Synthesis of Poly (ethylene glycol)-Based Saquinavir Prodrug

Bioconjugate Chem. 2004, 15, 1322−1333

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Synthesis of Poly(ethylene glycol)-Based Saquinavir Prodrug Conjugates and Assessment of Release and Anti-HIV-1 Bioactivity Using a Novel Protease Inhibition Assay Simi Gunaseelan,† Olivia Debrah,† Li Wan,† Michael J. Leibowitz,‡,|,§ Arnold B. Rabson,‡,| Stanley Stein,†,|,§ and Patrick J. Sinko*,†,|,§ Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, New Jersey 08854-0789, Center for Advanced Biotechnology and Medicine, Department of Molecular Genetics, Microbiology, and Immunology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854, Cancer Institute of New Jersey, New Brunswick, New Jersey 08903-2681, and New Jersey Center for Drug Targeting and Delivery, Piscataway, New Jersey 08854. Received May 12, 2004; Revised Manuscript Received September 23, 2004

Various poly(ethylene glycol)(PEG)-based prodrug conjugates of the HIV-1 protease inhibitor (PI) saquinavir (SQV) were prepared using several types of chemical groups potentially capable of modifying its pharmacokinetic properties. These prodrug conjugates included SQV-cysteine-PEG3400, SQVcysteine-PEG3400-biotin, SQV-cysteine(R.I.CK-Tat9) [a cationic retro-inverso-cysteine-lysine-Tat nonapeptide]-PEG3400, and SQV-cysteine(R.I.CK(stearate)-Tat9)-PEG3400. SQV was linked to cysteine to form a releasable SQV-cysteine ester bond in all of the conjugates. The amino group of the cysteine moiety provided an attachment site for a slower-degrading amide bond with Nhydroxysuccinimide-activated forms of PEG- and PEG-biotin. Disulfide bonds were used to attach the cationic peptides, R.I.CK-Tat9 and R.I.CK(stearate)-Tat9 to the cysteine moiety in order to provide cell-specific release. An assay was established and validated for measuring the activity of SQV and other protease inhibitors in biological samples. In this assay, cleavage of an internally quenched fluorescent substrate, Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gly-Lys(DABCYL)-Arg by HIV-1 protease was inhibited by SQV in a dose-dependent manner at concentrations of 0.05-0.5 µM. All prodrug conjugates were shown to be inactive in this assay until the ester bond was cleaved and active SQV was released. The prodrug reconversion half-lives in 0.1 N HCl, phosphate-buffered saline (PBS) at pH 7.4 and in spiked plasma at 37 °C were 9, 14, and 0.9 h, respectively. The anti-HIV-1 activity (ED50) of the PEG-based SQV prodrug conjugates was evaluated in MT-2 cells using an MTT assay. The activity of conjugated SQV was reduced (ED50 ) 900 nM) for the PEG only conjugate, but restored with the addition of biotin (ED50 ) 125 nM), R.I.CK-Tat9 (ED50 ) 15 nM), and R.I.CK(stearate)-Tat9 (ED50 ) 62 nM) as compared to maximum achievable anti-HIV-1 activity (unconjugated SQV, control, ED50 ) 15 nM), suggesting enhanced cellular uptake of conjugates. Cytotoxicity (LD50) was assessed for all prodrug conjugates using non-HIV-1 infected cells and was found to be in the micromolar range. The difference between the LD50 and ED50 suggests a favorable therapeutic index for the prodrug conjugates. In conclusion, these promising initial results demonstrate that the reconversion of the conjugate prodrugs was complete and that active SQV was released. Since the major delivery advantages of PEG prodrug conjugates can only be observed in vivo, issues of reconversion and elimination half-lives in plasma will have to be further studied in an in vivo model. The current results also demonstrate that the protease inhibition assay is a simple yet effective bioanalytical tool that can be used to assess the release and anti-HIV-1 activity of HIV-1 PIs from their prodrug forms.

INTRODUCTION

Acquired immunodeficiency syndrome (AIDS) is a degenerative disease of the immune and central nervous systems caused by the human immunodeficiency virus (HIV). HIV-1 protease is the target of the protease inhibitors, a class of drugs commonly used for AIDS therapy. SQV1 is the first HIV-1-protease inhibitor (PI) * To whom correspondence should be addressed. Tel: +1732-445-3831*213. Fax: +1-732-445-4271. E-mail: sinko@ rci.rutgers.edu. † Rutgers University. ‡ University of Medicine and Dentistry of New Jersey. | Cancer Institute of New Jersey. § New Jersey Center for Drug Targeting and Delivery.

to be approved by the U.S. Food and Drug Administration. However, therapeutic use of SQV suffers from problems of low absorptive and high secretory permeability, bioconversion to inactive metabolites, and poor solubility (1, 2). The oral bioavailability of SQV in clinical formulations is low and/or variable with limited penetration into the lymphatic and central nervous systems (CNS) (3-6). While its low and variable bioavailability is primarily attributed to metabolism by cytochrome P-450 3A, recent results published by our group (7, 8) and others (9) suggest that multiple membrane transporters may also contribute significantly to the observed behavior. The consequence of variable SQV bioavailability can be the development of viral resistance, ultimately resulting in therapeutic failure. Clearly, better drug

10.1021/bc0498875 CCC: $27.50 © 2004 American Chemical Society Published on Web 10/28/2004

Synthesis and Anti-HIV-1 Activity of Saquinavir Conjugates

delivery methods are required to enhance the bioavailability of SQV and to reduce the associated variability observed in the clinic. One common drug delivery approach to enhancing bioavailability is to utilize prodrugs. A ‘prodrug’ approach has the potential advantage of achieving greater drug bioavailability by altering the physicochemical properties of the drug, minimizing processes that would otherwise cause elimination or breakdown of the parent drug, or by increasing cellular uptake. Various ester PI prodrugs derived from SQV, indinavir, or nelfinavir connected to fatty acids, L-valine, L-tyrosine, PEG, or D-glucose (10, 11) have been reported. The most encouraging results were obtained with the L-valine PI prodrugs (12) which prompted the extension of this series to include L-leucine and L-phenylalanine along with new ester PI prodrugs derived from L-tyrosine or that are linked to diglycerides (13). Among the strategies explored up to now, the most successful has been the “hydrophilic” prodrug approach which has led to the discovery of fosamprenavir, a watersoluble phosphate ester prodrug of the sparingly watersoluble amprenavir that was approved in 2003. The absorption enhancement of amprenavir that is observed by using the fosamprenavir prodrug occurs because the water solubility of this poorly soluble drug is dramatically increased and net systemic absorption is greatly enhanced. Unfortunately, prodrugs are rapidly converted back to the parent drug in vivo either in the intestinal environment or on the first pass through the intestine and liver. Therefore, once a prodrug is “absorbed”, only the drug appears in the blood and the potential benefits of the prodrug are lost. Since nearly all PIs are faced with poor cellular uptake/retention, metabolism, and protein binding, the maximal therapeutic potential of these agents is not likely to be achieved using a classical prodrug approach since concentrations in target cells such as T4 helper lymphocytes or macrophages will be difficult to control. In the current report, macromolecular prodrug conjugates were designed to overcome several biopharmaceutical challenges associated with HIV-1 PIs and prodrugs in general including poor solubility, cellular uptake/ retention, metabolism/stability, and variable cellular drug concentrations. The conjugate scaffold used in the current study was based on PEG. PEGylation has been 1 Abbreviations: AcOEt, ethyl acetate; AIDS, acquired immunodeficiency syndrome; BOP, benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate; CDCl3, deuterated chloroform; CH2Cl2, methylene chloride; CNS, central nervous system; CPP, cell-penetrating peptide; CPT, camptothecin; Cys, cysteine; DCM, dichloromethane; Dde, 1-(4,4-dimethyl2,6-dioxocyclohex-1-ylidene)ethyl; DIEA, diisopropylethylamine; DIPC, 1,3-diisopropylcarbodiimide; DMAP, 4-(dimethylamino)pyridine; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; ED, effective dose; EDT, ethanedithiol; ESI-MS, electrospray ionization mass spectrometry; Et2O, diethyl ether; FBS, fetal bovine serum; Fmoc, fluorenylmethoxycarbonyl; FRET, fluorescence resonance energy transfer; Gly, glycine; HAART, highly active anti-retroviral therapy; HIV, human immunodeficiency virus; HOBt, N-hydroxybenzotriazole; LD, lethal dose; Lys, lysine; MALDI-TOF, matrix-assisted-laser-desorption ionization time-of-flight; MeOH, methanol; MOI, multiplicity of infection; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NHS, N-hydroxysuccinimide-; NMR, nuclear magnetic resonance; PBS, phosphate-buffered saline; PEG, poly(ethylene glycol); PI, protease inhibitor; PR, protease; R.I.CK-Tat9, retroinverso-cysteine-lysine-Tat nonapeptide; RP-HPLC, reversephase high-performance liquid-chromatography; SQV, saquinavir; TFA, trifluoroacetic acid; TIS, triisopropylsilane; TMS, tetramethylsilane; TP, 2-thiopyridine; Trt, trityl.

