An unexpected amide bond cleavage: poly (ethylene glycol) transport forms of vancomycin. 2.

PEG-amide vancomycin derivatives (V(3) position) have been synthesized and found to behave as prodrugs in vivo, demonstrating anti-microbial activity in mice when challenged with Staphylococcus aureus. The corresponding PEG-carbamate derivatives do not manifest this in vivo activity, although both classes of compounds have similar in vitro rat plasma stability. Thus, it appears that extra vascular cleavage of the amide bond can occur if the condition of extended circulation of the conjugate is met, resulting in the release of vancomycin.

A new platform for oligonucleotide delivery utilizing the PEG prodrug approach.

The oligonucleotide (oligo, ODN), Genasense (GS), an ODN currently waiting for FDA approval, was chosen as a model and modified with a 5' or 3' aminohexyl functionality (1 and 4, respectively) using solid-state synthesis. These amino derivatives were reacted with different releasable PEGs (rPEGs). The in vitro results of the PEG-modified oligos (Table 1) clearly show a substantial increase in rat plasma half-life and enhanced stability against a variety of nucleases, especially the predominant nuclease (PEII) in mammals, which is the main source of oligo degradation in cells. The advantage of using a PEG prodrug approach was further demonstrated by the pharmacokinetic (PK) results, which exhibited much greater C(max), plasma half-life, and area under the curve (AUC) for 3 compared to unmodified GS. A key step in the synthesis of ODN prodrug conjugates with a dye label was also accomplished successfully by employing dihydropyran derivatives of alcohols and acids as orthogonal protecting groups during the synthesis.

PEG prodrugs of 6-mercaptopurine for parenteral administration using benzyl elimination of thiols.

6-Mercaptopurine (6-MP) is an orally administered, water-insoluble purine analog that is effective against acute lymphatic leukemia. Oral absorption of 6-MP, however, is quite erratic, with only 16-50% of the administered dose reaching the blood. In this report, water-soluble parenterally administered poly(ethylene glycol) (PEG) prodrugs of 6-MP were synthesized using several chemical approaches that enabled the protection of the thiol group through a modification of the benzyl elimination (BE) system. In our earlier work on antimetabolites, it was found that branching of the PEG allowed greater loading of the active drug. This approach was also utilized within this work to give multiloaded systems. The resulting conjugates were stable in pH 7.4 PBS buffer as well as in rat plasma for extended periods. However, these conjugates did act as prodrugs in vivo and a number of PEG-6-MP constructs had significant (P < 0.05) activity in murine leukemia, as well as certain solid tumors, compared with unconjugated 6-MP in a solubilizing vehicle. The fact that some PEG-6-MP conjugates were stable during in vitro plasma dissociation assays, but demonstrated in vivo anticancer activity, suggests extravascular cleavage of the linking group. This work demonstrates that PEG conjugation is an effective means of solubilizing 6-MP for parenteral administration.

Poly(ethylene glycol) prodrugs of the CDK inhibitor, alsterpaullone (NSC 705701): synthesis and pharmacokinetic studies.

Two methods were devised to conjugate PEG to alsterpaullone (NSC 705701) via the N of the indole ring portion of the molecule. In the first approach, activation of the indole was accomplished by reaction with p-nitrophenyl chloroformate to produce a reactive carbamate that was then condensed with a mono blocked diamine to form a urea bond followed by deblocking and conjugation to PEG. The second route used the anion of the indole and produced a carbamate bond. Both compounds were highly water soluble, were stable in buffer, and released alsterpaullone in vitro and in vivo. Studies were conducted in mice to investigate the influence of PEGylation on the plasma pharmacokinetics of alsterpaullone. The total plasma clearance rate was decreased up to 32-fold, and the biological halflife lengthened up to 8-fold when alsterpaullone was injected i.v. as a PEG-conjugate and compared to injection of the unconjugated compound. The most pronounced effect on the pharmacokinetics of alsterpaullone was produced by a 40-kDa PEG urea-linked conjugate. When the 40- and 20-kDa urea-linked conjugates were administered by i.p. injection, high relative bioavailability (46% and 99%, respectively) of alsterpaullone was observed.

A new aliphatic amino prodrug system for the delivery of small molecules and proteins utilizing novel PEG derivatives.

A new amino PEG prodrug system, based entirely on aliphatic structures, has been designed using ester derivatives easily synthesized from N-modified bis-N-2-hydroxyethylglycinamides. Hydrolysis of the various promoiety bonds, in vivo, regenerated amine in a predictable manner. Thus, a novel new methodology for controlled release of amino-containing drugs, peptides, and proteins has been accomplished. This work demonstrates the usefulness of a PEG prodrug strategy that results in solubilization of insoluble amino-containing drugs and provides prodrugs with relatively long circulating half-lives. It can be appreciated that this novel system should also be applicable for nonpolymer-containing prodrugs as well.

Poly(ethylene glycol) transport forms of vancomycin: a long-lived continuous release delivery system.

The facile reaction of vancomycin with various PEG linkers, at the V(3) position, has been selectively accomplished by using an excess of base in DMF. Using rPEG as a blocking group for V(3) provides crystalline derivatives that can be further PEGylated to give pure V(3)-X(1) latentiated species (transport forms). V(3) tetrameric species were also prepared in order to increase the loading of drug on PEG. All PEG-vancomycin transport forms show significant antibacterial activity that is on the same order of native vancomycin. Significant increases in the AUC were observed for all PEG-vancomycin conjugates thus making them potential single dose therapies.

Synthesis, isolation, and characterization of 2'-paclitaxel glycinate: an application of the Bsmoc protecting group.

The relevance of the Bsmoc protecting group in the synthesis of moisture- and base-sensitive compounds has been demonstrated by the preparation of 2'-paclitaxel glycinate with use of the solid anhydrous base, 4-piperidinopiperidine in DCM solvent.

