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Medical applications of protein/peptide/oligonucleotide biotechnologyThe activities of this laboratory focus on developing novel approaches to diagnose and treat diseases. Unusual forms of diagnostic reagents and therapeutic agents are synthesized and then tested in collaborating laboratories. One example is a serodiagnostic ELISA for Lyme disease. a bacterial infection transmitted by ticks. This is important because Lyme disease must be treated early to avoid neurological complications. Only half of the victims develop a characteristic bull's eye rash. the other early symptoms are nonspecific and currently used immuoassays have high false positive and false negative rates. Using synthetic peptide libraries based on the sequences of the most immunogenic bacterial proteins. we selected the several most specific linear peptide epitopes by screening the libraries with Lyme disease sera and ruled out nonspecific (cross-reactive) epitopes by screening with normal sera. To achieve sensitivity for detection of low abundance antibodies in early stage disease. we prepared multivalent antigens having increased avidity for the antibodies. which were limited to IgM. The performance of this ELISA was exceptionally good. The concept of a multivalent carrier molecule was also applied to targeted drug delivery. We designed a form (a copolymer) of the FDA-approved polymer. poly(ethylene glycol) (PEG) having the following features: (i.) PEG has the dimensions of a molecular wire. (ii.) there are thiol groups placed at regular intervals along the PEG chain. (iii.) multiple copies of the same or different thiol-containing drugs can be linked via the bioreversible disulfide bond which should stay intact in the bloodstream but be cleaved after entering cells due to the relative concentrations of reduced glutathione. (iv.) multiple copies of a cell targeting agent can be appended to the PEG chain by non-reversible bonds. (v.) multiple copies of a cell uptake enhancer can also be appended to the carrier and (vi.) PEG is biocompatible. We successfully developed a targeted carrier for phagocytic cells based on their receptor for the bacterial-derived peptide. N-formyl-Met-Leu-Phe (fMLF). With 8 copies of fMLF per PEG chain. the Kd for the macrophage-like cell line. HL-60 was found to be 0.2 nM. whereas these cells were activated with an EC50 of 5 nM.. Thus. at a blood level of targeted carrier in the range of about 0.5 to 2 nM. essentially 100% of the receptors will have bound carrier but 0% will become activated. An initial in vivo study demonstrated uptake into mouse peritoneal macrophages. Since macrophages constitute the main reservoirs of the microorganisms that cause AIDS. tuberculosis and leishmaniasis. we next plan to deliver specific drugs in a manner that would concentrate the drug only where needed. thereby maximizing the therapeutic effect while minimizing adverse side reactions at other tissues. Of wider importance is our use of a nutrient-bearing carrier that utilizes a specific intestinal transporter. Drugs that otherwise have poor or variable oral bioavailability. such as peptides. can be reversibly linked to this nutrient-carrier and co-transported across the intestinal barrier. The same PEG-thiol copolymer has been used for making a gel that can provide sustained release of a drug. To a solution containing the copolymer and the drug (which must not have a free thiol group but can have disulfide bonds) is added a bifunctional cross-linking reagent. This mixture must be injected within about 1 minute after which it hardens into a gel. When injected into a host as a subcutaneous depot. it will release the drug uniformly over a period of weeks. The addition of increasing amounts of emulsified oil droplets to the copolymer solution will produce a gel having a release duration up to several months. Pharmacokinetic studies have been done in rabbits or rats with quinine (a small molecule drug). calcitonin (a 3 kDa peptide). pegylated forms of RANTES (an 8 kDa peptide) and erythropoietin (a 30 kDa glycoprotein). Interestingly. the rabbits had to be bled a few times to counteract the dangerously high levels of red blood cells induced by the erythropoietin. Among the advantages of this depot delivery system are (i.) non-fluctuating drug levels to avoid cycling from subtherapeutic to toxic levels. (ii.) elimination of patient noncompliance with the medication regimen and (iii.) localization of the depot to the site of the disease. There are numerous medical applications possible with the targeted carrier