Bioconjugate Chem., Vol. 15, No. 6, 2004 1323

shown to significantly enhance the biopharmaceutical properties (i.e., circulation half-life, aqueous solubility, etc.) of drugs resulting in more effective in vivo delivery (14-18). Enhancing cell association and/or uptake was also an important design element. This was addressed by using biotin (17,18), R.I.CK-Tat9 (19, 20), or R.I.CK(stearate)-Tat9 (21) to enhance cell uptake. In addition to being an effective cell-penetrating peptide (CPP), R.I.CK-Tat9 is also an anti-HIV-1 agent that acts on nonprotease targets (22-25) and may be useful in combination therapy. In this report, the design, synthesis, and initial characterization of prototype prodrug conjugates of SQV with PEG-based substituents are described. These substituents were linked to the drug using releasable (ester and disulfide) and nonreleasable (amide) bonds. For these prodrug conjugates, the anti-HIV-1 activity was assessed in HIV-infected MT-2 cells using an MTT assay and compared to the maximal possible in vitro therapeutic potential of unconjugated SQV. Also, a novel HIV-1 protease inhibition assay was used to monitor the release of active drug from the prodrug conjugates. EXPERIMENTAL PROCEDURES

Materials. Fluorenylmethoxycarbonyl (Fmoc)-amino acid derivatives, MBHA Rink amide resin, BOP (benzotriazol-1-yl-oxytris(dimethylamino)phosphonium hexafluorophosphate) and HOBt (N-hydroxybenzotriazole) were purchased from Calbiochem-Novabiochem Corp. (La Jalla, CA). Fmoc-PEG-NHS (MW 3400) and biotin-PEGNHS (MW 3400) were obtained from Nektar Therapeutics AL, Corp. (formerly Shearwater Corp.) (Huntsville, AL). Stearic acid, DIEA (diisopropylethylamine), aldrithiol, piperidine, and hydrazine were obtained from Sigma Chemicals (St. Louis, MO). Acetic anhydride and diethyl ether were purchased from Fischer Chemicals (Pittsburgh, PA). Dde (1-(4,4-dimethyl-2,6-dioxocyclohex1-ylidene)ethyl), EDT (ethanedithiol), TIS (triisopropyl silane), DMAP (4-(dimethylamino)pyridine), DIPC (1,3diisopropylcarbodiimide), anhydrous dimethylformamide (DMF), and anhydrous dimethyl sulfoxide (DMSO) were obtained from Aldrich Chemicals (Milwaukee, WI). Dichloromethane (DCM) obtained from Sigma was dried over calcium hydride and freshly distilled prior to use. Trifluoroacetic acid (TFA) obtained from Aldrich was distilled with H2SO4 before use. SQV from commercially purchased capsules (Inverase, Roche) was extracted with methanol. Chemical reactions were carried out under a nitrogen atmosphere. The purity of all new compounds was checked by mass spectrometry, TLC, and/or NMR. Spectral Analyses. UV spectra were recorded on a Shimadzu UV-1201 spectrophotometer. 1H and 13C NMR spectra were recorded on a Bruker ARX 300. Chemical shifts (δ) were expressed as parts per million (ppm) using tetramethylsilane (TMS) as the internal standard (δ ) 0.0 ppm). Coupling constants (J values) are expressed in Hz, and multiplicities are referred to as s (singlet), d (dublet), t (triplet), m (multiplet), bs (broad singlet), and td (triplet dublet). Electrospray ionization mass spectra (ESI-MS) were recorded on a Finnigan MAT TSQ 7000 equipped with an atmospheric pressure ionization source. This method used in positive mode detects either the (M + H)+ and/or (M + Na)+ signal. Mass spectrometry using matrix-assisted-laser-desorption-ionization timeof-flight (MALDI-TOF) was performed on a PE Biosystems Voyager System 6080. 1H/13C NMR and mass spectrometry (ESI-MS and/or MALDI-TOF) data fully confirm the signal assignments and structure of the materials. For atom numbering, see Scheme 1.

1324 Bioconjugate Chem., Vol. 15, No. 6, 2004 Scheme 1. Synthetic Pathway to SQV Prodrug Conjugatesa

a (i) 3 equiv of Fmoc-Cys(S-Trt)-COOH in CH Cl with DIPC/ 2 2 DMAP; (ii) 20% piperidine in CH2Cl2; (iii) TFA/CH2Cl2 (1:1); (iv) 2 equiv of biotin-PEG-NHS in CH2Cl2 with DIEA; (v) 2 equiv of Fmoc-PEG-NHS in CH2Cl2 with DIEA; (vi) 2 equiv of 2,2/dithiodipyridine in DMSO; (vii) 2 equiv of R.I.CK-Tat9 in DMSO; (viii) 2 equiv of R.I.CK(stearate)-Tat9 in DMSO.

Chromatography. All reactions were monitored by TLC analysis on precoated silica gel F254 plates (Merck) with detection by UV absorption and by charring with 50% methanol-sulfuric acid solution, KMnO4, or ninhydrin. Column chromatographic separations were carried out as flash chromatography using silica gel 60 (Merck, 70-230 mesh). Gel permeation chromatography was done on a Sephadex LH-20 column using dimethylformamide as solvent. The UV absorbance of each fraction obtained from gel permeation chromatography was detected at 239 nm, the absorption maximum for SQV (26). Peptide purification was done by HPLC on Vydac protein and peptide preparative column RP-C18 (10 µm)-packed column (2.2 × 25 cm) with detection at 220 nm, the maximum absorbance for peptides. The fractions obtained after HPLC purification were dried using a lyophilizer. Amino acid analysis was performed by cation exchange chromatography. Amino acid elution was accomplished by using a two buffer system initially eluting with 0.2 N sodium citrate, pH 3.28, followed by 1.0 N sodium citrate, pH 7.4. The amino acids were detected by on-line postcolumn reaction with ninhydrin. Derivatized amino acids were quantitated by their absorption at 570 nm wavelength. This procedure was performed on an automated Beckman system Gold HPLC amino acid analyzer. SQV-Cys Ester. Fmoc-Cys(S-Trt)-COOH (0.220 g, 0.375 mmol) was dissolved in 100 mL of anhydrous CH2Cl2 at room temperature, and to this solution was added DMAP (0.32 g, 0.265 mmol), DIPC (58.7 µL, 0.375 mmol), and SQV (0.074 g, 0.11 mmol) at 0 °C. The reaction

Gunaseelan et al.

