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Ganciclovir (GCV) is the first-line therapy against human cytomegalovirus (HCMV), a widespread infection that is particularly dangerous for immunodeficient individuals. Closely resembling deoxyguanosine triphosphate, the tri-phosphorylated metabolite of GCV (GCV-TP) is preferentially incorporated by the viral DNA polymerase, thereby terminating chain extension and, eventually, viral replication. However, the treatment outcome of GCV varies greatly among individuals, therefore warranting better understanding of its metabolism. Here we show that NUDT15, a Nudix hydrolase known to metabolize thiopurine triphosphates, can similarly hydrolyze GCV-TP through biochemical studies and co-crystallization of the NUDT15/GCV-TP complex. More critically, GCV efficacy was potentiated in HCMV-infected cells following NUDT15 depletion by RNAi or inhibition by an in-house-developed, nanomolar NUDT15 inhibitor, TH8321, suggesting that pharmacological targeting of NUDT15 is a possible avenue to improve existing anti-HCMV regimens. Collectively, the data further implicate NUDT15 as a broad-spectrum metabolic regulator of nucleoside analog therapeutics, such as thiopurines and GCV., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In the area of gene-directed enzyme prodrug therapy (GDEPT), using herpes simplex virus thymidine kinase (HSV- tk ) paired with prodrug ganciclovir (GCV) for cancer treatment has been extensively studied. It is a process involved with two steps whereby the gene (HSV- tk ) is first delivered to malignant cells. Afterward, non-toxic GCV is administered to that site and activated to cytotoxic ganciclovir triphosphate by HSV- tk enzyme expressed exogenously. In this study, we presented a one-step approach that both gene and prodrug were delivered at the same time by incorporating them with polymeric micellar nanovectors. GCV was employed as an initiator in the ring-opening polymerization of ε-caprolactone (ε-CL) to synthesize hydrophobic GCV-poly(caprolactone) (GCV-PCL), which was furthered grafted with hydrophilic chitosan to obtain amphiphilic polymer (GCV-PCL-chitosan) for the fabrication of self-assembled micellar nanoparticles. The synthesized amphiphilic polymer was characterized using Fourier transform infrared spectroscopy and proton nuclear magnetic resonance. Micellar prodrug nanoparticles were analyzed by dynamic light scattering, zeta potential, critical micelle concentration, and transmission electron microscopy. Polymeric prodrug micelles with optimal features incorporated with HSV- tk encoding plasmids were cultivated with HT29 colorectal cancer cells and anticancer effectiveness was determined. Our results showed that prodrug GCV and HSV- tk cDNA encoded plasmid incorporated in GCV-PCL-chitosan polymeric nanocarriers could be delivered in a one-step manner to HT-29 cells and triggered high cytotoxicity.

SARS-CoV-2, a member of the coronavirus family, is responsible for the current COVID-19 worldwide pandemic. We previously demonstrated that five nucleotide analogues inhibit the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), including the active triphosphate forms of Sofosbuvir, Alovudine, Zidovudine, Tenofovir alafenamide and Emtricitabine. We report here the evaluation of a library of nucleoside triphosphate analogues with a variety of structural and chemical features as inhibitors of the RdRps of SARS-CoV and SARS-CoV-2. These features include modifications on the sugar (2' or 3' modifications, carbocyclic, acyclic, or dideoxynucleotides) or on the base. The goal is to identify nucleotide analogues that not only terminate RNA synthesis catalyzed by these coronavirus RdRps, but also have the potential to resist the viruses' exonuclease activity. We examined these nucleotide analogues for their ability to be incorporated by the RdRps in the polymerase reaction and to prevent further incorporation. While all 11 molecules tested displayed incorporation, 6 exhibited immediate termination of the polymerase reaction (triphosphates of Carbovir, Ganciclovir, Stavudine and Entecavir; 3'-OMe-UTP and Biotin-16-dUTP), 2 showed delayed termination (Cidofovir diphosphate and 2'-OMe-UTP), and 3 did not terminate the polymerase reaction (2'-F-dUTP, 2'-NH 2 -dUTP and Desthiobiotin-16-UTP). The coronaviruses possess an exonuclease that apparently requires a 2'-OH at the 3'-terminus of the growing RNA strand for proofreading. In this study, all nucleoside triphosphate analogues evaluated form Watson-Crick-like base pairs. The nucleotide analogues demonstrating termination either lack a 2'-OH, have a blocked 2'-OH, or show delayed termination. Thus, these nucleotide analogues are of interest for further investigation to evaluate whether they can evade the viral exonuclease activity. Prodrugs of five of these nucleotide analogues (Cidofovir, Abacavir, Valganciclovir/Ganciclovir, Stavudine and Entecavir) are FDA-approved medications for treatment of other viral infections, and their safety profiles are well established. After demonstrating potency in inhibiting viral replication in cell culture, candidate molecules can be rapidly evaluated as potential therapies for COVID-19., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objectives: The implementation of dose-banding (DB) in centralised, pharmacy-based cytotoxic drug preparation units allows the preparation of standardised doses in series. The aim of this study was to evaluate the feasibility of DB for the prescribing of ganciclovir (GV) infusion solutions and to investigate the microbiological stability of dose-banded, automatically prepared ready-to-administer GV infusion bags by media-fill simulation tests and sterility tests., Methods: The frequency of prescription of GV doses was retrospectively analysed before and after implementing the DB scheme. Four dose-ranges or 'bands' and the corresponding standard doses (250, 300, 350, 400 mg) were identified. The maximum variance was set at ±10% of the individually prescribed dose. The aseptic preparation of a series of GV infusion bags was simulated with double strength tryptic soy broth as growth medium and prefilled 0.9% NaCl polyolefin infusion bags as primary packaging materials. The simulation process was performed with the APOTECAchemo robot on five consecutive days. In total, 50 infusion bags were filled, incubated and stored for 12 weeks at room temperature. The media-filled bags were visually inspected for turbidity after 2, 4, 8, 10 and 12 weeks. Following incubation, growth promotion tests were performed. During the simulation tests, airborne contamination was monitored with settle plates and microbial surface contamination with contact plates. Pooled sterility tests were performed for a series of 10 standard GV infusion bags after a 12-week storage period under refrigeration (2 °C-8 °C)., Results: After implementation of the DB scheme, about 60% of the prescribed GV doses were prepared as standard preparations by the robotic system. The number of different GV doses was reduced by 61.8% (76 vs 29). None of the 50 media-filled bags showed turbidity after a storage period of 12 weeks, indicating the absence of microorganisms. The environmental monitoring with settle/contact plates matched the recommended limits set for cleanroom Grade A zones, except in the loading area of the robot. Media fills used for the sterility tests remained clear during the incubation period, thereby revealing sterility. Positive growth promotion tests proved the process's reliability., Conclusions: A DB scheme for prescribing and preparation of standard GV infusion bags was successfully implemented. Microbiological tests of aseptic preparation of infusion bags in series by the APOTECAchemo robot revealed an adequate level of sterility and a well-controlled aseptic procedure. The sterility was maintained over extended storage periods, thereby encouraging extended beyond-use dating., Competing Interests: Competing interests: None declared., (© European Association of Hospital Pharmacists 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Because of poor ocular drug bioavailability, intravitreal injections have become the gold standard for drug delivery to the posterior eye. The prodrug approach can be used for optimizing the biopharmaceutical properties of intravitreal drugs. The preclinical screening of prodrugs' properties, such as hydrolysis and bioconversion, should be conducted in a resource-efficient way for an extensive set of synthesized compounds with validated methods. Our objective was to explore cassette dosing in in vitro prodrug hydrolysis and bioconversion studies in buffer, vitreous, and retinal pigment epithelium (RPE) homogenate for rapid medium-throughput screening. Moreover, our aim was to correlate the prodrug structure with hydrolytic behavior. We synthesized 18 novel ganciclovir prodrugs and first studied their hydrolysis in aqueous buffer and porcine vitreous in vitro with cassette dosing for 35 h. A method for vitreous homogenate pH equilibration to a physiological level by using buffer and incubation under 5% carbon dioxide was validated. The hydrolysis of the prodrugs was evaluated in porcine RPE homogenate in vitro with cassette dosing, and five prodrugs were assayed individually to examine their bioconversion into ganciclovir in RPE after 2 h. Lastly, the prodrugs' binding to melanin was studied in vitro. The prodrugs showed a wide spectrum of hydrolysis rates, ranging from a few percentages to 100% in the vitreous and RPE; in general, hydrolysis in RPE was faster than in vitreous. Prodrugs with long carbon chains and disubstitution showed lability in the tissue homogenates, whereas prodrugs with branched carbon chains and aromatic groups were stable. All five prodrugs chosen for the bioconversion study in RPE were hydrolyzed into ganciclovir, and their hydrolytic behavior matched results from the cassette mix experiment, supporting the cassette mix approach for hydrolysis and bioconversion studies. None of the prodrugs bound highly to melanin (<50% bound). In conclusion, cassette dosing proved useful for the rapid screening of prodrug hydrolysis and bioconversion properties. Analyzing several compounds simultaneously can complicate the analytics, and thus, choosing the compounds of the cassette mix should be done carefully to avoid mutual interference of the compounds with the results. The methodology and results of the work are applicable in ocular drug research and prodrug design.

