Technical note: Evaluation of the dose enhancement effect for a novel transmission-type x-ray tube using the Monte Carlo method.

PURPOSE: Transmission-target x-ray tubes generate more x-rays than reflection thick-target x-ray tubes. A transmission x-ray tube combined with radiosensitizers has a better radiation enhancement effect. This study investigated the feasibility of using a transmission x-ray tube with radiosensitizers in clinical radiotherapy and its effect on radiation dose enhancement. METHODS: This study used MCNP6.2 to simulate the model of a transmission x-ray tube and Co-60 beam.   The radiation enhancement effect of radiosensitizers was examined with iodine-127 (I-127), radioiodinated iododeoxyuridine (IUdR), and gold nanoparticles (GNPs). RESULTS: The study results showed that the dose enhancement factor (DEF) of the transmission x-ray tube with GNPs was 10.27, which was higher than that of I-127 (6.46) and IUdR (3.08). The DEF of the Co-60 beam with GNPs, I-127, and IUdR was 1.23, 1.19, and 1.2, respectively. The Auger electron flux of the transmission x-ray tube with GNPs was 1.19E+05 particles/cm2 . CONCLUSIONS: This study found that a transmission x-ray tube with appropriate radiosensitizers could produce a high rate of Auger electrons to fulfill the radiation enhancement effect, and this procedure has the potential to become a radiotherapy modality.

Suzuki-Miyaura Coupling, Heck Alkenylation, and Amidation of DMTr-Protected 5-Iodo-2'-Deoxyuridine via Palladium-catalyzed Reactions.

Modification of nucleosides via cross-coupling processes has been carried out extensively on unprotected halonucleosides to produce functionalized nucleosides that are often developed for incorporation into oligonucleotides or used as fluorescent probes. This approach requires protection of the 5'-OH with the 4,4'-dimethoxytrityl (DMTr) group, which is complicated and a common cause of reaction failure. Here we report a method for direct functionalization of 5'-O-DMTr-5-iodo-2'-deoxyuridine via Suzuki-Miyaura cross-coupling, Heck alkenylation, and carboamidation. This approach facilitates rapid synthesis of a variety of C5-substituted 5'-O-DMTr-2'-deoxyuridine derivatives. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of the SerrKap palladacycle complex Basic Protocol 2: Suzuki-Miyaura coupling of 5'-O-DMTr-5-iodo-2'-deoxyuridine using SerrKap palladacycle Basic Protocol 3: Heck coupling of 5'-O-DMTr-5-iodo-2'-deoxyuridine using SerrKap palladacycle Basic Protocol 4: Heck coupling of 5'-O-DMTr-5-iodo-2'-deoxyuridine with Ruth linker using Pd(OAc)2 /PTABS Basic Protocol 5: Carbonylative amidation of 5'-O-DMTr-5-iodo-2'-deoxyuridine using Pd(OAc)2 /PTABS.

Use of the Pyrimidine Analog, 5-Iodo-2'-Deoxyuridine (IdU) with Cell Cycle Markers to establish Cell Cycle Phases in a Mass Cytometry Platform.

The regulation of cell cycle phase is an important aspect of cellular proliferation and homeostasis. Disruption of the regulatory mechanisms governing the cell cycle is a feature of a number of diseases, including cancer. Study of the cell cycle necessitates the ability to define the number of cells in each portion of cell cycle progression as well as to clearly delineate between each cell cycle phase. The advent of mass cytometry (MCM) provides tremendous potential for high throughput single cell analysis through direct measurements of elemental isotopes, and the development of a method to measure the cell cycle state by MCM further extends the utility of MCM. Here we describe a method that directly measures 5-iodo-2'-deoxyuridine (IdU), similar to 5-bromo-2´-deoxyuridine (BrdU), in an MCM system. Use of this IdU-based MCM provides several advantages. First, IdU is rapidly incorporated into DNA during its synthesis, allowing reliable measurement of cells in the S-phase with incubations as short as 10-15 minutes. Second, IdU is measured without the need for secondary antibodies or the need for DNA degradation. Third, IdU staining can be easily combined with measurement of cyclin B1, phosphorylated retinoblastoma protein (pRb), and phosphorylated histone H3 (pHH3), which collectively provides clear delineation of the five cell cycle phases. Combination of these cell cycle markers with the high number of parameters possible with MCM allow combination with numerous other metrics.