mixture was allowed to warm to room temperature and stirred overnight. The product was washed with 0.1 N HCl, dried over MgSO4, and evaporated under reduced pressure to yield the solid product. The crude residue was chromatographed [silica gel chromatography (eluent: AcOEt)] yielding, 63 mg (85% recovery) of SQV-Cys(Trt)Fmoc ester as a white powder. TLC (AcOEt/MeOH: 90/ 10, v/v, UV detection): Rf ) 0.75. ESI-MS (m/z): 1238.5 (M + H)+; 1260.5 (M + Na)+. The Fmoc-protecting group was removed by dissolving the SQV-Cys(Trt)Fmoc ester (50 mg) in a mixture of CH2Cl2 and piperidine (80:20) and stirring at room temperature for 2 h. Solvent was evaporated under reduced pressure and the product subjected to silica gel chromatography (eluent: AcOEt) to yield 45 mg (90% recovery) of SQV-Cys(Trt) ester as a white solid. TLC (AcOEt/MeOH: 90/10, v/v, UV detection): Rf ) 0.80. ESI-MS (m/z): 1016.5 (M + H)+; 1038.5 (M + Na)+. The protecting group (Trt) was removed by incubating SQV-Cys(Trt) ester (49 mg) for 3 h in a mixture of CH2Cl2 and TFA (trifluoroacetic acid) (50:50). The product was recrystallized from cold ether (Et2O) and dried under vacuum overnight to give 40 mg (82% recovery) of SQV-Cys ester as a white powder. TLC (AcOEt/MeOH: 90/10, v/v, UV detection): Rf ) 0.85. ESIMS (m/z): 774.5 (M + H)+; 796.5 (M + Na)+. 1H NMR (CDCl3): δ 9.19 (d, J 7.2 Hz, H11); 8.24 (d, J 8.8 Hz, H7); 8.14 (d, J 8.5 Hz, H6); 8.12 (d, J 8.0 Hz, H1); 7.82 (d, J 8.1 Hz, H4); 7.74 (td, J 1.5 Hz, 8.1 Hz, H2); 7.59 (td, J 1.5 Hz, 8.1 Hz, H3); 7.51 (d, J 8.8 Hz, H17); 6.89-7.13 (m, H21-25); 6.50 (s, H15); 5.76 (bs, H39); 5.33 (m, H26); 4.81 (m, H12); 4.37 (m, H18); 4.12 (m, H43); 2.61-2.98 (m, H13/19/27/29/30/35/36/37); 2.44 (d, J 8.6 Hz, H44); 2.30 (t, J 7.6 Hz, H45); 1.92 (t, J 7.5 Hz, H46); 1.341.81 (m, H31-34); 1.22 (s, H41). 13C NMR (CDCl3): δ 173.7, 173.4 (C14/16); 170.4 (C38); 164.8 (C10); 155.6 (C42); 149.1 (C8); 146.8 (C9); 137.5 (C5); 137.1 (C6); 129.8-130.1 (C1/4); 129.4 (C20); 129.2 (C22/24); 128.5 (C21/25); 128.2 (C2); 127.7 (C3); 126.5 (C23); 118.7 (C7); 73.4 (C26); 70.8 (C37); 59.9 (C29); 57.1 (C27); 51.8 (C12); 51.3 (C43); 51.0 (C40); 49.8 (C18); 37.4 (C13); 36.1 (C45); 35.8 (C30); 35.5 (C19); 33.2 (C35); 30.8 (C36); 28.8 (C41); 30.7, 26.2, 25.8, 20.7 (C31-34). SQV-Cys-PEG3400. SQV-Cys ester (12.5 mg, 0.016 mmol) and Fmoc-PEG-NHS (3400 molecular mass of PEG) [105.0 mg, 0.031 mmol] were added to CH2Cl2 (1.25 mL), and DIEA (12.5 µL) was added to adjust the pH to basic. The reaction was stirred for 3 h at room temperature. The product was recrystallized from cold ether and dried under vacuum overnight. The product obtained as a white powder and after Fmoc removal was subjected to gel permeation chromatography using a Sephadex LH20 column in DMF, and absorbance was monitored at 239 nm. Two separate peaks were obtained as shown in Figure 1. The first peak corresponded to the purified SQV-Cys-PEG3400 and the second peak to the unreacted SQV-Cys ester. The fractions of first peak were combined and the solvent removed under reduced pressure to obtain SQV-Cys-PEG3400 with 75-80% recovery. Mass spectrometry of the conjugate showed a polydisperse peak at the expected molecular weight [m/z(%) 4044.9 Da] using MALDI-TOF (Figure 2). 1H NMR (CDCl3): δ 9.18 (d, J 7.6 Hz, H11); 8.27 (d, J 8.3 Hz, H7); 8.16 (d, J 8.2 Hz, H6); 8.11 (d, J 8.0 Hz, H1); 7.86 (d, J 8.2 Hz, H4); 7.73 (td, J 1.6 Hz, 8.3 Hz, H2); 7.64 (td, J 1.6 Hz, 8.3 Hz, H3); 7.51 (d, J 8.6 Hz, H17); 7.34 (d, J 8.1 Hz, H44); 6.85-7.15 (m, H21-25); 6.52 (s, H15); 5.70 (bs, H39); 5.30 (m, H26); 4.82 (m, H12); 4.32 (m, H18); 4.20 (m, CH2CH2); 4.11 (m, H43); 3.57 [bs, CH2(OCH2CH2)n]; 2.63-3.05 (m, H13/19/27/29/30/35/36/37);


Figure 1. Gel permeation chromatogram showing separation of SQV-Cys-PEG3400 from unreacted SQV-Cys ester using Sephadex LH-20 column in DMF. The absorbance of each fraction was detected at a wavelength of 239 nm, the absorption maximum for SQV.

2.41 [s, NH2]; 2.33 (t, J 7.4 Hz, H45); 1.92 (t, J 7.6 Hz, H46); 1.36-1.81 (m, H31-34); 1.22 (s, H41). 13C NMR (CDCl3): δ 173.5, 173.1 (C14/16); 172.5 (CH2CONH); 170.6 (C38); 165.1 (C10); 155.6 (C42); 149.3 (C8); 146.5 (C9); 137.8 (C5); 137.2 (C6); 129.8-130.1 (C1/4); 129.6 (C20); 129.2 (C22/24); 128.6 (C21/25); 128.1 (C2); 127.8 (C3); 126.5 (C23); 118.4 (C7); 73.1 (C26); 72.5 (OCH2CH2)n; 70.5 (C37); 59.2 (C29); 57.9 (C27); 55.5 (CH2CH2CO); 52.5 (CH2CH2CO); 51.9 (C12); 51.3 (C43); 51.3 (C40); 49.5 (C18); 37.9 (C13); 36.1 (C45); 35.7 (C30); 35.3 (C19); 33.0 (C35); 30.5 (C36); 28.5 (C41); 30.3, 26.2, 25.0, 20.8 (C3134). SQV-Cys-PEG3400-Biotin. The above process, when applied to NHS-PEG-biotin (3400 molecular mass of PEG) and SQV-Cys ester, followed by gel permeation chromatography in DMF, yielded SQV-Cys-PEG3400biotin as a white powder with 70% recovery. Mass spectrometry of this conjugate using MALDI-TOF showed a polydisperse peak at 4059.6 Da (m/z) identical to the expected molecular weight. 1H NMR (CDCl3): δ 9.13 (d,