Valganciclovir (VGC) is an orally available mono-valyl ester pro drug of the nucleoside analog (NA) ganciclovir (GCV) used to treat cytomegalovirus (CMV). Congenital CMV infection in the newborn is associated with progressive sensorineural hearing loss; however, effective CMV therapy with VGC can improve audiologic outcomes. Ongoing studies to demonstrate the effect of VGC in this setting are hampered by a poor understanding of the pharmacology of VGC and GCV in newborns, and the low blood volumes that can be safely collected from this population. We describe a simple method for determining systemic GCV concentrations using dried blood spot (DBS) samples. GCV was extracted from a single 6 mm punch via sonication in methanol, then quantified using liquid chromatography-tandem mass spectrometry. The assay was accurate and precise in the dynamic range of 10-10,000 ng/mL. GCV concentrations determined in DBS agreed well with GCV concentrations observed in serum. The assay was successfully applied to patient samples, and will be used to support pharmacokinetic studies in an ongoing clinical trial of VGC in infants with CMV-mediated hearing loss., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The 3,9-dihydro-3-[(2-hydroxyethoxy)methyl]-6-(4-methoxyphenyl)-9-oxo-5 H -imidazo[1,2- a ]-purine (6-(4-MeOPh)-TACV) was selected to assess the enzymatic stability of the tricyclic acyclovir derivatives from the imidazo[1,2- a ]-purine group. The parent compound and its esters (acetyl, isobutyryl, pivaloyl, nicotinic, ethoxycarbonyl) were subjected to kinetic studies and compared with the stability of analogous acyclovir (ACV) esters. The enzymatic hydrolysis was observed in vitro in a medium of 80% human plasma in the absence and presence of porcine liver esterase (PLE). The tests were carried out at 37 °C. To determine the kinetic parameters (k obs. , t 0.5 ) of the observed reaction, the validated HPLC-UV method in the reversed phase was used. The HPLC-MS/MS method was used to identify the degradation products under the tested conditions. In summary, it was found that 6-(4-MeOPh)-TACV esters are more susceptible to esterase metabolism than ACV esters. It was confirmed by HPLC-MS/MS that in the plasma, the main product of their hydrolysis is 6-(4-MeOPh)-TACV and not ACV, which confirms that their antiviral activity observed in vitro does not result from ring degradation.

АBSTRACTEsters of the antiherpetic drugs ganciclovir, penciclovir with the bile acids (cholic, chenodeoxycholic and deoxycholic) and amino acid esters of acyclovir were generated and evaluated for their in vitro antiviral activity against herpes simplex viruses type 1 and type 2 (HSV-1, HSV-2). The antiviral assays demonstrated that modified analogs of ACV and PCV are less active compared to the initial substances against HSV-1and HSV-2. CC50 for ganciclovir-deoxycholate corresponded to the CC50 of the other analogs and its activity is lower than ganciclovir. Obtained results show that tested modification do not improve bioavailability of nucleoside analogs in cells.

Ganciclovir (GCV) is one of the most widely used antiviral drugs for the treatment of cytomegalovirus (CMV) retinitis. In this context, the aim of this study was to design in situ thermosensitive hydrogels for GCV ocular delivery by intravitreal injection to achieve sustained drug release behavior and improved ocular bioavailability in the treatment of CMV retinitis. A thermosensitive poly-(β-butyrolactone-co-lactic acid)-polyethylene glycol-poly (β-butyrolactone-co-lactic acid) (PBLA-PEG-PBLA) triblock copolymer was synthesized by ring-opening polymerization and characterization. The GCV-loaded PBLA-PEG-PBLA in situ hydrogels (15%, w/w) were then prepared with drug concentration at 2 mg·mL -1 and the gelation temperatures, rheological properties, in vitro degradation and syringeability of in situ hydrogels for intravitreal injection were also investigated. Membraneless dissolution model was used to explore drug release behavior of PBLA-PEG-PBLA in situ hydrogel. The results indicated that more than 45 and 85% of GCV can be released within 24 and 96 h, respectively, which was verified by a non-Fickian diffusion mechanism. In vivo ocular pharmacokinetics study showed that area under drug-time curve (AUC) and half-life of PBLA-PEG-PBLA in situ hydrogel was higher (AUC was 61.80 μg·mL -1 ·h (p < .01) and t 1/2 was 10.29 h in aqueous humor; AUC was 1008.66 μg·mL -1 ·h (p < .01) and t 1/2 was 13.26 h (p < .01) in vitreous) than GCV injection with extended therapeutic activity. Based on obtained results, it was concluded that the thermosenstive PBLA-PEG-PBLA in situ hydrogel is a promising carrier of GCV for intravitreal injection.

To assess the pharmaceutical quality of eight commercially available generic products of ganciclovir injection produced in China with original brand product (Cymevene, Roche, Switzerland). Tests were performed according to China Pharmacopoeia 2015 and Import Drug Registration Standard introduced by CFDA. Items including characteristics of the packing and reconstituted solution, pH, visible particles, content of active and related substances, sterility and bacterial endotoxin were all carried out based on the standard laboratory operating rules and requirements. Seven of all tested domestically produced generics of ganciclovir for injection failed to reach the in vitro quality requirements in comparison with the original brand product Cymevene. Three generics failed to meet the standards for pH of an aqueous solution. One out of eight generic products fell outside the specifications for API content. All generics showed impurities, whose levels were generally greater than observed in the brand product. One generic product was identified an endotoxin contamination. In addition, six generic products failed to reach the quality requirements of water content, which should be under 3%. Most tested ganciclovir products failed to meet the pharmaceutical quality standards for original brand product. Important items like pH, endotoxin contamination, content of API and impurities could cause clinical attention, as they directly affect the therapeutic efficacy and patient tolerance.

Background: Cytomegalovirus (CMV) retinitis is an opportunistic infection that primarily affects immunocompromised individuals. Intravitreal ganciclovir injection monotherapy or in combination with systemic anti-CMV therapy are effective treatments for CMV retinitis. Crystallization of ganciclovir after intravitreal injection is extremely rare. Only two cases had been reported in literature. Crystallization in only one eye after bilateral injections had not been reported before. We hereby report a case of intraocular ganciclovir crystallization in one eye after bilateral intravitreal injections, and repeated crystallization in the same eye after repeated injections., Case Presentation: A 79-year-old patient had bilateral cytomegalovirus retinitis and received bilateral intravitreal ganciclovir injections of 2.5 mg in 0.05 ml sterile water. Fundus examination after injection showed formation of needle-shaped, golden-yellow crystals in the vitreous of right eye but not in left eye. The crystals dissolved spontaneously. Repeated bilateral intravitreal ganciclovir injections 4 days later resulted in repeated crystallization of ganciclovir in right eye but not in left eye. The crystals dissolved spontaneously and completely after 5 minutes. Visual acuity remained unchanged and intraocular pressure was normal., Conclusions: Intraocular ganciclovir crystallization could occur after intravitreal injections. It is important to perform fundus examination after injection. The crystals may dissolve rapidly and vitrectomy may not be necessary. Our case suggested intraocular ganciclovir crystallization is an idiosyncratic phenomenon, subjects to distinctive intraocular environment which could be different between two eyes of the same patient. The susceptible intraocular environment could be persistent leading to repeated crystallization.

Ganciclovir is synthetic nucleoside analog of guanine closely related to acyclovir but has greater activity against cytomegalovirus. This comprehensive profile on ganciclovir starts with a description of the drug: nomenclature, formulae, chemical structure, elemental composition, and appearance. The uses and application of the drug are explained. The methods that were used for the preparation of ganciclovir are described and their respective schemes are outlined. The methods which were used for the physical characterization of the dug are: ionization constant, solubility, X-ray powder diffraction pattern, crystal structure, melting point, and differential scanning calorimetry. The chapter contains the spectra of the drug: ultraviolet spectrum, vibrational spectrum, nuclear magnetic resonance spectra, and the mass spectrum. The compendial methods of analysis of ganciclovir include the United States Pharmacopeia methods. Other methods of analysis that were reported in the literature include: high-performance liquid chromatography alone or with mass spectrometry, electrophoresis, spectrophotometry, voltammetry, chemiluminescence, and radioimmunoassay. Biological investigation on the drug includes: pharmacokinetics, metabolism, bioavailability, and biological analysis. Reviews on the methods used for preparation or for analysis of the drug are provided. The stability of the drug in various media and storage conditions is reported. More than 240 references are listed at the end of the chapter., (© 2018 Elsevier Inc. All rights reserved.)