Enhancement radiation-induced apoptosis in C6 glioma tumor-bearing rats via pH-responsive magnetic graphene oxide nanocarrier.

5-iodo-2-deoxyuridine (IUdR) has been demonstrated to induce an appreciable radiosensitizing effect on glioblastoma patients, but due to the short circulation half-life times and failure to pass through the blood-brain barrier (BBB), its clinical use is limited. Accordingly, in this study, we used magnetic graphene oxide (NGO/SPIONs) nanoparticles coated with PLGA polymer as a dynamic nanocarrier for IUdR and, evaluated its sensitizing enhancement ratio in combination with a single dose X-ray at clinically megavoltage energies for treatment of C6 glioma rats. Nanoparticles were characterized using Zetasizer and TEM microscopy, and in vitro biocompatibility of nanoparticles was assessed with MTT assay. IUdR/MNPs were intravenously administered under a magnetic field (1.3 T) on day 13 after the implantation of C6 cells. After a day following the injection, rats exposed with radiation (8 Gy). ICP-OES analysis data indicated an effective magnetic targeting, leading to remarkably improved penetration through the BBB. In vivo release analysis with HPLC indicated sustained release of IUdR and, prolonged the lifespan in plasma (P < .01). In addition, our findings revealed a synergistic effect for IUdR/MNPs coupled with radiation, which significantly inhibited the tumor expansion (>100%), prolonged the survival time (>100%) and suppressed the anti-apoptotic response of glioma rats by increasing Bax/Bcl-2 ratio (2.13-fold) in compared with the radiation-only. In conclusion, besides high accumulation in targeted tumor sites, the newly developed IUdR/MNPs, also exhibited the ability of IUdR/MNPs to significantly enhance radiosensitizing effect, improve therapeutic efficacy and increase toxicity for glioma-bearing rats.

Phase I and Pharmacology Study of Ropidoxuridine (IPdR) as Prodrug for Iododeoxyuridine-Mediated Tumor Radiosensitization in Advanced GI Cancer Undergoing Radiation.

PURPOSE: Iododeoxyuridine (IUdR) is a potent radiosensitizer; however, its clinical utility is limited by dose-limiting systemic toxicities and the need for prolonged continuous infusion. 5-Iodo-2-pyrimidinone-2'-deoxyribose (IPdR) is an oral prodrug of IUdR that, compared with IUdR, is easier to administer and less toxic, with a more favorable therapeutic index in preclinical studies. Here, we report the clinical and pharmacologic results of a first-in-human phase I dose escalation study of IPdR + concurrent radiation therapy (RT) in patients with advanced metastatic gastrointestinal (GI) cancers. PATIENTS AND METHODS: Adult patients with metastatic GI cancers referred for palliative RT to the chest, abdomen, or pelvis were eligible for study. Patients received IPdR orally once every day × 28 days beginning 7 days before the initiation of RT (37.5 Gy in 2.5 Gy × 15 fractions). A 2-part dose escalation scheme was used, pharmacokinetic studies were performed at multiple time points, and all patients were assessed for toxicity and response to Day 56. RESULTS: Nineteen patients were entered on study. Dose-limiting toxicity was encountered at 1,800 mg every day, and the recommended phase II dose is 1,200 mg every day. Pharmacokinetic analyses demonstrated achievable and sustainable levels of plasma IUdR ≥1 µmol/L (levels previously shown to mediate radiosensitization). Two complete, 3 partial, and 9 stable responses were achieved in target lesions. CONCLUSIONS: Administration of IPdR orally every day × 28 days with RT is feasible and tolerable at doses that produce plasma IUdR levels ≥1 µmol/L. These results support the investigation of IPdR + RT in phase II studies.