J 7.3 Hz, H11); 8.26 (d, J 8.3 Hz, H7); 8.15 (d, J 8.3 Hz, H6); 8.11 (d, J 8.0 Hz, H1); 7.85 (d, J 8.1 Hz, H4); 7.76 (td, J 1.6 Hz, 8.3 Hz, H2); 7.66 (td, J 1.6 Hz, 8.3 Hz, H3); 7.48 (d, J 8.4 Hz, H17); 7.34 (d, J 8.1 Hz, H44); 7.24 (s, NH-CO-NH); 7.20 [s, NH(CH2CH2O)n]; 6.84-7.13 (m, H21-25); 6.52 (s, H15); 5.70 (bs, H39); 5.34 (m, H26); 4.84 (m, H12); 4.35 (m, H18); 4.13 (m, biotin aliphatic chain); 4.11 (m, H43); 3.56 [bs, (OCH2CH2)n]; 2.61-3.08 (m, H13/19/27/29/30/35/36/37); 2.33 (t, J 7.4 Hz, H45); 1.92 (t, J 7.6 Hz, H46); 1.35-1.84 (m, H31-34); 1.26 (s, H41). 13C NMR (CDCl3): δ 175.8 (NHCONH); 173.6, 173.1 (C14/16); 172.5, 172.9 [CONH (CH2CH2O)nCONH]; 170.6 (C38); 164.9 (C10); 155.7 (C42); 149.6 (C8); 146.3 (C9); 137.9 (C5); 137.2 (C6); 129.8-130.1 (C1/4); 129.5 (C20); 129.2 (C22/24); 128.6 (C21/25); 128.3 (C2); 127.9 (C3); 126.8 (C23); 118.5 (C7); 73.1 (C26); 72.5 (OCH2CH2)n; 70.4 (C37); 59.7 (C29); 57.9 (C27); 55.5 (biotin); 52.5 (CH2CH2CO); 51.9 (C12); 51.5 (C43); 51.0 (C40); 49.5 (C18); 37.5 (C13); 36.1 (C45); 35.8 (C30); 35.5 (C19); 33.1 (C35); 30.9 (C36); 28.8 (C41); 30.7, 26.3, 25.8, 20.6 (C31-34). SQV-Cys(R.I.CK-Tat9)-PEG3400. Aldrithiol (2,2′dithiodipyridine, 1.6 mg, 0.007 mmol) was dissolved in 2.0 mL of DMSO, and to this solution was added 12 mg (0.004 mmol) of SQV-Cys-PEG3400 having a free thiol group on cysteine. The reaction was stirred at room temperature. The completion of the reaction was monitored by measuring the absorbance of 2-thiopyridine (TP) release at 343 nm (27) until the absorbance value remained constant. Solvent was evaporated under reduced pressure to yield 9.5 mg (79% recovery) of SQVCys(TP)-PEG3400 as a white powder. The retro-inversocysteine-lysine-Tat nonapeptide (R.I.CK-Tat9), N-acetylCKRRRQRRKKR-NH2 was synthesized manually on a MBHA Rink Amide resin by Fmoc chemistry in the presence of coupling activating reagents, BOP and HOBt. The two additional residues at the N-terminus, Cys and Lys, were reserved for further conjugations. For acetylation, the assembled peptide on the solid support was allowed to react with acetic anhydride in the presence of BOP, HOBt, and 1% DIEA for 6 h. The peptide was cleaved from the support and deprotected by treatment

Figure 2. MALDI-TOF (m/z) spectrum of SQV-Cys-PEG3400 showing a polydisperse peak at 4044.9 Da.

1326 Bioconjugate Chem., Vol. 15, No. 6, 2004

with a mixture of 94% TFA, 2.5% water, 2.5% EDT, and 1% TIS for 3 h at room temperature. The crude product was obtained after ether precipitation. The R.I.CK-Tat9 peptide was purified (>95%) by preparative C18 reversephase high-performance liquid chromatography (RPHPLC). The fractions obtained after HPLC purification were lyophilized to obtain the dry purified peptide. HPLC: 0.05% TFA (v/v) in water (mobile phase A) and 0.05% TFA (v/v) in a mixture of 10% water, 20% 2-propanol, and 70% acetonitrile (mobile phase B), linear gradient (t ) 0 min, 20% B, 80% A; t ) 5 min, 20% B, 80% A; t ) 35 min, 50% B, 50% A; t ) 36 min, 100% B, 0% A, t ) 40 min stop), flow rate ) 3 mL/min, UV detection ) 220 nm, tR (retention time) ) 25 min. ESIMS: 1613 (M + H)+, 807.4 [(M + 2H)/2]+, 538.5 [(M + 3H)/3]+. R.I.CK-Tat9 peptide concentration was determined by amino acid analysis. R.I.CK-Tat9 (10.0 mg, 0.006 mmol) was dissolved in 2.0 mL of DMSO, and to this solution was added 10.2 mg (0.003 mmol) of SQVCys(TP)-PEG3400. The reaction was stirred to completion at room temperature and monitored at 343 nm to measure TP release. Solvent was evaporated under reduced pressure to yield a white microcrystalline powder. This product was subjected to gel permeation chromatography using a Sephadex LH-20 column in DMF, yielding a single sharp peak (absorbance monitored at 239 nm). The fractions of this peak were combined, and the solvent was removed under reduced pressure to obtain SQV-Cys(R.I.CK-Tat9)-PEG3400 with 65% recovery. Mass spectrometry of the conjugate demonstrated a polydisperse peak at the expected molecular weight [m/z(%) 5447.6 Da] using MALDI-TOF. SQV-Cys(R. I.CK(Stearate)-Tat9)-PEG3400. The synthesis of R.I.CK(stearate)-Tat9 [N-acetyl-CK(-stearate)RRRQRRKKR] entailed the same procedure as R.I.CKTat9 except for attachment of fatty acid, stearic acid was coupled to the side chain of the N-terminal lysine residue. To allow this reaction, this N-terminal lysine residue had the -Dde protecting group, which was selectively removed from the resin-bound peptide using 2% hydrazine in DMF. Thereafter, 3 equiv of stearic acid were dissolved in DMF with coupling agents HOBt and BOP and reacted with peptide on the solid support for 3 h at room temperature. After cleavage from the MBHA Rink amide resin, the peptide was precipitated in precooled ether. The crude peptide was then subjected to HPLC purification, lyophilized, and determined by amino acid analysis and mass spectrometry. HPLC: 0.05% TFA (v/v) in water (mobile phase A) and 0.05% TFA (v/v) in a mixture of 10% water, 20% 2-propanol, and 70% acetonitrile (mobile phase B), linear gradient (t ) 0 min, 50% B, 50% A; t ) 5 min, 50% B, 50% A; t ) 35 min, 100% B, 0% A; t ) 40 min, 100% B, 0% A, t ) 41 min stop), flow rate ) 3 mL/ min, UV detection ) 220 nm, tR (retention time) ) 32 min. ESI-MS: 1880 (M + H)+, 940.1 [(M + 2H)/2]+, 627.2 [(M + 3H)/3]+. The above process when applied to R.I.CK(stearate)-Tat9 and SQV-Cys(TP)-PEG3400, followed by gel permeation chromatography using Sephadex LH-20 in DMF, yielded SQV-Cys(R.I.CK(stearate)-Tat9)PEG3400 as a white microcrystalline powder with 68% recovery. Mass spectrometry of this conjugate using MALDI-TOF showed a polydisperse peak at 5716.6 Da (m/z) identical to the expected molecular weight. Protease Inhibition Assay. An assay developed by Matayoshi and co-workers that is based on intramolecular fluorescence resonance energy transfer (FRET) was adapted and modified (28). The assay uses quenched fluorogenic substrates having a peptide sequence derived from a natural processing site for HIV-1 protease (PR).