Utility of poly(ethylene glycol) conjugation to create prodrugs of amphotericin B.

This paper reports on the synthesis, safety, and efficacy of a series of water-soluble derivatives of poly(ethylene glycol) (PEG)-conjugated amphotericin B (AmB). PEG 40 000 attached to the sugar amino group of AmB via labile carbamate and carbonate linkages was examined. The synthetic program conducted for this investigation provided a series of disubstituted PEG-AmB derivatives which had in vitro PEG half-life of hydrolyses rates in rat plasma varying between 1 and 3 h. Importantly, all conjugates demonstrated less than 6% hydrolysis following 24 h incubation in pH 7.4 phosphate buffer at 25 degrees C and showed solubilities greater than 46 mg/mL in aqueous solutions. The solubility of AmB in the conjugates increased up to approximately 200 times compared to unmodified AmB in saline. As a major finding, this investigation demonstrated that conjugation of PEG to AmB could produce conjugates that were significantly (6x) less toxic than AmB-deoxycholate and maintained, or even had enhanced, in vivo antifungal activity.

Tripartate poly(ethylene glycol) prodrugs of the open lactone form of camptothecin.

Two PEG prodrugs utilizing conjugation of PEG through the C-21 acid functionality as well as the C-17 OH group of CPT hydroxy-amide open forms were synthesized and characterized. Both of these open lactone tripartate prodrugs were shown to be water soluble and highly effective in MX-1 mouse xenograph studies. Indirect evidence implies that the initial ester or carbonate bond breaking is esterase mediated in the first step of the cascade of CPT release.

Controlled release of proteins from their poly(ethylene glycol) conjugates: drug delivery systems employing 1,6-elimination.

Several tripartate releasable PEG linkers (rPEG) that can provide anchimeric assistance to hydrolysis (cyclization prodrugs) were prepared and, after conjugation to lysozyme demonstrated rapid cleavage in rat plasma compared to nonassisted, permanently bound PEG. By varying the chemical structure and adding steric hindrance, the half-life of the protein conjugates can be adjusted from slow to very fast. The pharmacokinetics (PK) of regeneration of native protein, from various rPEG conjugates can, for the first time, be easily followed in the rat using green fluorescent protein. The PK in mice was also determined for rPEG-Interleukin 2 (rPEG-IL-2) conjugates in vivo using an ELISA assay. Thus, a systematic study of rPEGylated proteins, either in vivo or in vitro during processing, has been investigated based on regeneration of native protein. The employment of releasable PEG polymers substantially broadens the applications of PEGylation drug delivery technology by introducing the benefits of controlled release of native protein therapeutics.

Effective drug delivery by PEGylated drug conjugates.

The current review presents an update of drug delivery using poly(ethylene glycol) (PEG), that focuses on recent developments in both protein and organic drugs. Certainly the past 10 years has resulted in a renaissance of the field of PEG drug conjugates, initiated by the use of higher molecular weight PEGs (M(w)>20,000), especially 40,000 which is estimated to have a plasma circulating t(1/2) of approximately 10 h in mice. This recent resuscitation of small organic molecule delivery by high molecular weight PEG conjugates was founded on meaningful in vivo testing using established tumor models, and has led to a clinical candidate, PEG-camptothecin (PROTHECAN), an ester based prodrug currently in phase II trials. Additional applications of high molecular weight PEG prodrug strategies to amino containing drugs are presented: similar tripartate systems based on lower M(w) PEG and their use with proteins is expounded on. The modification of a benzyl elimination tripartate prodrug specific for mercaptans is presented, and its successful application to 6-mercaptopurine giving a water soluble formulation is discussed. Recent novel PEG oligonucleotides and immunoconjugates are also covered. Clinical results of FDA approved PEGylated proteins are also presented.

Selective phenolic acylation of 10-hydroxycamptothecin using poly (ethylene glycol) carboxylic acid.

Selective acylation of the phenolic hydroxyl group of 10-hydroxycamptothecin has been accomplished using phenyl dichlorophosphate. Additional modification of the 10-OH as an ether permits a 20-acyl derivative to be synthesized. This result along with data from a 6-hydroxyquinoline model strongly suggests that powerful intermolecular hydrogen bonding exists in the parent molecule.

Anticancer drug delivery systems: N4-acyl poly(ethyleneglycol) prodrugs of ara-C. I. Efficacy in solid tumors.

A systematic study of N(4) amino PEG-prodrugs of ara-C (1) was conducted and provided a series of disubstituted amides, as well as a carbamate derivative. These conjugates showed hydrolysis half lives in rat plasma from about 1 h to 3 days, but were stable for >24 h in phosphate buffer, pH 7.4. In an LX-1 solid lung tumor model some of the PEG prodrugs exhibited superior activity to ara-C when compared on a molar basis. One problematic issue that was identified in this investigation was the need to increase the loading of ara-C onto PEG in order to avoid highly viscous solutions.

Anticancer drug delivery systems: multi-loaded N4-acyl poly(ethylene glycol) prodrugs of ara-C. II. Efficacy in ascites and solid tumors.

The synthesis of branched PEG (40,000) acids has been achieved using aspartic acid (Asp) and AspAsp dendrons. Complete conjugation of these dendritic acids with cytosine arabinoside (ara-C) was achieved by the use of spacers that allowed a greater separation of the branches to accommodate several large ara-C molecules in proximity to each other. The tetrameric and octameric PEG-ara-C amide prodrugs were much more effective in the treatment of solid and ascites tumors compared to the native drug. The greater loading of the PEG backbone appears to have achieved a minimum threshold concentration for the therapeutic delivery of ara-C.