Mass Cytometric Cell Cycle Analysis.

The regulated proliferation of cells is a critical factor in tumor progression, antineoplastic therapies, immune system regulation, and the cellular developmental of multicellular organisms. While measurement of cell cycle state by fluorescent flow cytometry is well established, mass cytometry allows the cell cycle to be measured along with large numbers of other antigens enabling characterization of the complex interactions between the cell cycle and wide variety of cellular processes. This method describes the use of mass cytometry for the analysis of cell cycle state for cells from three different sources: in vitro cultured cell lines, ex vivo human blood or bone marrow, and in vivo labeling of murine tissues. The method utilizes incorporation of 5-Iodo-2'-deoxyuridine (IdU), combined with measurement of phosphorylated retinoblastoma protein (pRb), Cyclin B1, and phosphorylated Histone H3 (pHH3). These measurements can be integrated into a gating strategy that enables clear separation of all five phases of the cell cycle.

Osteogenic differentiation of skeletal muscle progenitor cells is activated by the DNA damage response.

Heterotopic ossification (HO) is a pathological condition characterized by the deposition of mineralized tissue in ectopic locations such as the skeletal muscle. The precise cellular origin and molecular mechanisms underlying HO are still debated. In our study we focus on the differentiation of mesoangioblasts (MABs), a population of multipotent skeletal muscle precursors. High-content screening for small molecules that perturb MAB differentiation decisions identified Idoxuridine (IdU), an antiviral and radiotherapy adjuvant, as a molecule that promotes MAB osteogenic differentiation while inhibiting myogenesis. IdU-dependent osteogenesis does not rely on the canonical BMP-2/SMADs osteogenic pathway. At pro-osteogenic conditions IdU induces a mild DNA Damage Response (DDR) that activates ATM and p38 eventually promoting the phosphorylation of the osteogenesis master regulator RUNX2. By interfering with this pathway IdU-induced osteogenesis is severely impaired. Overall, our study suggests that induction of the DDR promotes osteogenesis in muscle resident MABs thereby offering a new mechanism that may be involved in the ectopic deposition of mineralized tissue in the muscle.

Evaluation of combined effect of hyperthermia and ionizing radiation on cytotoxic damages induced by IUdR-loaded PCL-PEG-coated magnetic nanoparticles in spheroid culture of U87MG glioblastoma cell line.

PURPOSE: Glioblastoma multiform (GBM) is the most prevalent and aggressive type of primary brain tumor. None of the current conventional treatment methods has improved treatment considerably. Therefore, in this study the effect of magnetic nanoparticles coated with poly (caprolactone)-poly (ethylene glycol) (PCL-PEG) as a 5-iodo 2'deoxyuridine (IUdR) carrier in the presence of hyperthermia and 6 MV (megavoltage) X-ray radiation, were investigated in a spheroid model of U87MG glioblastoma cell line using colony formation assay. MATERIALS AND METHODS: First, the human glioblastoma cell line U87MG was cultured as a spheroid using the liquid overlay technique. Spheroids on day 10 with 100 mm diameters were treated with 1 µM IUdR or nanoparticles as IUdR carriers for one volume doubling time (VDT) of spheroids (67 h) and hyperthermia at 43 °C for 1 h, and then irradiated with 2 Gy of 6 MV X-ray in different groups. Finally, the effect of treatment on colony-forming ability was obtained by colony formation and alkaline assay. RESULTS: Our results revealed that hyperthermia in combination with radiation could significantly reduce the colony number of glioblastoma spheroid cells treated with IUdR or nanoparticles as IUdR carriers. However, the extent of reduction in colony number following treatment with IUdR-loaded nanoparticles in combination with hyperthermia and then X-ray radiation was significantly more than free IUdR. CONCLUSION: According to this study, PCL-PEG-coated magnetic nanoparticles are effective delivery vehicles for IUdR into cells. Moreover, they can act as a radiosensitizer and thermosensitizer in the treatment of the glioblastoma cell line.