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The quenched fluorogenic substrate 1 (obtained from Sigma) was Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-IleVal-Gly-Lys(DABCYL)-Arg, where EDANS is the fluor and DABCYL the quencher. Recombinant HIV-1 PR was obtained from the NIH AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases. Incubation of recombinant HIV-1 PR with the fluorogenic substrate at 37 °C results in specific cleavage of a tyrosine-proline bond, causing an increase in fluorescence due to elimination of intramolecular quenching. In the presence of an inhibitor such as SQV, the fluorescence signal is expected to decrease with increasing concentration of inhibitor. Cleavage by the HIV-1 PR occurs at the pseudo-phenylalanine-proline bond in SQV and at the tyrosine-proline bond in substrate 1. Thus, inhibition of HIV-1 PR by SQV in the presence of substrate 1 is observed as a decrease in fluorescence intensity. Therefore, the release of SQV is measured resulting from the hydrolysis of the prodrug SQV-Cys ester. Steady-state fluorescence data were obtained on a Cytofluor Spectrofluorometer (excitation at 340 nm, emission at 490 nm). All data were corrected with blanks. Solutions of HIV-1 PR and substrate 1 were prepared in the HIV-1 protease assay buffer at pH 4.7 using published procedures (28). In all of the experiments, the protease was added last to keep the incubation times consistent. Determination of Hydrolysis Kinetics of SQVCys Ester. The fluorogenic protease inhibition assay was used to measure the hydrolysis kinetics of the prodrug, SQV-Cys ester. The chemical stability of the ester was determined in 0.1 N HCl at 37 °C, and the biological stability was determined in PBS at pH 7.4 and in plasma both measured at 37 °C. Initially, different SQV concentrations (0.05-0.5 µM) were taken separately in 0.1 N HCl, in PBS at pH 7.4, and in plasma. Plasma samples (90 µL) were spiked with 10 µL of various concentrations of SQV dissolved in DMSO. For the determination of SQV, plasma samples were deproteinated with 200 µL of ice cold MeOH. The samples were vortexed followed by centrifugation to precipitate the plasma proteins. The clear supernatant was then dried down and reconstituted in protease assay buffer. SQV samples taken in 0.1 N HCl were neutralized with sodium bicarbonate before fluorescence measurements. A fixed amount of substrate 1 (110 µM) and HIV-1 PR (35 nM) were added, and the reaction was carried out for 60 min in the HIV protease assay buffer at pH 4.7 (Figure 3). From the measurement of fluorescence versus SQV concentration, calibration curves were obtained in 0.1 N HCl, in PBS at pH 7.4, and in spiked plasma (Figure 4). Thereafter, the prodrug, SQV-Cys ester (0.15 µM) was incubated separately in 0.1 N HCl, in PBS at pH 7.4, and in spiked plasma at 37 °C, respectively. Aliquots were taken at different time points and measuring the protease inhibitory activity using the fluorogenic assay assessed the release of SQV, resulting from ester bond hydrolysis. Each measurement was done in triplicate. All fluorescence signals were converted to moles of SQV using the calibration curves derived in each medium (Figure 4) to obtain a plot of percentage of released SQV versus incubation time (Figure 5). The rate constant (k) was obtained from the linear plot of ln[(SQV)t - (SQV)∞] versus incubation time, t (h), where [SQV]t ) concentration of SQV at different incubation times, t, and [SQV]∞ ) concentration of SQV at the last time point, when hydrolysis is complete (Figure 6). The half-life (t1/2) for ester hydrolysis was estimated from the relation t1/2 ) 0.693/k where k is the slope of the linear plot.


Figure 3. Kinetics of HIV-1 protease (35 nM) cleavage of fluorescence-quenched substrate 1 (110 µM) carried out in the presence of various concentrations of SQV in PBS, pH 7.4 at 37 °C.


Figure 5. Release of SQV by hydrolysis of the prodrug, SQVCys ester (0.15 µM) in PBS, pH 7.4, at 37 °C, using protease assay. Inhibition of protease by released SQV was determined at 37 °C. The ester was hydrolyzed to release SQV as monitored by steady-state fluorescence. The fluorescence measured due to the released SQV was converted to moles of SQV using the calibration curves from Figure 4. All measurements were done in triplicate. Inset. Release of SQV by hydrolysis of SQV-Cys ester (0.15 µM) in plasma at 37 °C.

Figure 4. Calibration curve of fluorescence intensity of substrate 1 at 30 min as a function of various concentrations of SQV in PBS, pH 7.4 at 37 °C. Inset. Calibration curve in plasma at 37 °C.

Antiviral Assay. The in vitro antiviral activity of poly(ethylene glycol)(PEG)-based SQV prodrug conjugates was determined using a MTT-based HIV-1 susceptibility assay that was performed using MT-2 cells infected with the HIV-1 strain LAV/LAI (29). In this assay, the maximal potential antiviral efficacy was assessed by using unconjugated SQV. SQV remained in the test media for the entire duration of the in vitro test unlike the in vivo situation where SQV is rapidly cleared from cells and the body. Therefore, the in vitro antiviral activity of all conjugates was compared to the maximum achievable effect. The cytotoxicity of the prodrug conjugates was evaluated in parallel to their antiviral activity and was based on the viability of noninfected cells as monitored by the MTT method. The MT-2 cell line is an HTLV-1-transformed human T-cell leukemia cell line isolated from cord blood lymphocytes and cocultured with cells of patients with adult T-cell leukemia. The MT-2 cells were grown in RPMI 1640 DM (Dutch modification) medium, supplemented with 20% FBS, 1% w/v penstrep, and 1% w/v L-glutamine. The cells were maintained at 37 °C in a humidified atmosphere of 5% CO2 in air. Every 2-3 days, the growth medium was changed, and

Figure 6. Plot of ln[(SQV)t-(SQV)∞] (derived from Figure 5) versus incubation time (t) of SQV-Cys ester in PBS, pH 7.4 at 37 °C. The rate constant (k) is the slope of this linear plot. The half-life (t1/2) for the ester hydrolysis was calculated using the relation t1/2 ) 0.693/k. Inset. Plot of ln[(SQV)t - (SQV)∞] versus incubation time (t) of SQV-Cys ester in plasma at 37 °C.

the cells were diluted to 2 × 105 cells/mL and recultured. The cultured MT-2 cells were infected using a multiplicity of infection (MOI) of 0.01 (1 viral particle per 100 cells) causing the death of 90% of the cells 5 days later. The tested prodrug conjugates were diluted with RPMI 1640 medium and were added to the cultured MT-2 cells after viral infection. Dosage of each prodrug conjugate was given in terms of SQV concentration, and this was determined by absorbance of prodrug conjugate at 239 nm (λmax of SQV) with a calibration standard of SQV. Each prodrug conjugate was tested in triplicate for its antiviral activity. Cell viability was measured by the colorimetric MTT test, which is based on the capacity of living cells to reduce the yellow colored MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to blue formazan by mitochondrial dehydrogenases. The


quantity of formazan produced (absorbance at 540 nm) is directly proportional to the number of living cells. The percentage protection achieved by different concentrations of the tested prodrug conjugate in HIV-1-infected cells after 5 days of incubation at 37 °C was calculated using the formula:

[(ATC)HIV - (AUC)HIV]/[(AUC)NHIV - (AUC)HIV] × 100 where (ATC)HIV is the absorbance measured for treated HIV-1-infected cells, (AUC)HIV is the absorbance measured for untreated HIV-1-infected cells, and (AUC)NHIV is the absorbance for untreated noninfected cells. The dose achieving 50% protection according to the above formula is defined as the 50% effective dose (ED50). The prodrug conjugate ED50 values were determined from the curves of percentage protection versus prodrug conjugate concentration. For cytotoxicity, the effect of the prodrug conjugates on cell viability was measured in noninfected cells using the colorimetric MTT test after 5 days of incubation at 37 °C with various concentrations of the tested compounds. The percentage cytotoxicity of the tested prodrug conjugates was calculated using the formula:

[(ATC)NHIV]/[(AUC)NHIV] × 100 where (ATC)NHIV is the absorbance measured for treated noninfected cells. The dose achieving 50% toxicity is defined as the 50% lethal dose (LD50). The prodrug conjugate LD50 values were determined from the curves of the percentage of living cells against prodrug conjugate concentration. RESULTS