Ganciclovir (GCV; systematic name: 2-amino-9-{[(1,3-dihydroxypropan-2-yl)oxy]methyl}-6,9-dihydro-1H-purin-6-one), C 9 H 13 N 5 O 4 , an antiviral drug for treating cytomegalovirus infections, has two known polymorphs (Forms I and II), but only the structure of the metastable Form II has been reported [Kawamura & Hirayama (2009). X-ray Struct. Anal. Online, 25, 51-52]. We describe a successful preparation of GCV Form I and its crystal structure. GCV is an achiral molecule in the sense that its individual conformers, which are generally chiral objects, undergo fast interconversion in the liquid state and cannot be isolated. In the crystalline state, GCV exists as two inversion-related conformers in Form I and as a single chiral conformer in Form II. This situation is similar to that observed for glycine, also an achiral molecule, whose α-polymorph contains two inversion-related conformers, while the γ-polymorph contains a single conformer that is chiral. The hydrogen bonds are exclusively intermolecular in Form I, but both inter- and intramolecular in Form II, which accounts for the different molecular conformations in the two polymorphs.

DNA-binding properties of an antiviral drug, valganciclovir (valcyte) was studied by using emission, absorption, circular dichroism, viscosity, differential pulse voltammetry, fluorescence techniques, and computational studies. The drug bound to calf thymus DNA (ct-DNA) in a groove-binding mode. The calculated binding constant of UV-vis, K a , is comparable to groove-binding drugs. Competitive fluorimetric studies with Hoechst 33258 showed that valcyte could displace the DNA-bound Hoechst 33258. The drug could not displace intercalated methylene blue from DNA double helix. Furthermore, the induced detectable changes in the CD spectrum of ct-DNA as well as changes in its viscosity confirm the groove-binding mode. In addition, an integrated molecular docking was employed to further investigate the binding interactions between valcyte and calf thymus DNA.

Embryonic trisomy leads to abortion or congenital genetic disorders in humans. The most common autosomal chromosome abnormalities are trisomy of chromosomes 13, 18, and 21. Although alteration of gene dosage is thought to contribute to disorders caused by extra copies of chromosomes, genes associated with specific disease phenotypes remain unclear. To generate a normal cell from a trisomic cell as a means of etiological analysis or candidate therapy for trisomy syndromes, we developed a system to eliminate a targeted chromosome from human cells. Chromosome 21 was targeted by integration of a DNA cassette in HeLa cells that harbored three copies of chromosome 21. The DNA cassette included two inverted loxP sites and a herpes simplex virus thymidine kinase (HSV-tk) gene. This system causes missegregation of chromosome 21 after expression of Cre recombinase and subsequently enables the selection of cells lacking the chromosome by culturing in a medium that includes ganciclovir (GCV). Cells harboring only two copies of chromosome 21 were efficiently induced by transfection of a Cre expression vector, indicating that this approach is useful for eliminating a targeted chromosome.

A hydroxyethylcellulose-poly(acrylic acid) (HEC-PAA) lyomatrix was developed for ganciclovir (GCV) intestine targeting to overcome its undesirable degradation in the stomach. GCV was encapsulated within the HEC-PAA lyomatrix prepared by lyophilization. Conventional tablets were also prepared with identical GCV concentrations in order to compare the GCV release behavior from the lyomatrix and tablets. GCV incorporation (75.12%) was confirmed using FTIR, DSC, and TGA. The effect of GCV loading on the microstructure properties of the lyomatrix was evaluated by SEM, AFM, and BET surface area measurements. The in vitro drug release study showed steady and rapid release profiles from the GCV-loaded lyomatrix compared with the tablet formulation at identical pH values. Minimum GCV release was observed at acidic pH (≤40%) and maximum release occurred at intestinal pH values (≥90%) proving the intestinal targeting ability of the lyomatrix. Kinetic modeling revealed that the GCV-loaded lyomatrix exhibited zero-order release kinetics (n = 1), while the tablets were best described via the Peppas model. Textural analysis highlighted enhanced matrix resilience and rigidity gradient (12.5%, 20 Pa) for the GCV-loaded lyomatrix compared to the pure (7%, 9.5 Pa) HEC-PAA lyomatrix. Bench-top MRI imaging was used to confirm the mechanism of GCV release behavior by monitoring the swelling and erosion rates. The swelling and erosion rate of the tablets was not sufficient to achieve rapid zero-order GCV release as with the lyomatrix. These combined results suggest that the HEC-PAA lyomatrix may be suitable for GCV intestinal targeting after oral administration.

Purpose: The objective of this study is to investigate cellular uptake of prodrug-loaded nanoparticle (NP). Another objective is to study bioconversion of stereoisomeric dipeptide prodrugs of ganciclovir (GCV) including L-Val-L-Val-GCV (LLGCV), L-Val-D-Val-GCV (LDGCV) and d-Val-l-Val-GCV (DLGCV) in human corneal epithelial cell (HCEC) model., Methods: Poly( D,L -lactic-co-glycolic acid) (PLGA) NP encapsulating prodrugs of GCV were formulated under a double emulsion method. Fluorescein isothiocyanate isomer-PLGA conjugates were synthesized to fabricate biocompatible fluorescent PLGA NP. Intracellular uptake of FITC-labeled NP was visualized by a fluorescent microscope in HCEC cells., Results: Fluorescent PLGA NP and non-fluorescent NP display similar hydrodynamic diameter in the range of 115-145 nm with a narrow particle size distribution and zeta potentials around -13 mV. Both NP types showed identical intracellular accumulation in HCEC cells. Maximum uptake (around 60%) was noted at 3 h for NP. Cellular uptake and intracellular accumulation of prodrugs are significantly different among three stereoisomeric dipeptide prodrugs. The microscopic images show that NPs are avidly internalized by HCEC cells and distributed throughout the cytoplasm instead of being localized on the cell surface. Following cellular uptake, prodrugs released from NP gradually bioreversed into parent drug GCV. LLGCV showed the highest degradation rate, followed by LDGCV and DLGCV., Conclusion: LLGCV, LDGCV and DLGCV released from NP exhibited superior uptake and bioreversion in corneal cells.

Poly(d,l-lactic-co-glycolic acid) (PLGA) nanoparticles (NP) of Val-Val dipeptide monoester prodrugs of ganciclovir (GCV) including L-Val-L-Val-GCV (LLGCV), L-Val-D-Val-GCV (LDGCV) and D-Val-L-Val-GCV (DLGCV) were formulated and dispersed in thermosensitive PLGA-PEG-PLGA polymer gel for the treatment of herpes simplex virus type 1 (HSV-1)-induced viral corneal keratitis. Nanoparticles containing prodrugs of GCV were prepared by a double-emulsion solvent evaporation technique using various PLGA polymers with different drug/polymer ratios. Nanoparticles were characterized with respect to particle size, entrapment efficiency, polydispersity, drug loading, surface morphology, zeta potential and crystallinity. Prodrugs-loaded NP were incorporated into in situ gelling system. These formulations were examined for in vitro release and cytotoxicity. The results of optimized entrapment efficiencies of LLGCV-, LDGCV- and DLGCV-loaded NP are of 38.7 ± 2.0%, 41.8 ± 1.9%, and 45.3 ± 2.2%; drug loadings 3.87 ± 0.20%, 2.79 ± 0.13% and 3.02 ± 0.15%; yield 85.2 ± 3.0%, 86.9 ± 4.6% and 76.9 ± 2.1%; particle sizes 116.6 ± 4.5, 143.0 ± 3.8 and 134.1 ± 5.2 nm; and zeta potential -15.0 ± 4.96, -13.8 ± 5.26 and -13.9 ± 5.14 mV, respectively. Cytotoxicity studies suggested that all the formulations are non-toxic. In vitro release of prodrugs from NP showed a biphasic release pattern with an initial burst phase followed by a sustained phase. Such burst effect was completely eliminated when NP were suspended in thermosensitive gels with near zero-order release kinetics. Prodrugs-loaded PLGA NP dispersed in thermosensitive gels can thus serve as a promising drug delivery system for the treatment of anterior eye diseases.