Comparative gene expression analysis after exposure to 123I-iododeoxyuridine, γ- and α-radiation-potential biomarkers for the discrimination of radiation qualities.

Gene expression analysis was carried out in Jurkat cells in order to identify candidate genes showing significant gene expression alterations allowing robust discrimination of the Auger emitter 123I, incorporated into the DNA as 123I-iododeoxyuridine (123IUdR), from α- and γ-radiation. The γ-H2AX foci assay was used to determine equi-effect doses or activity, and gene expression analysis was carried out at similar levels of foci induction. Comparative gene expression analysis was performed employing whole human genome DNA microarrays. Candidate genes had to show significant expression changes and no altered gene regulation or opposite regulation after exposure to the radiation quality to be compared. The gene expression of all candidate genes was validated by quantitative real-time PCR. The functional categorization of significantly deregulated genes revealed that chromatin organization and apoptosis were generally affected. After exposure to 123IUdR, α-particles and γ-rays, at equi-effect doses/activity, 155, 316 and 982 genes were exclusively regulated, respectively. Applying the stringent requirements for candidate genes, four (PPP1R14C, TNFAIP8L1, DNAJC1 and PRTFDC1), one (KLF10) and one (TNFAIP8L1) gene(s) were identified, respectively allowing reliable discrimination between γ- and 123IUdR exposure, γ- and α-radiation, and α- and 123IUdR exposure, respectively. The Auger emitter 123I induced specific gene expression patterns in Jurkat cells when compared with γ- and α-irradiation, suggesting a unique cellular response after 123IUdR exposure. Gene expression analysis might be an effective tool for identifying biomarkers for discriminating different radiation qualities and, furthermore, might help to explain the varying biological effectiveness at the mechanistic level.

Milker's nodule: an occupational infection and threat to the immunocompromised.

Milker's nodule virus, also called paravaccinia virus, is a DNA virus of the parapoxvirus genus transmitted from infected cows to humans. It results from contact with cattle, cattle by-products or fomites. Classified as an occupational disorder, those at risk of exposure include farmers, butchers and agricultural tourists. The viral infection begins 5-15 days after inoculation as an erythematous-purple, round nodule with a clear depressed centre and a surrounding erythematous ring. While familiar to those in farming communities, the presence of the nodule may be concerning to others, particularly the immunosuppressed. Milker's nodules are self-limited in immunocompetent individuals and heal without scarring within 8 weeks. Another member of the Parapoxvirus genus, the orf virus, is also transmitted from animals to humans by direct contact. While complications are rare, haematopoietic stem cell transplant recipients are at risk of graft-versus-host disease, as the parapoxvirus may trigger these complications in immunocompromised individuals. In addition, paravaccinia may serve as the antigen source for the development of erythema multiforme. The unique structure and replication process of viruses in the Poxvirus family, while includes the Parapoxvirus genus, have been a focus for treatment of infections and cancer. Manipulation of these viruses has demonstrated promising therapeutic possibilities as vectors for vaccines and oncologic therapy.

Evaluation of the combined effect of NIR laser and ionizing radiation on cellular damages induced by IUdR-loaded PLGA-coated Nano-graphene oxide.

