proliferation that are overexpressed in most breast cancers. Therefore, we hypothesized that malignant breast masses could be imaged and quantitated externally by PET with a dual-specificity probe that targets both CCND1 mRNA and IGF1R. METHODS: We designed a CCND1-specific peptide nucleic acid (PNA) hybridization sequence (CTGGTGTTCCAT), separated by a C-terminal spacer to a cyclized IGF1 peptide analog (d-Cys-Ser-Lys-Cys), for IGF1R-mediated endocytosis. On the N-terminus we attached a chelator (1,4,7-tris(carboxymethylaza)cyclododecane-10-azaacetyl [DO3A]) for the positron-emitting nuclide (64)Cu. We administered the [(64)Cu]CCND1-IGF1 analog radiohybridization probes, as well as sequence controls, by tail vein to immunocompromised female NCr mice bearing human MCF7 estrogen-dependent, receptor-positive xenografts. We imaged the mice by PET and CT 4 and 24 h later, and measured tissue distribution of the radiohybridization probes. RESULTS: We observed 8 +/- 2-fold higher PET intensity in the center of the breast cancer xenografts than in the contralateral tissues at 24 h after injection of the [(64)Cu]CCND1-IGF1 analog radiohybridization probe. IGF1 blocking yielded significantly weaker images (P < 0.05) relative to the tumor-free side at 24 h after injection, as did a PNA mismatch probe, a peptide mismatch probe, and free (64)CuCl(2). CONCLUSION: These results are consistent with our hypothesis for radiohybridization PET of overexpressed CCND1 mRNA, dependent on IGF1R-mediated endocytosis, in suspect masses. Early noninvasive detection of initial cancerous transformation, as well as invasive or recurrent breast cancer, with dual-specificity radiohybridization probes, might enable molecularly targeted staging, stratification, and choice of therapy.