Background: The objective of this randomized, prospective and controlled study was to investigate the ability of a closed-system transfer device (CSTD; BD-Phaseal) to reduce the occupational exposure of two isolators to 10 cytotoxic drugs and compare to standard compounding devices., Methods and Findings: The 6-month study started with the opening of a new compounding unit. Two isolators were set up with 2 workstations each, one to compound with standard devices (needles and spikes) and the other using the Phaseal system. Drugs were alternatively compounded in each isolator. Sampling involved wiping three surfaces (gloves, window, worktop), before and after a cleaning process. Exposure to ten antineoplastic drugs (cyclophosphamide, ifosfamide, dacarbazine, 5-FU, methotrexate, gemcitabine, cytarabine, irinotecan, doxorubicine and ganciclovir) was assessed on wipes by LC-MS/MS analysis. Contamination rates were compared using a Chi2 test and drug amounts by a Mann-Whitney test. Significance was defined for p<0.05. Overall contamination was lower in the "Phaseal" isolator than in the "Standard" isolator (12.24% vs. 26.39%; p < 0.0001) although it differed according to drug. Indeed, the contamination rates of gemcitabine were 49.3 and 43.4% (NS) for the Standard and Phaseal isolators, respectively, whereas for ganciclovir, they were 54.2 and 2.8% (p<0.0001). Gemcitabine amounts were 220.6 and 283.6 ng for the Standard and Phaseal isolators (NS), and ganciclovir amounts were 179.9 and 2.4 ng (p<0.0001)., Conclusion: This study confirms that using a CSTD may significantly decrease the chemical contamination of barrier isolators compared to standard devices for some drugs, although it does not eliminate contamination totally.

Background: The poor ocular bioavailability of the conventional eye drops is due to lack of corneal permeability, nasolacrimal drainage and metabolic degradation. To overcome this issue, drug encapsulated in mucoadhesive polymer based ocular microspheres have the advantages of improved drug stability, easy administration in liquid form, diffuse rapidly and better ocular tissue internalization., Methods: The ganciclovir chitosan microspheres (GCM) were prepared by modified water-in-oil emulsification method. The formulation was optimized and characterized by investigating in vitro release study, release kinetics, XRD and microspheres stability. Ocular irritancy, in vivo ocular pharmacokinetic parameters and histopathology study was evaluated in Wistar rats. The use of pharmacokinetic/pharmacodynamic indices and simulation process was carried out to further ensure clinical applicability of the formulation., Results: The in vitro release study showed initial burst (nearly 50 %) in first few minutes and followed Fickian (R(2) = 0.9234, n-value = 0.2329) type of diffusion release mechanism. The XRD and stability studies showed favorable results. The Wistar rat eyes treated with GCM showed significant increase in ganciclovir AUC (~4.99-fold) and Cmax (2.69-fold) in aqueous humor compared to ganciclovir solution and delay in Tmax. The Cmax/MIC90, AUC0-24/MIC90, AUC above MIC90 and T above MIC90 were significantly higher in GCM group. The aqueous humor concentration-time profile of ganciclovir in GCM and ganciclovir solution was simulated with every 28.1 and 12.8 h, respectively. The simulated concentration-time profile shows that in duration of 75 h, the ganciclovir solution require six ocular instillations compared to three ocular instillations of the GCM formulation. The photomicrograph of GCM and ganciclovir solution treated rat retina showed normal organization and cytoarchitecture., Conclusions: Correlating with in vitro data, the formulation showed sustained drug release along with improved intraocular bioavailability of ganciclovir in Wistar rats.

The success of conventional suicide gene therapy for cancer treatment is still limited because of lack of efficient delivery methods, as well as poor penetration into tumor tissues. Mesenchymal stem cells (MSCs) have recently emerged as potential vehicles in improving delivery issues. However, these stem cells are usually genetically modified using viral gene vectors for suicide gene overexpression to induce sufficient therapeutic efficacy. This approach may result in safety risks for clinical translation. Therefore, we designed a novel strategy that uses non-viral gene vector in modifying MSCs with suicide genes to reduce risks. In addition, these cells were co-administrated with prodrug-encapsulated liposomes for synergistic anti-tumor effects. Results demonstrate that this strategy is effective for gene and prodrug delivery, which co-target tumor tissues, to achieve a significant decrease in tumor colonization and a subsequent increase in survival in a murine melanoma lung metastasis model. Moreover, for the first time, we demonstrated the permeability of MSCs within tumor nests by using an in vitro 3D tumor spheroid model. Thus, the present study provides a new strategy to improve the delivery problem in conventional suicide gene therapy and enhance the therapeutic efficacy. Furthermore, this study also presents new findings to improve our understanding of MSCs in tumor-targeted gene delivery., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Theranostics is emerging as a popular strategy for cancer therapy; thanks to the development of nanotechnology. In this work, we have combined an HSV-TK/GCV suicide gene system and near-infrared quantum dots, as the former is quite effective in liver cancer treatment and the latter facilitates tumor imaging. A folate-modified theranostic liposome (FL/QD-TK) was developed, which is composed of an HSV-TK suicide gene covalently coupling with near-infrared fluorescent CdSeTe/ZnS core/shell quantum dots. The liver cancer-targeting and biosafety of FL/QD-TK were studied in vitro and in vivo. FL/QD-TK exhibited highly specific tumor imaging and strong inhibition of the folate receptor-overexpressed Bel-7402 mouse xenografts without systematic toxicity. This study may shed light on gene delivery and targeted cancer therapy.

Ganciclovir (GCV) is a nucleoside analogue with antiviral activity against herpes viral infections, and the most widely used antiviral to treat cytomegalovirus infections. However, the low bioavailability and short half-life of GCV necessitate the development of a carrier for sustained delivery. In this study, guanosine-based GCV was used as the initiator directly in ring-opening polymerization of ε-caprolactone (ε-CL) to form hydrophobic GCV-poly(caprolactone) (GCV-PCL) which was then grafted with hydrophilic chitosan to form amphiphilic copolymers for the preparation of stable micellar nanoparticles. Successful synthesis of GCV-PCL and GCV-PCL-chitosan were verified by 1H-NMR analysis. Self-assembled micellar nanoparticles were characterized by dynamic light scattering and zetasizer with an average size of 117 nm and a positive charge of 24.2 mV. The drug release kinetics of GCV was investigated and cytotoxicity assay demonstrated that GCV-tagged polymeric micelles were non-toxic. Our results showed that GCV could be used directly in the initiation of ring-opening polymerization of ε-CL and non-toxic polymeric micelles for GCV delivery can be formed.

The development of deoxynucleoside triphosphate (dNTP)-based drugs requires a quantitative understanding of any inhibition, activation, or hydrolysis by off-target cellular enzymes. SAMHD1 is a regulatory dNTP-triphosphohydrolase that inhibits HIV-1 replication in human myeloid cells. We describe here an enzyme-coupled assay for quantifying the activation, inhibition, and hydrolysis of dNTPs, nucleotide analogues, and nucleotide analogue inhibitors by triphosphohydrolase enzymes. The assay facilitates mechanistic studies of triphosphohydrolase enzymes and the quantification of off-target effects of nucleotide-based antiviral and chemotherapeutic agents., (Copyright © 2015, Arnold et al.)

Prediction of the in vivo performance of the drug product from the in vitro studies is the major challenging job for the pharmaceutical industries. From the current regulatory perspective, biorelevant dissolution media should now be considered as quality control media in order to avoid the risk associated. Physiological based pharmacokinetic models (PBPK) coupled with biorelevant dissolution medium is widely used in simulation and prediction of the plasma drug concentration and in vivo drug performance. The present investigation deals with the evaluation of biorelevant dissolution media as well as in vivo drug performance by PBPK modelling using STELLA® simulation software. The PBPK model was developed using STELLA® using dissolution kinetics, solubility, standard gastrointestinal parameters and post-absorptive disposition parameters. The drug product selected for the present study includes Linezolid film-coated immediate-release tablets (Zyvox), Tacrolimus prolonged-release capsules (Advagraf), Valganciclovir tablets (Valcyte) and Mesalamine controlled-release capsules (Pentasa) each belonging to different biopharmaceutics classification system (BCS). The simulated plasma drug concentration was analyzed and pharmacokinetic parameters were calculated and compared with the reported values. The result from the present investigation indicates that STELLA® when coupled with biorelevant dissolution media can predict the in vivo performance of the drug product with prediction error less than 20% irrespective of the dosage form (immediate release versus modified release) and BCS Classification. Thus, STELLA® can be used for in vivo drug prediction which will be helpful in generic drug development.

