Bioconjugate Chem. 2005, 16, 758−766
758
A New Platform for Oligonucleotide Delivery Utilizing the PEG Prodrug Approach Hong Zhao,* Richard B. Greenwald, Prasanna Reddy, Jing Xia, and Ping Peng Enzon Pharmaceuticals Inc., 20 Kingsbridge Road, Piscataway, New Jersey 08854. Received August 16, 2004; Revised Manuscript Received May 24, 2005
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 Cmax, 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.
INTRODUCTION
Poly(ethylene glycol) (PEG) permanently bonded to oligodeoxynucleotides, generally as phosphate esters, has been synthesized by Bonora (1), Burchovich (2), Drioli (3) and several other groups to stabilize the rapidly metabolized oligo while enhancing, hopefully, cellular uptake (for reviews on this subject see Chirila (4) and Bonora (5)). PEG coupling to the 3′ and 5′ terminal positions showed more than a 10-fold increase in exonuclease stability, while maintaining in vitro activity (6). However anti-sense activity in vivo has yet to be demonstrated. This may in part be due to the inability of solid-phase methods to produce sufficient quantities of materials to test. Other cases of oligos with 3′ or 5′ modifications including dyes, cholesterol (7), or other polymers (8) have shown enhanced antisense binding activities in vitro (9-12). Recently Agrawal et al. (13) have shown that conjugation of dyes, nucleotides, or oligomers through the 5′-position suppresses immunostimulatory activity of CpG containing ODNs, while conjugation at the 3′-position does not. Construction of a pure, high molecular weight PEG prodrug by using the available NH2 moiety obtained by introduction of an amino alkyl side chain at either the 3′ or 5′ terminal positions should provide a long-lived species and enhance oligo bioavailability using this longcirculating prodrug form. Kawaguchi (14) used a hexylamine linker at the 5′ terminus during the automated synthesis of a 15 base pair ODN and then permanently conjugated the basic NH2 group to a low molecular weight (10 000 Da) branched PEG derivative, 2,4-(O-methoxypoly(ethylene glycol))-6-chloro-S-triazine. Unfortunately, purity was not defined, and a comparison of half-lives in human plasma in the presence of S1 nuclease demonstrated that the PEGylated mixture had marginally greater stability, while the free oligo with a phosphorothioate backbone showed the greatest stability. No PEGylation of the latter was reported. * To whom correspondence should be addressed. Phone: (732) 980-4902. Fax: (732) 885-2950. E-mail:
[email protected].
PEG conjugation has been shown to enhance cellular uptake in vitro (15), thus making a PEG prodrug strategy particularly appealing since the native drug can be generated after cellular accumulation. The use of high molecular weight PEG (>20 000 Da) can control circulating half-life in plasma, while the linkage can be designed to release native oligo into the cell. In fact, application of this concept using phosphorothioate-based ODNs conceivably can provide constructs with outstanding stability. Therefore, to evaluate the effect of ODN-PEG conjugation on circulating half-life, drug delivery, and cellular uptake, we initiated a program that was based on the use of an aminohexyl modification at the 3′- and 5′-postions of different oligos to facilitate the reaction sequences. Preparation for future cellular uptake investigations of these PEG conjugates was also addressed by synthesizing a heterobifunctional rPEG with a releasable FITC conjugated oligo on one terminus and a fluorescent dye (Texas Red) on the distal end. CHEMISTRY
The oligo, 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) using a solid-state synthesis. This amino derivative was reacted with releasable PEG (rPEG) based on 1,6-benzyl elimination (BE) (16) as shown in Schemes 1-3, and this reaction resulted in 50-80% isolated yield of the desired PEG prodrugs, 3, 5, 7, 9, and 11. Conjugation with the bicin aliphatic linker (16) (17) proceeded readily and also gave similar yields to the BE aromatic product, 17 (Scheme 5). Unreacted oligo is easily recovered from these conjugation reactions and can be recycled to increase the overall yield. This is in contrast to the liquid-phase synthesis of a PEGylated mercaptoalkyl ester where the final yield of substituted oligo was reported (11) to be only 3%. As was the case with other small molecules, by variation of the trigger different rates of release could be obtained (16). In addition to 1, which contains a phosphorothioate backbone, another model 4-mer oligo