Glioma is one of the most common malignant cancers of the central nervous system (CNS). Radiatherapy and chemotherapy may be used to slow the growth of tumors that cannot be removed with surgery. The current study developed a combination therapy tool using Nanographene oxide (NGO) functionalized with poly lactic-co-glycolic acid (PLGA) as a carrier of 5-iodo-2-deoxyuridine (IUdR) for glioma cancer treatment. U87MG cells were treated in different groups with IUdR, PLGA-coated Nanographene oxide (PLGA-NGO), IUdR-loaded PLGA-coated Nanographene oxide (IUdR-PLGA-NGO), 2Gy 6MV X-ray radiation, and near-infrared region (NIR) laser radiation. PLGA-NGO showed excellent biocompatibility, high storage capacity for IUdR and high photothermal conversion efficiency. It was effectively employed to create cell damage in the U87MG cell line in the presence of X-ray (6 MV) and NIR laser. Moreover, IUdR-PLGA-NGO+X-ray+NIR laser significantly reduced the plating efficiency of the cells in comparison with IUdR-PLGA-NGO+X-ray and IUdR-PLGA-NGO+NIR laser. Furthermore, Prussian blue staining showed that IUdR-PLGA-NGO-SPIONs were delivered into glioblastoma cells. The PLGA-NGO loaded with IUdR under NIR and X-ray radiation exhibited the highest cytotoxicity toward U87MG cells when compared with other treatment methods, indicating efficient radio-photothermal targeted therapy.

Cell Cycle Analysis by Mass Cytometry.

The regulated progression of cells through the cell cycle during proliferation is a critical factor in tumor progression, anti-neoplastic therapy response, immune system regulation, and developmental biology. While flow cytometric measurement of cell cycle progression is well established, mass cytometry assays allow the cell cycle to be measured along with up to 39 other antigens enabling characterization of the complex interactions between the cell cycle and a wide variety of cellular processes. This method describes the use of mass cytometry for the analysis of cell cycle state for cells from three different sources: in vitro cultured cell lines, ex vivo human blood or bone marrow, and in vivo labeling and ex vivo analysis of murine tissues. The method utilizes incorporation of 5-Iodo-2'-deoxyuridine (IdU), combined with measurement of phosphorylated retinoblastoma protein (pRb), cyclin B1, and phosphorylated histone H3 (p-HH3). These measurements can be integrated into a gating strategy that allows for clear separation of all five phases of the cell cycle.

Enhancement of IUdR Radiosensitization by Low-Energy Photons Results from Increased and Persistent DNA Damage.

Low-energy X-rays induce Auger cascades by photoelectric absorption in iodine present in the DNA of cells labeled with 5-iodo-2'-deoxyuridine (IUdR). This photoactivation therapy results in enhanced cellular sensitivity to radiation which reaches its maximum with 50 keV photons. Synchrotron core facilities are the only way to generate such monochromatic beams. However, these structures are not adapted for the routine treatment of patients. In this study, we generated two beams emitting photon energy means of 42 and 50 keV respectively, from a conventional 225 kV X-ray source. Viability assays performed after pre-exposure to 10 µM of IUdR for 48h suggest that complex lethal damage is generated after low energy photons irradiation compared to 137Cs irradiation (662KeV). To further decipher the molecular mechanisms leading to IUdR-mediated radiosensitization, we analyzed the content of DNA damage-induced foci in two glioblastoma cell lines and showed that the decrease in survival under these conditions was correlated with an increase in the content of DNA damage-induced foci in cell lines. Moreover, the follow-up of repair kinetics of the induced double-strand breaks showed the maximum delay in cells labeled with IUdR and exposed to X-ray irradiation. Thus, there appears to be a direct relationship between the reduction of radiation survival parameters and the production of DNA damage with impaired repair of these breaks. These results further support the clinical potential use of a halogenated pyrimidine analog combined with low-energy X-ray therapy.

DNA Fiber Spreading Assay to Test HDACi Effects on DNA and Its Replication.

DNA fiber spreading assay is an invaluable technique to visualize and follow the spatial and temporal progress of individual DNA replication forks. It provides information on the DNA replication progress and its regulation under normal conditions as well as on replication stress induced by environmental genotoxic agents or cancer drugs. The method relies on the detection of incorporated thymidine analogues during DNA synthesis in the S phase of the cell cycle by indirect immunofluorescence. Here, we describe the procedure established in our laboratories for sequential pulse labeling of human cells with 5-chloro-2'-deoxyuridine (CldU) and 5-iodo-2'-deoxyuridine (IdU), cell lysis, and DNA fiber spreading on slides and sequential immunodetection of the incorporated thymidine analogues by primary antibodies recognizing specifically CldU or IdU alone. We describe also the laser scanning imaging, classification, and measurement of the detected DNA fiber tracks. The obtained quantitative data can be evaluated statistically to reveal the immediate or long-term effects of DNA-damaging agents, DNA repair inhibitors, and epigenetic modulators like HDAC inhibitors on DNA replication in normal and tumor cells.