Many antiviral and anticancer drugs are nucleoside analogs that target polymerases and cause DNA chain termination. Interestingly, ganciclovir (GCV), the first line of therapy for human cytomegalovirus (HCMV) infections, induces chain termination despite containing the equivalent of a 3'-hydroxyl group. Certain HCMV GCV resistance (GCV(r)) mutations, including ones associated with treatment failures, result in substitutions in the 3'-5' exonuclease (Exo) domain of the catalytic subunit of the viral DNA polymerase (Pol). To investigate how these mutations confer resistance, we overexpressed and purified wild-type (WT) HCMV Pol and three GCV(r) Exo mutants. Kinetic studies provided little support for resistance being due to effects on Pol binding or incorporation of GCV-triphosphate. The mutants were defective for Exo activity on all primer templates tested, including those with primers terminating with GCV, arguing against the mutations increasing excision of the incorporated drug. However, although the WT enzyme terminated DNA synthesis after incorporation of GCV-triphosphate and an additional nucleotide (N+1), the Exo mutants could efficiently synthesize DNA to the end of such primer templates. Notably, the Exo activity of WT Pol rapidly and efficiently degraded N+2 primer templates to N+1 products that were not further degraded. On N+1 primer templates, WT Pol, much more than the Exo mutants, converted the incoming deoxynucleoside triphosphate to its monophosphate, indicative of rapid addition and removal of incorporated nucleotides ("idling"). These results explain how GCV induces chain termination and elucidate a previously unidentified mechanism of antiviral drug resistance.

The susceptibilities of gammaherpesviruses, including Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and animal rhadinoviruses, to various nucleoside analogs was investigated in this work. Besides examining the antiviral activities and modes of action of antivirals currently marketed for the treatment of alpha- and/or betaherpesvirus infections (including acyclovir, ganciclovir, penciclovir, foscarnet, and brivudin), we also investigated the structure-activity relationship of various 5-substituted uridine and cytidine molecules. The antiviral efficacy of nucleoside derivatives bearing substitutions at the 5 position was decreased if the bromovinyl was replaced by chlorovinyl. 1-β-D-Arabinofuranosyl-(E)-5-(2-bromovinyl)uracil (BVaraU), a nucleoside with an arabinose configuration of the sugar ring, exhibited no inhibitory effect against rhadinoviruses but was active against EBV. On the other hand, the fluoroarabinose cytidine analog 2'-fluoro-5-iodo-aracytosine (FIAC) showed high selectivity indices against gammaherpesviruses that were comparable to those of brivudin. Additionally, we selected brivudin- and acyclovir-resistant rhadinoviruses in vitro and characterized them by phenotypic and genotypic (i.e., sequencing of the viral thymidine kinase, protein kinase, and DNA polymerase) analysis. Here, we reveal key amino acids in these enzymes that play an important role in substrate recognition. Our data on drug susceptibility profiles of the different animal gammaherpesvirus mutants highlighted cross-resistance patterns and indicated that pyrimidine nucleoside derivatives are phosphorylated by the viral thymidine kinase and purine nucleosides are preferentially activated by the gammaherpesvirus protein kinase., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

This study compares the performance of a reference method of HPLC to Raman spectroscopy (RS) for the analytical quality control (AQC) of therapeutic objects. We assessed a model consisting of a widely used antiviral drug, i.e., ganciclovir, which was compounded in a medical device and then transferred in a vacuum glass vial prior to analyses. As the aim of the alternative RS method is to replace the destructive, time-consuming HPLC method, requiring sample preparation, it needs to be demonstrated that RS performs at least as good as the HPLC method. Therefore, the two methods were validated by calculating the accuracy profile and provided excellent results for the analytical validation key criteria, i.e., trueness, precision and accuracy, ranging from 0.8 to 10mg/mL. The Spearman and Kendall correlation tests (p-value<1.10-15) and the statistical studies performed on the graphs confirm a strong correlation in the results between RS and the standard HPLC under the experimental conditions. These results confirmed the potential of this option for future applications, owing to its analytical and practical quality and its contributions to the safety of the medication circuit. This method also greatly contributes to the protection of caregivers and their working environment., (Copyright © 2014 Elsevier B.V. All rights reserved.)

This study pragmatically characterized the micromechanical and physical stability of a poly(lactic-co-glycolic acid) (PLGA)-based ganciclovir (GCV)-loaded donut-shaped minitablet (DSMT) device for intraocular implantation. Thermal and spectroscopic analysis was performed on various drug-polymer permutations. Porositometric profiles were quantitatively analyzed coupled with qualitatively SEM imaging. The tensile strength (TS) and fracture energy (FE) of the device was also determined pre- and post-γ-sterilization. Inimitably, chemometric and molecular modeling provided a supportive confirmatory tool for establishing fundamental correlative suppositions between the transitioned surface morphology and the micromechanical stability after γ-irradiation. Isotherm plot volumes ranged between -0.028 ± 0.022 and 0.110 ± 0.005 m(2)/g for pre- and post-sterilized devices, respectively, revealing a microporous alteration in porosity. Pre-sterilized devices had larger pores (BJHa=286.22 vs. 192.49 Å) and lower FE (151.301 ± 6.089 N/m) and TS (26.396 ± 1.062 N) values while sterilized devices had crystalline matrices that facilitated the superiorly controlled drug release kinetcs obtained. DSC thermograms displayed the characteristic disordered crystallization of GCV and hydration exotherms resulting from ionization during γ-irradiation. FTIR spectrograms showed fingerprint molecular imprints of GCV and axial stretching of hybridized carbons of PLGA with no subversive drug-polymer interactions after γ-irradiation. Integration of the results inveterately revealed that compression and subsequent γ-irradiation of the device affected desirable micromechanical and solid-state stability behavior.

The efficient syntheses of 5-(2-hydroxyethyl)- and 5-(3-hydroxypropyl)-substituted pyrimidine derivatives bearing 2,3-dihydroxypropyl, acyclovir-, ganciclovir- and penciclovir-like side chains are reported. A synthetic approach that included the alkylation of an N-anionic-5-substituted pyrimidine intermediate (method A) provided the target acyclonucleosides in significantly higher overall yields in comparison to those obtained by method B using sylilation reaction. The phosphorylation assays of novel compounds as potential substrates for thymidine kinase of herpes simplex virus type 1 (HSV-1 TK) showed that solely pyrimidine 5-substituted acyclonucleosides with a penciclovir-like side chain acted as a fraudulent substrates of HSV-1 TK. Moreover, the uracil derivative with penciclovir-like side chain with less bulky 2-hydroxyethyl substituent at C-5 proved to be a better substrate than the corresponding one with a 3-hydroxypropyl substituent. Therefore, this acyclonucleoside was selected as a lead compound for the development of a positron emission tomography HSV-1 TK activity imaging agent.

Human cytomegalovirus (HCMV) is a prevalent virus that infects up to 90% of the population. The goal of this research is to determine if small molecular prodrug substrates can be developed for a specific HCMV encoded protease and thus achieve site-specific activation. HCMV encodes a 256 amino acid serine protease that is responsible for capsid assembly, an essential process for herpes virus production. The esterase activity of the more stable HCMV A143T/A144T protease mutant was evaluated with model p-nitrophenol (ONp) esters, Boc-Xaa-ONp (Ala, Leu, Ile, Val, Gln, Phe at the Xaa position). We demonstrate that the A143T/A144T mutant has esterase activity toward specific small ester compounds, e.g., Boc-L-Ala-ONp. Mono amino acid and dipeptide prodrugs of ganciclovir (GCV) were also synthesized and evaluated for hydrolysis by the A143T/A144T protease mutant in solution. Hydrolysis of these prodrugs was also evaluated in Caco-2 cell homogenates, human liver microsomes (HLMs), and rat and human plasma. For the selectivity potential of the prodrugs, the hydrolysis ratio was evaluated as a percentage of prodrug hydrolyzed by the HCMV protease over the percentages of prodrug hydrolyses by Caco-2 cell homogenates, HLMs, and human/rat plasma. A dipeptide prodrug of ganciclovir, Ac-l-Gln-l-Ala-GCV, emerged as a potential selective prodrug candidate. The results of this research demonstrate that targeting prodrugs for activation by a specific protease encoded by the infectious HCMV pathogen may be achievable.

Background: Chitosan has been investigated as a promising nonviral vector. However, several problems still remain, such as a relatively low transfection efficiency and instability under physiological conditions. We previously demonstrated that a chondroitin sulfate (CS) coating enhanced the transfection efficiency and physicochemical stability of plasmid DNA (pDNA)/chitosan complexes in vitro. In the present study, the effects of coating pDNA/chitosan complexes with CS on the stability in freeze-dry rehydration processes and gene expression in vivo were investigated., Methods: Freeze-drying storage at -20 °C, 4 °C, or room temperature, freezing storage at -20 °C, or liquid storage at 4 °C or room temperature, were examined for preservation conditions of pDNA/chitosan/CS ternary complexes by a gel retardation assay, measurements of sizes and zeta potentials, and a luciferase assay. Moreover, to determine the transfection efficiency of the ternary complexes in vivo, suicide gene therapy was carried out in Huh-7-implanted mice using herpes simplex virus thymidine kinase coding pDNA and ganciclovir., Results: The freeze-dried pDNA/chitosan/CS ternary complexes showed sufficient cell transfection ability in vitro and in vivo. In addition, ternary complexes were associated with a significant suppression of tumor growth and a histopathologically high anti-tumor effect by intratumoral injection to tumor-bearing mice., Conclusions: The CS coating enhanced the preservation stability of the pDNA/chitosan complexes after freeze-drying-rehydration and their transgene expression in vivo., (Copyright © 2013 John Wiley & Sons, Ltd.)

