In Vitro Analysis of Defibrotide’s (DF) Uptake by the Human Cell, as Well as DF’s Spesificity for the Viral Envelope Proteins of HIV-! HIV-3, and of Spesific HIV, with Conformatory Data Regarding the Dose Dependency of DF’s Pharmacological Paleiotropism.
Authors: Oral Arsinur D. , Wagner Mark 
Published: April 2020

Abstract
To assess DF’s uptake by the human cell, DF was labeled with a photo-activable analogue of biotin. Cellular uptake started with cell surface receptors to be ultimately localized in the human nucleus.This was confirmed with image analysis utilizing a cold CCD camera.The biologic activity after labelling was considered as being preserved based on prior data with other labeled oligonucleotides.Fig. not shown here clearly displays that nuclear uptake has been in all cases directly proportional to the concentration of DF with biotin The cellular uptake without biotin but with cyanine dye Cy5.18 confirmed the significant enhancement of the uptake with biotin-especially by lymphocytes.To further confirm and explore the above commented dose dependency of the cellular uptake of DF with DF’s therapeutic efficacy against HIV, serial experiments were run, PBMNC were prepared in the known manner, The white cells were then concentrated to a level of 2 million cells per 3 ml RPMI 1640 solution. Two subpopulations of cells were created: Con A stimulated versus unstimulated cell lines. The stimulated cell line showed more enhanced uptake of antibody dye, thereby demonstrating increased celllular expression of viral proteins -even in the face nonspesific immune stimulation (ConA). Subpopulations of unstimulated and stimulated cells were then incubated in the presence of discrete concentrations of DF, each successive assay employing successively higher concentrations of DF. Appropriate controls were run. Cell subpopulations were further stratified into 2 groups of intracellular versus extracellular antibody staining. The former group of cells were fixed with 70% ETOH, washed x2, and suspended in a solution containing 200 ml Hank’s solution, with 2 % FCS (Fetal Calf Serum),0.1% sodium azide, and 5 microliters of Alpha-HIV-Cy5.18. and then incubated for 45 min,to be resuspended finally in 1%paraformaldehyde.Cells reserved for surface staining were washed x2 with monoclonal wash with added 5 ml Alpha-HIV-Cy5.18, as well as 20 microliters surface glycoprotein monoclonal antibody solution of CD3-FITC and CD4-RPE. All prepared cells were analized using a Becton-Dickinson FACS 440 dual laser (argon/krypton)flowcytometry.The expression of HIV proteins was determined on a per cell basis. Flouresence was measured on a logorythmic scale but converted to linear scale for analysis. Mean Linear Flourescence Intensity was accepted to be proportional to the HIV protein expression.. Whether protein expression correlates with cell lysis and/or viability of the virus cannot be determined here.. However decreased expression of viral proteins with increasing concentration of DFand presence of ConA. was verifiable in each case. At .10 mg, 20mg, 30mg DF, respectively.protein expression decreased logorythmically. At 30 mg concentration of DF, however, in both ConA stimulated or unstimulated cell lines, protein expression leveledoff.-further escalation of doses not being pharmacologically effective.3 HIV patients from Phase II will be presented under separate cover. A self -regulating dose range between 50mg/kg----275 mg/kg/d)was also reproducably seen in human patients. And in all instances the spectrum of DF’s pharmacological paleiotropism qualitatively and quantitatively with escalation of doses.