Highly Effective Auger-Electron Therapy in an Orthotopic Glioblastoma Xenograft Model using Convection-Enhanced Delivery.

Glioblastoma, the most common and malignant primary brain tumor, always recurs after standard treatment. Therefore, promising new therapeutic approaches are needed. Short-range Auger-electron-emitters carry the ability of causing highly damaging radiation effects in cells. The aim of this study was to test the effect of [125I]5-Iodo-2'-deoxyuridine (125I-UdR, a radioactive Auger-electron-emitting thymidine analogue) Auger-therapy on immature glioblastoma spheroid cultures and orthotopic xenografted glioblastoma-bearing rats, the latter by means of convection-enhanced delivery (CED). Moreover, we aimed to determine if the therapeutic effect could be enhanced when combining 125I-UdR therapy with the currently used first-line chemotherapeutic agent temozolomide. 125I-UdR significantly decreased glioblastoma cell viability and migration in vitro and the cell viability was further decreased by co-treatment with methotrexate and/or temozolomide. Intratumoral CED of methotrexate and 125I-UdR with and without concomitant systemic temozolomide chemotherapy significantly reduced the tumor burden in orthotopically xenografted glioblastoma-bearing nude rats. Thus, 100% (8/8) of the animals survived the entire observation period of 180 days when subjected to the combined Auger-chemotherapy while 57% (4/7) survived after the Auger-therapy alone. No animals (0/8) treated with temozolomide alone survived longer than 50 days. Blood samples and post-mortem histology showed no signs of dose-limiting adverse effects. In conclusion, the multidrug approach consisting of CED of methotrexate and 125I-UdR with concomitant systemic temozolomide was safe and very effective leading to 100% survival in an orthotopic xenograft glioblastoma model. Therefore, this therapeutic strategy may be a promising option for future glioblastoma therapy.

Radiation damage to single stranded oligonucleotide trimers labelled with 5-iodopyrimidines.

The radiolysis of deoxygenated aqueous solution containing trimeric oligonucleotides labelled with iodinated pyrimidines and Tris-HCl as the hydroxyl radical scavenger leads to electron attachment to the halogenated bases that mainly results in single strand breaks. The iodinated trimers are 2-fold more sensitive to solvated electrons than the brominated oligonucleotides, which is explained by the barrier-free dissociation of the iodinated base anions. The present study fills the literature gap concerning the chemistry triggered by ionizing radiation in the iodinated pyrimidines incorporated into DNA.

Melanoma-targeted delivery system (part 2): Synthesis, radioiodination and biological evaluation in B16F0 bearing mice.

Here we report the synthesis and radiolabelling with iodine-125 of a melanoma-selective prodrug (17a*) and its parent drug IUdR. The in vivo and ex vivo biodistributions of [(125)I](17a*) and [(125)I]IUdR were evaluated in a model of melanoma B16F0-bearing mice. The pharmacokinetic profile of [(125)I](17a*) suggests rapid release of the active drug [(125)I]IUdR after i.v. administration of [(125)I](17a*). Preliminary metabolism studies in dedicated compartments (i.e. blood, urine and tumour) yielded results consistent with this hypothesis.

Effects of resveratrol and methoxyamine on the radiosensitivity of iododeoxyuridine in U87MG glioblastoma cell line.