Background: Until the availability of ganciclovir ophthalmic gel in 2009, the only option for treating herpes simplex (HSV) keratitis in the USA has been trifluridine (TFT), a compound with tolerability issues related to its nonselective inhibition of DNA replication in both normal cells and virus-infected cells. Ganciclovir has selective pharmacologic activity on viral thymidine kinase and a lower potential for toxicity to healthy human cells. Our objective was to evaluate safety and efficacy findings reported with the use of ganciclovir ophthalmic gel, both for HSV keratitis and other potential clinical indications., Methods: Clinical and preclinical data with ganciclovir were identified through a comprehensive electronic search of PubMed and Medline, using the search terms ganciclovir, ganciclovir 0.15% ophthalmic gel, acyclovir, acyclovir ointment 3%, herpes simplex keratitis, treatment of herpes simplex keratitis, and adenoviral keratoconjunctivitis. The authors were also granted access to previously unpublished ganciclovir surveillance safety data from Bausch & Lomb, Inc., Results: No clinical data comparing ganciclovir ophthalmic gel to 1% trifluorothymidine (TFT) for HSV keratitis could be identified. Four international, randomized, multicenter clinical trials have demonstrated that ganciclovir gel is at least as effective as acyclovir ointment for the treatment of HSV keratitis. Ganciclovir gel was better tolerated, with lower rates of blurred vision, eye irritation, and punctate keratitis. Recent data also indicate it may hold promise as a treatment for adenoviral keratoconjunctivitis. Worldwide safety surveillance data collected over the past 10-15 years in over 30 countries suggests an extremely low rate of spontaneously reported adverse events with ganciclovir ophthalmic gel., Conclusions: Current data suggest that ganciclovir ophthalmic gel has similar efficacy as acyclovir ointment for the treatment of HSV keratitis and is better tolerated. Clinical head-to-head studies comparing ganciclovir and TFT would be of great interest, especially for US physicians.

Human Cytomegalovirus is a commonly identified herpesvirus that establishes a state of latent infection in the majority of the population by adulthood. A coordinated immune response involving both the innate and adaptive immune system prevents active viral replication and disease. Cellular immunity appears particularly important to control of viremia requiring both a CMV-specific CD4+ and CD8+ T cell response. Solid organ transplant recipients are particularly susceptible to CMV related disease due to the immunosuppression necessary to prevent organ rejection, with patients receiving T cell depleting therapies being at highest risk. The deleterious outcomes of CMV in organ transplant recipients result from both direct cytopathic and indirect immune-modulatory effects of CMV viral replication. The recognition of the negative effects of CMV in solid organ transplantation has resulted in the routine prophylaxis of organ recipients with antiviral nucleoside analogues. The appropriate duration of therapy is still controversial although guidelines do exist. The ability to assay an individual immune response to CMV should allow for tailored duration of therapy in the future.

Encapsulation of Ganciclovir in lipophilic vesicular structure may be expected to enhance the oral absorption and prolong the existence of the drug in the systemic circulation. So the purpose of the present study was to improve the oral bioavailability of Ganciclovir by preparing nanosized niosomal dispersion. Niosomes were prepared from Span40, Span60, and Cholesterol in the molar ratio of 1:1, 2:1, 3:1, and 3:2 using reverse evaporation method. The developed niosomal dispersions were characterized for entrapment efficiency, size, shape, in vitro drug release, release kinetic study, and in vivo performance. Optimized formulation (NG8; Span60:Cholesterol 3:2 molar ratio) has shown a significantly high encapsulation of Ganciclovir (89±2.13%) with vesicle size of 144±3.47 nm (polydispersity index [PDI]=0.08). The in vitro release study signifies sustained release profile of niosomal dispersions. Release profile of prepared formulations have shown that more than 85.2±0.015% drug was released in 24 h with zero-order release kinetics. The results obtained also revealed that the types of surfactant and Cholesterol content ratio altered the entrapment efficiency, size, and drug release rate from niosomes. In vivo study on rats reveals five-time increment in bioavailability of Ganciclovir after oral administration of optimized formulation (NG8) as compared with tablet. The effective drug concentration (>0.69 µg/mL in plasma) was also maintained for at least 8 h on administration of the niosomal formulation. In conclusion, niosomes can be proposed as a potential oral delivery system for the effective delivery of Ganciclovir.

Objectives: The aim of this work was to develop a simple, sensitive and selective LC/MS/MS method for the assay of valganciclovir and ganciclovir in human plasma., Design and Methods: Sample preparation involved solid phase extraction on mix mode cation exchanger. Separation was performed on Chromolith RP18e column using water, trifluoroacetic acid (1M, pH 4.4) and methanol (29.9:0.1:70, v/v) as mobile phase. Both analytes were detected by electro spray ionization mass spectrometry in positive ion multiple reaction monitoring mode., Results: CCs with good linearties having r≥0.9990 and ≥0.9992 were obtained in the range of 5-800ng/mL and 70-11,200ng/mL for valganciclovir and ganciclovir, respectively. The extraction recoveries were around 85% for both the analytes., Conclusion: The method provided a simple and selective procedure that can be easily used for the evaluation of the pharmacokinetic profile of valganciclovir and ganciclovir in human plasma., (Copyright © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

This manuscript deals with the effect of a counterion on the glass transition temperature for lyophilization of ganciclovir salts. Salt forms of ganciclovir, namely, sodium, potassium, rubidium, and cesium salts, were prepared by an in situ technique and analyzed by modulated differential scanning calorimetry (MDSC) for the determination of the critical process parameter for lyophilization. Nonionized ganciclovir and its salt forms showed a glass transition (T(g)') in the reversing MDSC signal, confirming their amorphous nature. T(g)' of the nonionized ganciclovir and ganciclovir sodium, potassium, rubidium, and cesium salts followed the order: sodium salt (-34.94°C) > nonionized ganciclovir (-40.15°C) > potassium salt (-46.23°C) > rubidium salt (-49.95°C) > cesium salt (-53.62°C). The analysis of the freezable water content for ganciclovir and its salts showed the trend: pure water > nonionized ganciclovir > potassium salt ∼ sodium salt > rubidium salt > cesium salt. This showed that a majority of water in the salts is present as an unfrozen fraction, thus leading to a lowering of T(g)' because of the plasticizing effect of unfrozen water. Density functional theory (DFT) further suggested a positive contribution of the strength of intra- and intermolecular force of interactions to the T(g)' value, with a higher intramolecular and intermolecular force of interaction leading to a higher T(g)'.

In rats we examined the effects of some common excipients on the intestinal absorption of ganciclovir (GCV), a BCS-III drug and substrate of P-gp, by assessing its in vitro transfer from mucosa to serosa and in situ transepithelial permeation. In vitro, all selected excipients (concentration range 0.1-1% [w/v]) could increase the transport amount of GCV in the everted gut sac model. Whereas enhancement by F-68 demonstrated regional differences like verapamil, PEG-400, Tween-80 and EL-35 exhibited no regional differences. In situ studies were performed by an improved perfusion model, single-pass perfusion with whole small intestine, to determine more accurately the permeability of lipophobic compounds. The permeability of GCV was significantly increased by all excipients. The effects of EL-35 and F-68 were dose-dependent but those of PEG-400 and Tween-80 were not. The results suggest that enhancements of intestinal absorption of GCV by these excipients are probably due to inhibition of P-gp-mediated drug efflux. It could be deduced from their different properties that both blocking binding sites of P-gp and altering membrane fluidity were involved in their P-gp-inhibition. The former mechanism might be involved for F-68, while the latter one might account for the effects of PEG-400, Tween-80 and EL-35., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Recombinant silk-elastin-like protein polymers (SELPs) are well-known for their highly tunable properties on both the molecular and macroscopic hydrogel levels. One specific structure of these polymers, SELP-815K, has been investigated as an injectable controlled delivery system for the treatment of head and neck cancer via a gene-directed enzyme prodrug therapy (GDEPT) approach. Due to its pore size and gelation properties in vivo, SELP restricts the distribution and controls the release of therapeutic viruses for up to one month. It has been shown that SELP-mediated delivery significantly improves therapeutic outcome of the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) system in xenograft models of human head and neck cancer. However little is known about potential benefits of this approach with regard to toxicity in the presence of a fully intact immune system. The studies presented here were designed to assess the change in toxicity of the SELP-mediated viral delivery compared to free viral injection in a non-tumor-bearing immune competent mouse model. Toxicity was assessed at 1, 2, 4, and 12 weeks via body weight monitoring, complete blood count (CBC), and blood chemistry. It was found that in the acute and subacute phases (weeks 1-4) there is significant toxicity in groups combining the virus and the prodrug, and matrix-mediated gene delivery with SELP demonstrates a reduction in toxicity from the 2 week time point through the 4 week time point. At the end of the subchronic phase (12 weeks), signs of toxicity had subsided in both groups. Based on these results, recombinant SELPs offer a significant reduction in toxicity of virus-mediated GDEPT treatment compared to free virus injection in the acute and subacute phases.