and the depot delivery systems. Selected PublicationsSundaram RK, Kasinathan C, Stein S, Sundaram P. (2008) Detoxification depot for beta-amyloid peptides. Curr Alzheimer Res. 5(1):26-32. Wan L, Zhang X, Pooyan S, Palombo MS, Leibowitz MJ, Stein S, Sinko PJ. (2008) Optimizing size and copy number for PEG-fMLF (N-formyl-methionyl-leucyl-phenylalanine) nanocarrier uptake by macrophages. Bioconjug Chem. 19(1):28-38. Wan L, Pooyan S, Hu P, Leibowitz MJ, Stein S, Sinko PJ. (2007) Peritoneal macrophage uptake, pharmacokinetics and biodistribution of macrophage-targeted PEG-fMLF (N-formyl-methionyl-leucyl-phenylalanine) nanocarriers for improving HIV drug delivery. Lalloo A. Chao P. Hu P. Stein S. Sinko PJ. (2006) Pharmacokinetic and pharmacodynamic evaluation of a novel in situ forming poly(ethylene glycol)-based hydrogel for the controlled delivery of the camptothecins. J Control Release. 112(3):333-42. Wan L. Zhang X. Gunaseelan S. Pooyan S. Debrah O. Leibowitz MJ. Rabson AB. Stein S. Sinko PJ. (2006) Novel multi-component nanopharmaceuticals derived from poly(ethylene) glycol. retro-inverso-Tat nonapeptide and saquinavir demonstrate combined anti-HIV effects. AIDS Res Ther. 3:12. Dharap SS. Chandna P. Wang Y. Khandare JJ. Qiu B. Stein S. Minko T. (2006) Molecular targeting of BCL2 and BCLXL proteins by synthetic BCL2 homology 3 domain peptide enhances the efficacy of chemotherapy. J Pharmacol Exp Ther. 316(3):992-8. Paranjpe PV. Stein S. Sinko PJ. (2005) Tumor-targeted and activated bioconjugates for improved camptothecin delivery. Anticancer Drugs. 16(7):763-75. Zhang X. Wan L. Pooyan S. Su Y. Gardner CR. Leibowitz MJ. Stein S. Sinko PJ. (2004) Quantitative assessment of the cell penetrating properties of RI-Tat-9: evidence for a cell type-specific barrier at the plasma membrane of epithelial cells. Mol Pharm. 1(2):145-55. Gunaseelan S. Debrah O. Wan L. Leibowitz MJ. Rabson AB. Stein S. Sinko PJ. (2004) Synthesis of poly(ethylene glycol)-based saquinavir prodrug conjugates and assessment of release and anti-HIV-1 bioactivity using a novel protease inhibition assay. Bioconjug Chem. 15(6):1322-33. Paranjpe PV. Chen Y. Kholodovych V. Welsh W. Stein S. Sinko PJ. (2004) Tumor-targeted bioconjugate based delivery of camptothecin: design. synthesis and in vitro evaluation. J Control Release. 100(2):275-92. Zhang G. Leibowitz MJ. Sinko PJ. Stein S. (2003) Multiple-peptide conjugates for binding beta-amyloid plaques of Alzheimer's disease. Bioconjug Chem. 14(1):86-92. Qiu. B.. Stefanos. S.. Ma. J.. Lalloo. A.. Perry. B. A.. Leibowitz. M. J.. Sinko. P. J. and Stein. S. (2003) A hydrogel prepared by in situ cross-linking of a thiol-containing polyethylene glycol based polymer: A new biomaterial for protein drug delivery. Biomaterials 24:11-18. Zhang. G.. Leibowitz. M. J. Sinko,P. and Stein. S. (2003). Multiple peptide conjugates for binding b-amyloid plaques of Alzheimer's disease. Bioconjugate Chemistry 14:86-92. Dharap. S. S.. Qiu. B.. Williams. G. C.. Sinko. P.. Stein. S. and Minko. T. (2003) Molecular targeting of drug delivery systems to ovarian cancer by BH3 and LHRH peptides. J. Controlled Release 91:61-73. Pooyan. S.. Qiu. B.. Chen. M.. Fong. D.. Leibowitz. M. J. and Stein. S. (2002). Conjugates bearing multiple formyl-methionyl peptides display enhanced binding to but not activation of phagocytic cells. Bioconjugate Chemistry 13:216-223. Minko. T.. Paranjpe,P.. Qiu. B.. Lallo. A.. Won,R.. Stein. S.and Sinko. P. J. (2002) Enhancing of the anticancer efficacy of camptothecin by biotinylated poly(ethylene glycol)vehicle in sensitive and multidrug resistant human ovarian carcinoma cells. Cancer Chemotherapy and Pharmacology 50:143-150. Ramanathan. S. . Qiu. B. . Pooyan. S.. Zhang. G.. Leibowitz. M. J.. Stein. S.. and Sinko. P. (2001) Targeted PEG-based bioconjugates enhance the cellular uptake and transport of a HIV-1 TAT nonapeptide. J. Controlled Release 77:199-213. Ramanathan S. Pooyan S. Stein S. Prasad PD. Wang J. Leibowitz MJ. Ganapathy V. Sinko PJ. (2001) Targeting the sodium-dependent multivitamin transporter (SMVT) for improving the oral absorption properties of a retro-inverso Tat nonapeptide. Pharm Res 18:950-6 Tung CH. Stein S. (2000) Preparation and applications of peptide-oligonucleotide conjugates. Bioconjug Chem 11:605-18 Qiu B. Brunner M. Zhang G. Sigal L. Stein S. (2000) Selection of continuous epitope sequences and their incorporation into poly(ethylene glycol)-peptide conjugates for use in serodiagnostic immunoassays: application to Lyme disease. Biopolymers 55:319-33 |
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