Analytical Biochemcial Analysis of Commerical Defibrotide - Sequence Analysis of the Nucleotidic Fractions Via Delayed Extraction Matrix Assissted Laser Desorption / Ionization Time of Flight Mass Spectrometry.

Introduction: Defibrotide (DF), now in clinical trials in Veno-Occlussive Disease (VOD), and Multi-Organ Failure (MOF) as an anti-thrombotic agent is also recognized as a modulator of phosphorilated nucleotides of ATP, ADP, NAD/NADH and cAMP..Thus far the mechanism of its polypharmacology, and the.identity of its biologically active components are not known.The precursor molecule is a double stranded mammalian DNA. And the final commercial product is a mixture of nucleotidic fractions which have formed by the ability of the molecule to form intra-molecular bonding., with random sequences such as:P1-5, (dAp)12–24, (dGp) 10–20, (dTp)13–26, (dCp)10–20, Wherein, P=phosphoric radical; dAp= deoxyadenylic monomer; dGp= deoxyguanylic monomer; dTp= deoxythymidylic monomer; dCp= deoxycytidilic monomer.(US Patent # 4,985,552,Crinos). This report aims to investigate for the first time the sequence spesificity, and the reproducability of the nucleotidic fractions of DF. and to define a structural base for DF as a global modulator of the signalling network.

Method:.Materials from two commercial forms of DF, Prociclide and Noravid in solution and capsule forms, were separated in each case by HPLC on a Vydac, C8 column using 0.1% trifluoroacetate in water. 513 mass spectra peaks were collected, freeze dried, preserved on ice and subjected to Voyager Elite Biospectrometry Workstation using the technique of delayed extraction matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry..A blank run representing DNA matrix was done in each case on MALDI-TOF to detect the low MW particles that were not related to the sample. The peaks were superimposed on the HPLC profiles of mono-, di-, tri-phosphates of T,C,G, A via crossmatching the MW, together with retention times as described above.260,000 crossmatches were done.

Statistics: The min, max, mean and median mass values were stratified into four respective batches of DF, capsule and liquid forms, and two commercial labels The standard deviations (SD) were calculated in each of the above stratifications with the “n-1” method.

Results: HPLC profiles contained more than one molecular species with MWs ranging from <300 to <1500 Da. and were not superimposable. A representative panel would include deoxyadenylic and deoxycytidylic monomers, guanine and other nucleotides of 3–25 bases, such as dC,dA, G, dGMP, AMP, oligonucleotides with 3–5 bases such as UTP, dTTP, CTP, ATP, dGTP, cTMP, CMP, cGMP, and dAMP. Since the study was only directed at bases, deoxy-bases and mono-and di-, and tri-nucleotides of </= 560 Da (the calculated MW of alkali sodium salt of ATP), statistical calculations were carried out only for these fractions. When compared to capsules, liquid DF showed double number of peaks of ATP, ADP, AMP, GTP, GDP, and GMP. The standard deviations (STD) on the nucleotides in the various liquid forms of Defibrotide were much narrower, ranging between (+ / −) 0–1.4, capsule forms showing less consistency with STD’s of (+ / −)0–2.1

Conclusion: The sequence analysis of the nucleotidic fractions of <560 D MW displayed presence of cAMP, ATP, ADP, GTP, GDP,,GMP, CTP,CMP,CDP, TTP,TDP,TMP reproducably between batches. DF maybe a direct and indirect supplier of messenger nucleotides. This structural analysis may support this hypothesis generating report presenting DF as the prototype global modulator of the signalling network as a mechanism of its polypharmacology.