The purpose of this study was to evaluate the combination effect of resveratrol and methoxyamine on radiosensitivity of iododeoxyuridine in spheroid culture of U87MG glioblastoma cell line using colony formation and alkaline comet assays. Spheroids on day-20 with 350 µm diameters were treated with 20 µM resveratrol and/or 6 mM methoxyamine and/or 1 µM iododeoxyuridine for one volume doubling time (67 h), and then irradiated with 2 Gy gamma-radiation ((60)Co) in different groups. After treatment, viability of the cells, colony forming ability and DNA damages were obtained by blue dye exclusion, colony formation and alkaline comet assay, respectively. Our results showed that methoxyamine and resveratrol could significantly reduce colony number and induce the DNA damages of glioblastoma spheroid cells treated with iododeoxyuridine in combination with gamma-rays. Therefore, methoxyamine as base excision repair inhibitor and resveratrol as hypoxia inducible factor 1-alpha inhibitor in combination with iododeoxyuridine as radiosensitizer enhanced the radiosensitization of glioblastoma spheroid cells.

Melanoma-targeted delivery system (part 1): design, synthesis and evaluation of releasable disulfide drug by glutathione.

Here we describe the design and synthesis of a prodrug developed for pigmented melanoma therapy, consisting of a Melanin-Targeting Probe (MTP) conjugated to 5-iodo-2'-deoxyuridine (IUdR) with a reduction-sensitive pre-determined breaking point. Compared with the non-cleavable conjugate (17b), prodrug (17a) bearing a self-immolative disulfide linker achieved complete release of IUdR within 20 min in the presence of reducing agents such as DTT or glutathione. Analytical results also showed that prodrug (17a) was more sensitive than parent non-cleavable conjugate (17b) for a concentration range of glutathione similar to that found in the intracellular compartment of tumours.

A systematic review and meta-analysis to compare the efficacy of acyclovir 3% ophthalmic ointment to idoxuridine in curing herpetic keratitis by Day 7 of treatment.

BACKGROUND: This objective of the review and analysis is to demonstrate that acyclovir (ACV) 3% ophthalmic ointment is superior to idoxuridine (IDU) in treating herpetic keratitis (HK) presenting as dendritic and geographic ulcer sub-types. DATA SOURCES: Publications in human subjects were identified by searching the Ovid MEDLINE database through April 2011, combining medical subject headings (MESH) "Keratitis, Herpetic/" AND "Acyclovir/" limiting by the key words "topical" OR "ointment" and also restricted to MESH "Administration, Topical/" OR "Ointments/". The results were cross checked with the references used in the Cochrane Database Syst Rev. 1:1-134, 2009 and GlaxoSmithKline clinical documents related to acyclovir. STUDY SELECTION: Randomized, double-masked studies in subjects diagnosed with HK with head to head comparator arms of ACV ophthalmic ointment and topical IDU that had actual or calculable healing rates at Day seven. DATA EXTRACTION: Data independently extracted from identified articles by two authors of this manuscript. DATA SYNTHESIS: Data from seven randomized, controlled trials (RCT) evaluating 432 subjects that met inclusion criteria (214 were treated with ACV and 218 were treated with IDU) and had Day seven healing rates calculable. All sub-classified lesions were identified as either dendritic ulcers (n = 185) or geographic ulcers (n = 35). The Cochran-Mantel-Haenszel (CMH) method in Biometrics 10:417-51, 1954 and JNCI 22:719-48, 1959, controlling for study, was performed as the primary analysis using SAS v9. Homogeneity was assessed using Breslow-Day-Tarone (BDT) test in IARC 1:1-32, 1980 and Biometrika 72:91-5, 1985. The analysis was performed with outliers removed to assess their impact. RESULTS: ACV showed statistically significant greater odds of healing HK at Day seven in all subjects (Odds Ratio 3.95, 95% CI2.60, 6.00, p < 0.0001), in dendritic ulcers (Odds Ratio 4.22, 95% CI: 2.14, 8.32; p < 0.0001) and geographic ulcers (Odds Ratio 5.31, 95% CI: 1.09, 25.93; p = 0.0244). CONCLUSION: ACV 3% ophthalmic ointment is a valuable intervention for dendritic and geographic corneal ulcers. ACV and IDU were generally well tolerated in the studies reviewed.