10.1021/bc049804k CCC: $30.25 © 2005 American Chemical Society Published on Web 06/30/2005
New Platform for Oligonucleotide Delivery
Bioconjugate Chem., Vol. 16, No. 4, 2005 759
Scheme 1
Scheme 2
was prepared with a normal phosphate ester backbone, modified as the PEG prodrug (15, Scheme 4), and encouragingly, also demonstrated much greater in vitro stabilities (Table 1). Cellular uptake studies of an rPEG-oligo conjugate should characterize the species produced using confocal microscopy analysis. To accomplish such a study, a heterobifunctional molecule would be required to provide two different types of substitutions at the termini of the PEG linker. Since the rPEG that was used in the first part of this investigation had a MW of 20 000 Da, it was decided to fix the variable of MW so that direct comparisons could be made. The synthesis of 35, the first example of a high molecular weight (hMW) PEG heterobifunctional prodrug, was accomplished by the series of reactions shown in Schemes 6-8. Noteworthy in these sequences is the use of dihydropyran to form orthogonal protecting groups for the distal hydroxy and carboxy groups. The hMW R,ω-amino carboxy PEG derivative 24 was prepared by condensing the commercially available 3400 Da heterobifunctional derivatives 18 and 19 in the presence of DMAP to give heterobifunctional 6800 Da 20, which was divided into two separate portions. The first segment was treated with TFA freeing the distal amino
group and producing the amino acid salt 21. The other functional unit was activated at the carboxy functionality by reaction with 2-mercaptothiazolidine (2-MT) to yield 22 in 90% yield. Reaction of 21 and 22 in the presence of DMAP resulted in the Boc-protected amino acid, 23, which had a MW of 13 600Da. Compound 23 was deblocked with TFA and then reacted with 22, which resulted in the hMW amino acid 24 (20 400 Da). It can be appreciated not only that further symmetrical condensations will double the MW each time but that the use of unequal MW portions will result in amino acids of intermediate molecular weights (MW). The introduction of the unsubstituted 1, 6-BE system was done as described earlier (18) for 2,6-dimethyl-4-hydroxybenzyl alcohol as shown in Scheme 7. Reaction of the carbonate of p-hydroxy benzaldehyde (25) with 24 formed the PEG aldehyde (26), which was reduced to the desired alcohol acid, 27, with sodium borohydride. Hussain and Truelove (19) have shown that the acyloxy derivatives of vinyl ethers (2-tetrahydropyranyl benzoate) undergo first-order hydrolysis with respect to the compound, independent of pH. However acetal derivatives of dihydropyran are only susceptible to acid-catalyzed hydrolysis (20). Since both types of dihydropyran deriva-
760 Bioconjugate Chem., Vol. 16, No. 4, 2005
Zhao et al.
Scheme 3
Scheme 4
tives are formed under the same conditions, we reasoned that reaction of the heterobifunctional PEG alcohol acid (27) with dihydropyran should form both types of protecting groups simultaneously. With dry DCM and TsOH as catalyst, 27 provided the desired di-protected PEG species, 28, in 95% yield. Dissolution of 28 in water at room temperature for 1 h removed the acyloxy ether but not the acetal moiety and provided an 88% yield of the desired monoprotected PEG acid, 29. Conjugation of the free carboxyl group with Texas Red (30) in the presence of EDC resulted in the formation of the stable amide (31) in quantitative yield (Scheme 8). Finally, removal of the acetal from 31 was done with 50% acetic acid and gave the 1,6-benzyl alcohol-Texas Red derivative 32 in 89%
yield. Activation of the OH group was done as described previously (16) by reaction with DSC to give 33, followed by condensation with the aminohexyl derivative of GS with FITC labeling at the 3′ terminus resulting in the desired bifunctional substituted 35. Inspection of 35 pinpoints the trigger (16) as an enzymatically labile phenolic carbamate (21, 22). The ODN is attached distally as a stable aliphatic carbamate that releases only after the trigger hydrolyzes. EXPERIMENTAL SECTION
General Procedures. All the conjugation reactions between PEG linkers and oligonucleotides were carried out in PBS buffer systems at various temperatures.
Bioconjugate Chem., Vol. 16, No. 4, 2005 761
New Platform for Oligonucleotide Delivery Scheme 5
Table 1. In Vitro Properties of PEG-Oligo Conjugatesa t1/2 t1/2 (buffer), (rat plasma), h h compd 1 3 5 7 9 11 13 15 17
>48 >48 >48 >48 >48 >48 >48 >48 >48
1.3 14.7 13.8 >24 6.5 4.1 >48 2.3 11
t1/2 (PEI), h 1.5 >24 >24 >24 >24 5.6 >24 0.25 18.7
t1/2 t1/2 (PEII), (nuclease P1), h min >24 >48 >48 >48 >48 >48 >48 >48 >24