Synthesis of Prodrug Conjugates. A series of SQV prodrug conjugates, SQV-Cys-PEG3400, SQV-CysPEG3400-biotin, SQV-Cys(R.I.CK-Tat9)-PEG3400, and SQV-Cys(R.I.CK(stearate)-Tat9)-PEG3400 was prepared and characterized. In all of these prodrug conjugates, the SQV hydroxyl group was covalently linked to a Cys carboxylic acid group by a releasable ester bond. The two other functional groups on Cys were then used for further derivatization. The thiol group of Cys was used to attach R.I.CK-Tat9 and R.I.CK(stearate)-Tat9 via a releasable disulfide bond. The amino group of Cys was used to attach PEG3400 and PEG3400-biotin via a more stable amide bond. In the synthesis of the SQV prodrug conjugates (Scheme 1), the active hydroxyl function of SQV was esterified with Fmoc-Cys(S-Trt)-COOH using DIPC/DMAP as coupling reagent. SQV-Cys ester was obtained with 82% yield after Fmoc removal with piperidine, followed by TFA-deprotection of the Trt group. The esterification of the hydroxyl of SQV after silica gel purification was unambiguously confirmed by ESI-MS and 1H and 13C NMR. Different types of prodrug conjugates were synthesized keeping the SQV-Cys ester as the common prodrug element. PEGylation (using Fmoc-PEG3400NHS and biotin-PEG3400- NHS) of the SQV-Cys ester was carried out using DIEA/DCM with the formation of relatively more stable amide linkages. The PEGylated products (SQV-Cys-PEG3400 and SQV-Cys-PEG3400biotin) were purified using gel permeation chromatography (Sephadex LH-20 column in DMF, UV 239 nm) (Figure 2) resulting in 70-80% yield. The formation of these products was confirmed by MALDI-TOF (Figure 3) and 1H and 13C NMR. In the synthesis of SQV-Cys(R.I.CK-Tat9)-PEG3400 and SQV-Cys(R.I.CK(stearate)-

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Tat9)-PEG3400, the thiol group of the Cys in SQV-CysPEG3400 was first activated with 2,2′-dithiodipyridine in DMSO to give SQV-Cys(TP)-PEG3400. With the addition of R.I.CK-Tat9 or R.I.CK(stearate)-Tat9 to the activated PEGylated form of SQV, TP release (343 nm) was monitored to confirm reaction completion. The formation of the respective SQV-Cys(R.I.CK-Tat9)PEG3400 and SQV-Cys(R.I.CK(stearate)-Tat9)-PEG3400 having reversible disulfide bonds in 65-68% yield range was confirmed by MALDI-TOF. Finally, significant increases in water solubility of these PEG-based SQV prodrug conjugates were observed relative to unconjugated SQV. Chemical and Biological Stability of the Prodrug, SQV-Cys, and Release of SQV. The HIV-1 PR inhibition assay was used for measuring the release of active (i.e., free) SQV resulting from the hydrolysis of the prodrug, SQV-Cys ester. This study was important since the level of HIV-1 inhibition by the prodrug conjugates depends on the apparent hydrolysis kinetics of SQV release. Since the SQV-Cys ester was the common intermediate for all of the synthesized prodrug conjugates, the in vitro stability of the prodrug was the initial focus of the investigation. The chemical stability of SQVCys in 0.1 N HCl and the biological stability in PBS at pH 7.4 and in spiked plasma were evaluated at 37 °C. In the absence of SQV, the incubation of fluorogenic substrate 1 with HIV-1 PR produced a 40-fold increase in fluorescence signal in a reaction that was approximately linear for 30 min (Figure 3). However, SQV inhibited this response in a dose-dependent manner at concentrations of 0.05-0.5 µM as shown in Figure 3. The calibration curves of fluorescence versus concentration of SQV in 0.1 N HCl, PBS at pH 7.4, and plasma were linear with a correlation coefficient (R2) of 0.9834 for 0.1 N HCl, 0.9779 for PBS at pH 7.4, and 0.9787 for plasma (Figure 4). SQV-Cys ester (0.15 µM) itself did not inhibit fluorescence, suggesting that the SQV prodrug was inactive. The inhibition of fluorescence signal increased with time of hydrolysis of SQV-Cys ester. Therefore, prodrug reconversion is necessary in order for SQV to exert protease inhibition. At the latest time point (31 h in the case of 0.1 N HCl, 47.5 h for PBS at pH 7.4, and 4 h in plasma) there was about a 90% inhibition of enzyme activity by free SQV (Figure 5). Since this level of inhibition is about the same as that produced by 0.15 µM parent drug (Figure 3), the reconversion to active drug was deemed essentially complete. Plots of ln[(SQV)t - SQV)∞] versus incubation time, t(h), were linear within the concentration range studied with correlation coefficients (R2) of 0.9939 (for 0.1 N HCl), 0.9834 (for PBS at pH 7.4), and 0.9916 (for plasma), indicating that the hydrolysis is a first-order process with respect to the prodrug (Figure 6). The half-lives of hydrolysis (t1/2) obtained from each of the media were calculated from these linear plots. The prodrug reconversion half-lives in 0.1 N HCl, in phosphate-buffered saline (PBS) at pH 7.4, and in spiked plasma at 37 °C were 9, 14, and 0.9 h, respectively. These studies demonstrate that SQV prodrug conjugates with their in-built SQV-Cys ester are inactive until the bond between SQV and cysteine is cleaved. Biological Activity of Prodrug Conjugates. The HIV-1 inhibition levels and cytotoxicities of PEG-based SQV prodrug conjugates were evaluated against HIV-1 in vitro in MT-2 cells according to an established antiviral assay (29). Since the active hydroxyl group of SQV is responsible for its protease inhibitory potency, it is essential that this functional group is free and accessible



Table 1. Anti-HIV-1 Activity (ED50) and Cytotoxicity (LD50) Data for SQV and Its Prodrugsa in MT-2 Cells Infected with HIV-1 Strain LAV/LAI (MOI of 0.01)b compound

ED50 (µM)

Rc

LD50 (µM)

therapeutic index LD50/ED50

SQV (MeSO3H) SQV-Cys-PEG3400 SQV-Cys-PEG3400-biotin SQV-Cys(R.I.CK-Tat9)-PEG3400 SQV-Cys(R.I.CK(stearate)-Tat9)-PEG3400

0.015 0.90 0.125 0.015 0.062

60 8.3 1 4.1

25 45 15 12.5 6.3

1667 50 120 833 102

a These data reflect the amount of parent drug that was released during the 5 day incubation at 37 °C. b Several assays of each compound were done with variations in the virus stock, dosage, and days of incubation. c R is the ED50 ratio of the prodrug to that of its parent drug, as previously described (10).

for antiviral activity. Thus, the level of HIV-1 inhibition of the prodrug conjugates would depend on their hydrolysis kinetics, which in turn reflects the release rate of the active parent drug, SQV. The antiviral activity of the SQV prodrug conjugates was determined by incubating HIV-1-infected MT-2 cells in the presence of different concentrations of prodrug conjugates for 5 days at 37 °C (Table 1). On the basis of the hydrolysis half-lives of the prodrug, SQV-Cys ester, and the antiviral activity results of the prodrug conjugates, it appears that most of the SQV was released during the 5-day incubation period. The appended putative cell uptake promoting groups (PEG3400, PEG3400-biotin, R.I.CK-Tat9, and R.I.CK(stearate)-Tat9) might have influenced the hydrolysis half-lives and/or cell uptake of the prodrug conjugates as measured by differences in antiviral activity (Table 1). The activity of SQV in prodrug conjugates compared to the maximal achievable antiviral efficacy (control, ED50 ) 15 nM) was reduced (ED50 ) 900 nM) for PEG alone, but restored with the addition of biotin (ED50 ) 125 nM), R.I.CK-Tat9 (ED50 ) 15 nM), or R.I.CK(stearate)-Tat9 (ED50 ) 62 nM). Similar results were obtained during repeated assays of each compound with variations in the virus stock, dosage, and days of incubation. The cytotoxicities of the SQV prodrug conjugates were measured by incubating noninfected MT-2 cells in the presence of different concentrations of prodrug conjugates for 5 days at 37 °C. The cytotoxicity (LD50) of all the tested conjugates was in the low micromolar range (6.3-45 µM). Results also show that all the SQV-based prodrug conjugates were less toxic than free SQV. The ratio of LD50/ED50 was used as an indicator of the in vitro therapeutic index and is shown in Table 1. DISCUSSION