Purpose: To delineate the vitreal pharmacokinetics of dipeptide monoester prodrugs of ganciclovir (GCV) with conscious rabbit model using ocular microdialysis and to compare with published results from anesthetized model., Methods: New Zealand albino male rabbit was selected as the animal model. Conscious animal ocular microdialysis technique with permanently implanted probes was employed to delineate the pharmacokinetics of GCV, L-valine-GCV (Val-GCV), and dipeptide monoester GCV prodrugs [val-val and L-glycine-val (Gly-Val)] after intravitreal administration., Results: This work employs conscious model to evaluate vitreal pharmacokinetic parameters and compares the results with previously published data from anesthetized animal, thereby demonstrating the effect of anesthesia on the vitreal disposition of dipeptide prodrugs of GCV. Results have revealed that area under curve (AUC), clearance, and last measured plasma concentration (C(last)) for all 4 compounds were significantly altered in a conscious animal relative to the anesthetized model, while mean residence time (MRT) was significantly reduced. However, the AUCs of regenerated Val-GCV and GCV from Gly-Val-GCV and Val-Val-GCV were found to be unchanged, suggesting higher ocular metabolism in conscious animals., Conclusion: This study for the first time delineates the vitreal pharmacokinetics of a GCV prodrug in conscious animals and compares the data with anesthetized animals. Lower vitreal exposure levels were obtained in case of conscious animal model; however, the elimination rates were not influenced by anesthesia.

Studies were carried out, for the first time, to investigate the charge-transfer reactions of ganciclovir as n-electron donor with the sigma-acceptor iodine and various pi-acceptors: 7,7,8,8-tetracyanoquinodimethane; tetracyanoethylene; 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; p-chloranilic acid; 2,3,5,6-tetrabromo-1,4-benzoquinone; 2,3,5,6-tetrachloro-1,4-benzoquinone and 2,4,7-trinitro-9-fluorenone. The formation of the colored charge-transfer complexes was utilized in the development of simple, rapid and accurate spectrophotometric methods for the analysis of ganciclovir in pure form as well as in its pharmaceutical formulation (capsules). Different variables affecting the reactions were studied and optimized. Under the optimum reaction conditions, linear relationships with good correlation coefficients (0.9993-0.9998) were found between the absorbance and the concentration of ganciclovir in the range of 2.0-240 microg mL(-1). For more accurate analysis, Ringbom optimum concentration range was found to be between 5.0 and 225 microg mL(-1). The limits of detection ranged from 0.36 to 2.45 microg mL(-1) and the limits of quantification ranged from 1.20 to 8.17 microg mL(-1). A Job's plot of the absorbance versus the molar ratio of ganciclovir to each of acceptors under consideration indicated (1:1) ratio. The proposed methods were applied successfully for simultaneous determination of ganciclovir in capsules with good accuracy and precision and without interferences from common additives. The recovery percentages ranged from 99.45+/-0.73% to 100.35+/-1.40%. The results were compared favourably with the reported method.

The thermodynamics of the drug-inhibitors acyclovir, ganciclovir, and 9-benzylguanine binding to human purine nucleoside phosphorylase (hsPNP) were determined from isothermal titration calorimetry as a function of the substrate phosphate ion (Pi) concentration from 0 to 0.125 M and temperature from 15 degrees C to 35 degrees C. At 25 degrees C and with an increase in the Pi concentration from 0 to 50mM, acyclovir binding becomes more entropically-driven and ganciclovir binding becomes more enthalpically-driven. At 25 degrees C, the tighter 9-benzylguanine binding reaction goes from an enthalpically-driven reaction in the absence of Pi to an entropically-driven reaction at 10 mM Pi, and the enthalpically-driven nature of the binding reaction is restored at 75 mM Pi. Since the dependencies of the driving-nature of the binding reactions on Pi concentration can be simulated by Pi binding to its catalytic site, it is believed that bound Pi affects the interactions of the side-chains with the ribose catalytic site. However, the binding constants are unaffected by change in the bound Pi concentration because of enthalpy-entropy compensation. The enzymatic activity of hsPNP was determined by an ITC-based assay employing 7-methylguanosine and Pi as the substrates. The heat of reaction determined from the assay increased by 7.5 kJ mol(-1) with increase in Pi concentration from 50 to 100mM and is attributed to weak binding of the Pi to a secondary regulatory site. Although the binding constants of acyclovir and ganciclovir at 20 microM hsPNP were in agreement with the inverse inhibition constants determined from the ITC enzyme inhibition assays at 60 nM, the binding constant of 9-benzylguanine, which interacts with Phe159 from an adjacent subunit, decreased from 5.62 x 10(5) M(-1) to 1.14 x 10(5) M(-1). This reduction in the 9-benzylguanine binding affinity along with a 7-fold increase in the specific activity of hsPNP at 14.5 nM results from partial dissociation of the hsPNP trimer into monomers below the 60 nM level.

Chemical modification to improve biopharmaceutical properties, especially oral absorption and bioavailability, is a common strategy employed by pharmaceutical chemists. The approach often employs a simple structural modification and utilizes ubiquitous endogenous esterases as activation enzymes, although such enzymes are often unidentified. This report describes the crystal structure and specificity of a novel activating enzyme for valacyclovir and valganciclovir. Our structural insights show that human valacyclovirase has a unique binding mode and specificity for amino acid esters. Biochemical data demonstrate that the enzyme hydrolyzes esters of alpha-amino acids exclusively and displays a broad specificity spectrum for the aminoacyl moiety similar to tricorn-interacting aminopeptidase F1. Crystal structures of the enzyme, two mechanistic mutants, and a complex with a product analogue, when combined with biochemical analysis, reveal the key determinants for substrate recognition; that is, a flexible and mostly hydrophobic acyl pocket, a localized negative electrostatic potential, a large open leaving group-accommodating groove, and a pivotal acidic residue, Asp-123, after the nucleophile Ser-122. This is the first time that a residue immediately after the nucleophile has been found to have its side chain directed into the substrate binding pocket and play an essential role in substrate discrimination in serine hydrolases. These results as well as a phylogenetic analysis establish that the enzyme functions as a specific alpha-amino acid ester hydrolase. Valacyclovirase is a valuable target for amino acid ester prodrug-based oral drug delivery enhancement strategies.

The objective of this study was to evaluate the effect of 2 independent formulation variables on the drug release from a novel doughnut-shaped minitablet (DSMT) in order to optimize formulations for intraocular drug delivery. Formulations were based on a 3(2) full-factorial design. The 2 independent variables were the concentration of Resomer (% wt/wt) and the type of Resomer grade (RG502, RG503, and RG504), respectively. The evaluated response was the drug release rate constant computed from a referenced marketed product and in vitro drug release data obtained at pH 7.4 in simulated vitreous humor. DSMT devices were prepared containing either of 2 model drugs, ganciclovir or foscarnet, using a Manesty F3 tableting press fitted with a novel central-rod, punch, and die setup. Dissolution data revealed biphasic drug release behavior with 55% to 60% drug released over 120 days. The inherent viscosity of the various Resomer grades and the concentration were significant to achieve optimum release rate constants. Using the resultant statistical relationships with the release rate constant as a response, the optimum formulation predicted for devices formulated with foscarnet was 70% wt/wt of Resomer RG504, while 92% wt/wt of Resomer RG503 was ideal for devices formulated with ganciclovir. The results of this study revealed that the full-factorial design was a suitable tool to predict an optimized formulation for prolonged intraocular drug delivery.