HIV-1 protease inhibitors are “peptoids” or “peptidomimetics” that bind and block the active site of the viral proteinase enzyme, thereby inhibiting maturation of the virus. HAART (highly active anti-retroviral therapy) regimens that utilize inhibitors of HIV-1-encoded protease combined with reverse transcriptase inhibitors result in a profound and sustained suppression of viral replication, reduce morbidity, and prolong life in patients with HIV-1 infection (30-32). Current guidelines recommend that initial treatment of all HIV-1-infected patients include the administration of an HIV-1-protease inhibitor (33). However, despite attaining nondetectable viral blood levels, the development of resistance and the inability to ultimately cure HIV-1 infection has raised considerable debate about evaluating treatment efficacy (34) and which drugs should be used in optimal combination therapy. The control of HIV-1 infection and the ultimate cure of AIDS will require the development of not only new and more potent therapeutic agents but also of novel pharmacodelivery strategies. The current report represents an initial effort in developing smarter drug delivery

strategies to overcome the many biopharmaceutical challenges associated with anti-HIV-1 therapeutic regimens. In the current study, macromolecular prodrug conjugates have been designed and evaluated. Classical prodrugs are typically designed to overcome problems with solubility, stability, or limited absorption. Some prodrugs of anti-HIV-1 agents have been reported in the literature (10, 11, 13, 35). However, these previous efforts have focused on improving absorption by improving the physicochemical properties of these drugs. The current work focuses on a prodrug conjugate approach that attempts to control the kinetics of release and cellular concentrations of SQV at the target sites and protects the “cargo” (i.e., SQV) until it reaches the site of action where it can be released. As a first step toward improving the cellular bioavailability and efficacy of SQV, various prototype prodrug conjuates were designed, based on their known pharmacological properties. PEG-based prodrug conjugates composed of the putative cell uptake enhancing agents, biotin, R.I.CK-Tat9, and R.I.CK(stearate)-Tat9 were synthesized (Scheme 1), characterized. and studied in order to test the hypothesis that improved delivery would result in an enhancement in the pharmacological properties of SQV. The various components of the conjugates were carefully selected. The rationale for this selection and the results obtained for each conjugate component will be discussed separately. (1) PEG was chosen as a scaffold based on its ability to promote an extended half-life in blood or extracellular fluid and to increase the solubility of SQV (14, 15). PEG conjugates are less toxic than free drug (16) and have also been found to prevent in vivo binding to plasma proteins, as well as inactivation and rapid elimination from the blood (36). The conjugation of PEG to SQV yielded a less active prodrug conjugate, SQV-Cys-PEG3400 (Table 1). In this conjugate, PEG is stably bound to Cys by an amide bond, while the carboxylic acid group of Cys has a reversible ester bond with SQV. The 60-fold lower activity of the conjugate compared to the parent drug could be due to the slow cleavage of the ester bond and/or cell uptake of the conjugate. (2) Biotin was incorporated into the conjugate to enhance the uptake of the conjugates into cells (17, 18) since the hydrophobicity of biotin is known to promote cell membrane adhesion and enhanced cellular penetration (37, 38). Indeed, about 8-fold greater antiviral activity was observed when biotin was appended to the distal end of the long PEG chain (Table 1). This phenomenon has been observed in other systems as well. For example, the biotinylated form of Tat protein shows better cell uptake and transactivating activity presumably due to increased hydrophobic interactions with the plasma membrane (39). We also observed this with a prodrug conjugate of the anticancer drug, camptothecin (CPT), in which an ester bond with the carboxylic acid group of glycine (Gly) has to be cleaved to generate the


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Scheme 2. Possible Metabolic Pathways Shown for SQV-Cys(R.I.CK-Tat9)-PEG3400a

a If the disulfide bond is cleaved first (i), then the product is still an inactive prodrug, which can be further cleaved at the ester bond (ii) to form the active drug, SQV. The remainder of the conjugate might be degraded (iii). Alternatively, if the ester bond is cleaved first, active drug SQV is released (iv). The remainder of the conjugate might be degraded (v). Released Tat might also contribute to antiviral activity (see Discussion).

parent drug (17). In that study, the Gly-PEG conjugate of CPT was about 12-fold more potent than CPT alone. However, an additional biotin group appended to the distal end of PEG, CPT-Gly-PEG3400-biotin, gave a further increase in potency of 5-fold, similar to the effect of biotin in the present study (Table 1). (3) R.I.CK-Tat9 was chosen for two reasons. First, it possesses anti-HIV-1 activity and may be useful in combination therapy. Tat analogues have been shown to exert an inhibitory effect by interacting with HIV-1 RNA (22, 23) or by binding to the CXCR4 receptor on CD4+ cells (24, 25). Second, polycationic peptides such as Tat9 have been reported to facilitate membrane penetration (19, 20). The R.I.CK (retro-inverso-cysteine-lysine)-Tat9 consisted of D-amino acids assembled in the reverse order of the natural L-amino acid Tat9 peptide, N-acetyl-L-Arg-L-Lys-L-LysL-Arg-L-Arg-L-Gln-L-Arg-L-Arg-L-Arg-NH2. Thus, R.I.CKTat9 represents the sequence of all D-amino acids: N-acetyl-D-Cys-D-Lys-D-Arg-D-Arg-D-Arg-D-Gln-D-Arg-DArg-D-Lys-D-Lys-D-Arg-NH2. R.I. peptides have similar shapes and charge distributions to the natural L-amino acid peptides but are more stable to proteases and retain pharmacological activity (40). The attachment of the Tat peptide by a reversible disulfide linkage to the Cys residue returns the prodrug conjugate to the full antiviral activity of the parent drug, SQV (Table 1). The prodrug conjugate containing Tat peptide could have an even greater interaction with the plasma membrane due to the eight basic amino acids, including five arginines, which could interact with the phosphate headgroups of the lipid bilayer. Thus, immobilization of the conjugate to the plasma membrane might then increase cell uptake, although other factors such as the effect of Tat peptide on hydrolysis rate may be involved in the antiviral activity. Furthermore, the Tat peptide and similar cationic peptides (23, 25) also participate in a relatively strong interaction with the chemokine receptor, CXCR4 on T-cells, which is inhibitory to HIV-1 infection (24, 25). As a result, SQV-Cys(R.I.CK-Tat9)-PEG3400 might possess two different antiviral activities that are additive

in function (Table 1). Further studies will have to be performed to determine the exact mechanism of enhancement. (4) Tat-like peptides appended to fatty acids such as stearic acid have been reported to possess a greater penetrating ability for DNA delivery into cells (21). In the present work, the stearate group was initially appended to the Tat peptide in order to combine the hydrophobic and cationic properties and thereby achieve even greater membrane affinity. Instead, the stearate adduct had less activity (Table 1). This could be due to an inhibitory effect on the binding of Tat peptide to the chemokine receptor. Improvements in the ability of cationic peptides to augment drug uptake continue to be published (41). (5) The various conjugate components are linked to the scaffold by bonds that control the site and rate of release of the components and SQV from the conjugate. This is an important consideration for improving the delivery properties of SQV since it was previously demonstrated that SQV conjugates with a stable carbamate bond between the hydroxyl group of SQV and an amino group of an amino acid linker did not possess antiHIV-1 activity (13). In contrast, a less stable ester bond with cysteine was used for the conjugates prepared for the current study resulting in releasable SQV conjugates. One mechanism of control utilized in the current studies was a labile ester bond present in the SQV-Cys ester that can be hydrolyzed by cellular esterases to regenerate the parent antiviral drug. Similar ester prodrugs of SQV have been reported (10, 11, 13, 42). The in vivo stability of ester bonds in conjugates with other drugs was previously reported by Greenwald and co-workers (14, 43-45). Using the fluorescence-based HIV-1 PR inhibition assay, the in vitro release of SQV was assessed (Figures 5, 6). The labile disulfide linkage used for conjugating R.I.CK-Tat9 compounds to SQV-Cys-PEG3400 can be cleaved by glutathione, a physiologically relevant reducing agent. This linkage might be especially useful for drug delivery into cells, due to the stronger reducing environment within cells than in extracellular fluids (46). Thus, a disulfide-linked conjugate is expected to be