Ophthalmic liposomes of ganciclovir (GCV) were prepared by the reverse phase evaporation method, and their ocular pharmacokinetics in albino rabbits were compared with those obtained after dosing with GCV solution. The in vitro transcorneal permeability of GCV liposomes was found to be 3.9-fold higher than that of the solution. After in vivo instillation in albino rabbits, no difference was found in the precorneal elimination rate of GCV from liposome vs solution dosing. The aqueous humor concentration-time profiles of both liposomes and solution were well described by 2-compartmental pharmacokinetics with first-order absorption. The area under the curve of the aqueous humor concentration-time profiles of GCV liposomes was found to be 1.7-fold higher than that of GCV solution. Ocular tissue distribution of GCV from liposomes was 2 to 10 times higher in the sclera, cornea, iris, lens, and vitreous humor when compared with those observed after solution dosing. These results suggested that liposomes may hold some promise in ocular GCV delivery.

The purpose of this investigation was to study the influences of absorption enhancers in increasing oral bioavailability of Ganciclovir (GAN) by assessing the transepithelial permeation across cell monolayers in vitro and bioavailability in rats in vivo. The permeation of GAN across Caco-2 and MDCK cell monolayers in the absence/presence of dimethyl-beta-cyclodextrin (DMbetaCD), chitosan hydrochloride (CH), sodium lauryl sulphate (SLS), and their combinations was studied for a 2-h period. GAN was administered to rats in absence/presence of absorption enhancers and drug contents in plasma were estimated. We found that the apparent permeability coefficient (Papp) of GAN in absence of absorption enhancers (control) were 0.261 +/- 0.072 x 10(-6) and 0.486 +/- 0.063 x 10(-6) cm/s in Caco-2 and MDCK cell monolayers, respectively, whereas in the presence of DMbetaCD, CH, SLS, and their combinations, Papp of GAN increased by 5- to 25-fold and 7- to 33-fold as compared to control in Caco-2 and MDCK cell monolayers, respectively. However, in rats, the maximum enhancement in bioavailability of GAN during coadministration of these absorption enhancers was only fivefold compared to GAN control. To conclude, the absorption enhancers-DMbetaCD, CH, SLS, and their combinations demonstrated significant improvement in transepithelial permeation and bioavailability of GAN., ((c) 2007 Wiley-Liss, Inc.)

To enhance the efficacy of ganciclovir/herpes simplex virus thymidine kinase (GCV/HSV-TK) suicide gene therapy for nasopharyngeal cancer KB, we developed long-circulating liposome-encapsulated GCV, and evaluated cytotoxicity in vitro and in vivo. PEGylated liposome-encapsulated GCV (PEG-GCV-lipo) was prepared by the freeze-thawing method. In vitro experiments demonstrated that GCV from liposomes was gradually released over a period of 3 days. The in vitro cytotoxicity of PEG-GCV-lipo was similar to that of GCV solution in human cervical carcinoma HeLa cells expressing HSV-TK. Pharmacokinetics studies in mice showed that, compared with GCV solution, intravenous and intraperitoneal injection of PEG-GCV-lipo (10 mg/kg) led to long circulation in plasma; the area under the curve was 36-fold or 32-fold higher than that of GCV solution, respectively. In GCV/HSV-TK suicide gene therapy, the HSV-TK gene complexed with nanoparticle vector was directly injected into KB xenografts, and PEG-GCV-lipo or GCV solution was injected intravenously in mice once a day (25 mg/kg/day every 2nd day, 4 times). PEG-GCV-lipo was significantly 3-fold more effective than GCV solution in inhibiting tumor growth and produced durable complete tumor remissions on day 11 after injection. These findings demonstrate that long-circulating liposome-encapsulated GCV is a new approach to drug carriers to enhance the efficacy of suicide gene therapy.

Primary effusion lymphoma (PEL) is a rare B-cell lymphoma caused by Kaposi's sarcoma-associated herpesvirus (KSHV). PEL is poorly responsive to standard cytotoxic chemotherapy and portends a poor survival. Consequently, new effective treatment options are urgently needed. It is known that KSHV encodes two lytic genes, ORF36 (phosphotransferase) and KSHV ORF21 (thymidine kinase), which can phosphorylate ganciclovir and azidothymidine, respectively. Here, we have explored whether these genes can be used as therapeutic targets for PEL. PEL arises in pleural spaces and other effusions that provide a hypoxic environment. Based on Northern blot analysis, exposure of PEL cells to hypoxia up-regulated the expression of both ORF36 and ORF21. Using a newly developed nonradioactive reverse-phase high-performance liquid chromatography/mass spectrometry method to separate and quantify the phosphorylated forms of ganciclovir and azidothymidine, we found that PEL cells exposed to hypoxia produced increased amounts of the toxic triphosphates of these drugs. Moreover, we found that hypoxia increased the cell toxicity of ganciclovir and azidothymidine in PEL cells but had no significant effect on the herpesvirus-negative cell line CA46. These findings may have clinical applicability in the development of effective therapies for PEL or other KSHV-related malignancies.

Transferrin modified pro-cationic liposomes were prepared and used to investigate the effect of targeting therapeutic genes to human hepatoma carcinoma cells in vitro. The main lipid CHETA, cholest-5-en-3beta-yl[2-[[4-[(carboxymethyl)dithio]-1-iminobutyl]amino]ethyl] carbamate (C36H61N3O4S2), was synthesized and used to prepare pro-cationic liposomes. The thymidine kinase (TK) gene loaded pro-cationic liposomes were prepared by first mixing the plasmid DNA and protamine together, and then incubating the resulted polyplexes with blank pro-cationic liposomes preformed by the thin film dispersion-sonication method. Transferrin (Tf) was adsorbed on the surface of pro-cationic liposomes via electrostatic interactions to form transferrin modified pro-cationic liposomes. Cellular association was measured by fluorimetry at excitation and emission wavelengths of 490 and 520 nm, respectively. The viability of TK gene infected cells following administration of ganciclovir (GCV) was investigated by MTT assay. The transferrin modified TK gene pro-cationic liposomes had a mean diameter of 240 +/- 12 nm and zeta potential of -24.10 +/- 2.5 mV (n = 3). The transmission electron microscopy image indicated that most of the liposomes were relatively regular and spherical with a condensed core inside. Cell-associated fluorescence of Tf-liposomes and unmodified liposomes (without transferrin) was 7.8 x 10(6), and 3.2 x 10(6) per milligram protein, respectively. Compared to Lipofectamine 2000 (Invitrogen, USA) the pro-cationic liposomes and transferrin modified pro-cationic liposomes had less cytotoxicity to cells. The transduced TK gene HepG2 cells were more sensitive to GCV than the un-transduced TK gene ones and the human normal Chang liver cells were not affected by the TK/GCV system mediated by procationic liposomes.

Purpose: The aim of this study is to investigate the roles of the lactide/glycolide ratio and prodrug derivatization on the in vitro release of ganciclovir and its lipophilic prodrug (GCV-monobutyrate) from PLGA microspheres for the treatment of cytomegalovirus (CMV) retinitis., Method: Two grades of PLGA, a higher lactide content PLGA 6535 [d,l-lactide:glycolide=65:35, MW 45,000-75,000Da] and lower lactide content PLGA 5050 [d,l-lactide:glycolide=50:50, MW 45,000-75,000Da] were employed to prepare GCV loaded microspheres. The effect of lipophilic prodrug derivatization was investigated by converting GCV to GCV-monobutyrate (GCVMB). Microspheres were prepared by the oil-in-oil (O/O) solvent evaporation method and characterized in vitro, by studying their surface/internal morphology, entrapment efficiency, particle size, drug release, true density and glass transition temperature. In vitro release data were analyzed by a model equation to estimate various parameters of the drug release curves., Results: The O/O solvent evaporation method generated a high drug payload of up to 91%. Higher entrapment efficiencies were observed in the case of hydrophilic drug (GCV) relative to the lipophilic prodrug (GCVMB). Loosely bound or surface adsorbed drug/prodrug molecules may have resulted in the very short period (about 6h) of the initial burst phase in all types of microspheres. GCV loaded microspheres utilized more time to release 50% (T(50) value) of entrapped drug than GCVMB microspheres. T(50) values estimated for GCVMB were shorter than those for GCV from microspheres with similar lactide/glycolide ratios. Lactide content in PLGA did not significantly alter GCVMB release relative to GCV release. The proposed model equation effectively estimated the drug release parameters (R(2)>0.98) with all drug/prodrug-PLGA combinations. SEM pictures have revealed that although both GCV and GCVMB microspheres were spherical but internal morphology was different, with former having uniform and dense whereas later have porous structures. Corroborating with internal morphologies, results revealed that true densities of GCV microspheres were relatively greater than corresponding GCVMB microspheres., Conclusion: The proposed method of preparation yields higher efficiency of drug entrapment for the hydrophilic drug. Prodrug entrapment into microspheres could result in longer residence time at the site of administration due to multiple processes involved in drug release at infected tissue. These processes include release from microspheres and enzymatic conversion of the prodrug to parent drug.