relatively stable in extracellular fluids or plasma but should be cleaved once inside cells. Previously, disulfide linkages have been successfully used by our group to link PEG-based polymers to a cysteine-containing peptide (46). However, cleavage of the disulfide bond is not sufficient to regenerate free SQV. Instead, an inactive prodrug, SQV-Cys-PEG3400, as well as free R.I.CK-Tat9 (or R.I.CK(stearate)-Tat9) are released (Scheme 2). For this particular situation, two consecutive release steps (i.e., cleavage of the disulfide bond followed later by ester bond cleavage) would be valuable since esterases are ubiquitous, and SQV release would occur readily in the blood stream. Hence, these prodrug conjugates would be no more effective than traditional prodrugs. However, the relative stability of the disulfide bond must be designed to protect the ester bond by stearic hindrance or by electron-donating effect and thereby ensure that SQV release would occur preferentially inside cells. A rapid release of SQV inside cells might result in cellular concentrations that are higher than the secretory Km for transporters such as P-gp and MRP2, resulting in prolonged and more effective cellular drug concentrations. The HIV-1 MTT assay was used to determine the maximal achievable antiviral activity of free SQV and to determine if all SQV was released from the conjugates in vitro. While the MTT assay format is good for assessing if the conjugates release active SQV, the assay does not yield other mechanistic information that might further suggest differences in the in vivo performance between the various conjugate forms or in comparison to free SQV. The reasons for the limitations relate to the closed nature of the test system and the 5-day time frame of the study. Released SQV is available for the entire 5 days of the assay. For the case of the control, free SQV is present during the entire study, and this therefore represents the maximal achievable antiviral activity. This limitation is not uncommon and was recently demonstrated for PEGylated R-interferon. PEGylated R-interferon is an example of a clinically useful drug conjugate for which an improvement in therapeutic value was not revealed by an in vitro assay (47). In that study, the receptor affinity and the specific activity of the PEGylated interferon were substantially lower (about 7%), but the half-life was increased almost 10-fold in vivo after subcutaneous injection. This would lead to a substantial increase in the “area under the curve” (i.e., blood level amounts), and, instead of injecting the patient 3 times each week, the PEGylated protein may be administered weekly, giving nearly constant drug levels (48). Furthermore, comparison of the two forms of interferon in a clinical trial of chronic hepatitis C (49) demonstrated greater efficacy with PEGylated interferon R-2a. While these examples further point out limitations in the in vitro assay, the assay is a reliable indicator of SQV activity and release. However in vivo studies are needed in order to evaluate delivery system performance and possible in vivo effectiveness. SUMMARY AND CONCLUSIONS

The current results suggest that SQV bioconjugates can be synthesized and active SQV can be released. This is critical since conjugated SQV is inactive. The in vitro anti-HIV-1 activity of all conjugates was evaluated against an unconjugated SQV control where the drug was 100% available during the entire anti-HIV-1 test. This rigorous test is unlike an in vivo situation where SQV is quickly cleared from target cells and the body; however, it gave an indication of the expected maximum thera-


peutic potential of SQV in this in vitro system. Appending various putative cell uptake enhancing moieties to the SQV conjugates produced variable effects on anti-HIV-1 activity, and in vitro activity was fully restored compared to nonconjugated SQV with one conjugate. Since conjugated SQV is inactive, the current results suggest that the appended moieties may be significantly modifying the release of SQV from the conjugate. Also, further work is required to ascertain the specific mechanism of enhancement for the Tat-based conjugate since Tat also possesses anti-HIV-1 activity. In conclusion, SQV conjugates were successfully synthesized and chemically characterized. They also show promise since anti-HIV-1 activity is not lost in vitro. However, the major benefits of conjugation are expected to be observed in vivo, and this is the focus of the next set of studies. ACKNOWLEDGMENT

This work was supported by NIH Grants AI 33789 and 51214. The following reagent was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: HIV-1HXB2 KIIA Protease, cat. no. 4375 from Drs. David Davis, Stephen Stahl, Paul Wingfield, and Joshua Kaufmann. We acknowledge Dr. Hsin-Ching Lin for assistance in establishment of the HIV assays. LITERATURE CITED (1) Aungst, B. J. (1999) P-Glycoprotein, Secretory Transport, and Other Barriers to the Oral Delivery of Anti-HIV Drugs. Adv. Drug Delivery Rev. 39, 105-116. (2) Sinko, P. J., Kunta, J. R., Usansky, H. H., and Perry, B. A. (2004) Differentiation of Gut and Hepatic First Pass Metabolism and Secretion of Saquinavir in Ported Rabbits. J. Pharmacol. Exp. Ther. 310, 359-366. (3) Chun, T. W., Stuyver, L., Mizell, S. B., Ehler, L. A., Mican, J. A., Baseler, M., Lloyd, A. L., Nowak, M. A., and Fauci, A. S. (1997) Presence of an Inducible HIV-1 Latent Reservoir During Highly Active Antiretroviral Therapy. Proc. Natl. Acad. Sci. U. S. A 94, 13193-13197. (4) Flexner, C. (1998) HIV-Protease Inhibitors. N. Engl. J. Med. 338, 1281-1292. (5) Pialoux, G., Fournier, S., Moulignier, A., Poveda, J. D., Clavel, F., and Dupont, B. (1997) Central Nervous System As a Sanctuary for HIV-1 Infection Despite Treatment With Zidovudine, Lamivudine and Indinavir. AIDS 11, 1302-1303. (6) Sawchuk, R. J., and Yang, Z. (1999) Investigation of Distribution, Transport and Uptake of Anti-HIV Drugs to the Central Nervous System. Adv. Drug Delivery Rev. 39, 5-31. (7) Su, Y., Zhang, X., and Sinko, P. J. (2004) Human Organic Anion-Transporting Polypeptide OATP-A (SLC21A3) Acts in Concert With P-Glycoprotein and Multidrug Resistance Protein 2 in the Vectorial Transport of Saquinavir in Hep G2 Cells. Mol. Pharm. 1, 49-56. (8) Williams, G. C., Liu, A., Knipp, G., and Sinko, P. J. (2002) Direct Evidence That Saquinavir Is Transported by Multidrug Resistance-Associated Protein (MRP1) and Canalicular Multispecific Organic Anion Transporter (MRP2). Antimicrob. Agents Chemother. 46, 3456-3462. (9) Jones, K., Bray, P. G., Khoo, S. H., Davey, R. A., Meaden, E. R., Ward, S. A., and Back, D. J. (2001) P-Glycoprotein and Transporter MRP1 Reduce HIV Protease Inhibitor Uptake in CD4 Cells: Potential for Accelerated Viral Drug Resistance? AIDS 15, 1353-1358. (10) Farese-Di Giorgio, A., Rouquayrol, M., Greiner, J., Aubertin, A. M., Vierling, P., and Guedj, R. (2000) Synthesis and Anti-HIV Activity of Prodrugs Derived From Saquinavir and Indinavir. Antivir. Chem. Chemother. 11, 97-110.

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BC0498875

Synthesis of Poly (ethylene glycol)-Based Saquinavir Prodrug

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