Doses Of Ganciclovir Research Articles

Antiviral prevention of sepsis induced cytomegalovirus reactivation in immunocompetent mice

Introduction Immunocompetent patients can reactivate latent cytomegalovirus (CMV) during critical illness and reactivation is associated with significantly worse outcomes. Prior to clinical trials in humans to prove causality, we sought to determine an optimal antiviral treatment strategy. Methods Mice latently infected with murine CMV (MCMV) received a septic reactivation trigger and were randomized to receive one of four ganciclovir regimens or saline. Lungs were evaluated for viral transcriptional reactivation and fibrosis after each regimen. Influences of ganciclovir on early sepsis-induced pulmonary inflammation and T-cell activation were studied after sepsis induction. Results All ganciclovir regimens reduced measurable MCMV transcriptional reactivation, and 10 mg/day for 7 or 21 days was most effective. Lower dose (5 mg/kg/day) or delayed therapy was associated with significant breakthrough reactivation. Higher doses of ganciclovir given early were associated with the lowest incidence of pulmonary fibrosis, and delay of therapy for 1 week was associated with significantly worse pulmonary fibrosis. Although bacterial sepsis induced activation of MCMV-specific pulmonary T-cells, this activation was not influenced by ganciclovir. Conclusion These results suggest that antiviral treatment trials in humans should use 10 mg/kg/day ganciclovir administered as early as possible in at-risk patients to minimize reactivation events and associated pulmonary injury.

Inhibition of B Virus (Macacine herpesvirus1) by Conventional and Experimental Antiviral Compounds

B virus infection of humans results in high morbidity and mortality in as many as 80% of identified cases. The main objective of this study was to conduct a comparative analysis of conventional and experimental antiviral drug susceptibilities of B virus isolates from multiple macaque species and zoonotically infected humans. We used a plaque reduction assay to establish the effective inhibitory doses of acyclovir, ganciclovir, and vidarabine, as well as those of a group of experimental nucleoside analogs with known anti-herpes simplex virus activity. Four of the experimental drugs tested were 10- to 100-fold more potent inhibitors of B virus replication than conventional antiviral agents. Drug efficacies were similar for multiple B virus isolates tested, with variations within 2-fold of the median effective concentration (EC(50)) for each drug, and each EC(50) was considerably lower than those for B virus thymidine kinase (TK) mutants. We observed no differences in the viral TK amino acid sequence between B virus isolates from rhesus monkeys and those from human zoonoses. Differences in the TK protein sequence between cynomolgus and pigtail macaque B virus isolates did not affect drug sensitivity except in the case of one compound. Taken together, these data suggest that future B virus zoonoses will respond consistently to conventional antiviral treatment. Further, the considerably higher potency of FEAU (2'-fluoro-5-ethyl-Ara-U) than of conventional antiviral drugs argues for its compassionate use in advanced human B virus infections.

Fusion enzymes containing HSV-1 thymidine kinase mutants and guanylate kinase enhance prodrug sensitivity in vitro and in vivo

Herpes Simplex Virus thymidine kinase (HSVTK) with ganciclovir (GCV) is currently the most widely used suicide gene/prodrug system in cancer gene therapy. A major limitation in this therapy is the inefficient activation of GCV by HSVTK to its active antimetabolites. We previously described two strategies to overcome this limitation: 1) generation of HSVTK mutants with improved GCV activation potential and 2) construction of a fusion protein encoding HSVTK and mouse guanylate kinase (MGMK), the second enzyme in the GCV activation pathway. As a means to further enhance GCV activation, two MGMK/HSVTK constructs containing the HSVTK mutants (mutant 30 and SR39) were generated and evaluated for their tumor and bystander killing effects in vitro and in vivo. One fusion mutant, MGMK/30, demonstrates significant reduction in IC50 values of approximately 12,500-fold, 100-fold, and 125-fold compared to HSVTK, mutant 30 or MGMK/HSVTK, respectively. In vitro bystander analyses reveal that 5% of MGMK/30-expressing cells are sufficient to induce 75% of tumor cell killing. In an xenograft tumor model, MGMK/30 displays the greatest inhibition of tumor growth at a GCV concentration (1mg/kg) that has no effect on wild type HSVTK-, MGMK/HSVTK-, or mutant 30-transfected cells. Another fusion construct, MGMK/SR39, sensitizes rat C6 glioma cells to GCV by 2,500-fold or 25-fold compared to HSVTK or MGMK/HSVTK, respectively. In vitro analyses show similar IC50 values between cells harboring SR39 and MGMK/SR39, although MGMK/SR39 appears to elicit stronger bystander killing effects where 1% of MGMK/SR39-transfected cells result in 60% cell death. In a xenograft tumor model, despite observable tumor growth inhibition, no statistical significance in tumor volume was detected between mice harboring SR39- and MGMK/SR39-transfected cells when dosed with 1mg/kg GCV. However, at a lower dose of GCV (0.1mg/kg), MGMK/SR39 appears to have slightly greater tumor growth inhibition properties compared to SR39 (P≤0.05). In vivo studies indicate that both mutant fusion proteins display substantial improvements in bystander killing in the presence of 1mg/kg GCV, even when only 5% of the tumor cells are transfected. Such fusion mutants with exceptional prodrug converting properties will allow administration of lower and non-myelosuppressive doses of GCV concomitant with improved tumor killing and as such are promising candidates for translational gene therapy studies.

Hematologic complications of anti‐CMV therapy in solid organ transplant recipients

Cytomegalovirus (CMV) infection complicates the post-operative course of patients receiving solid organ transplants. While ganciclovir has significantly reduced the direct effects of CMV infection, some patients cannot tolerate the optimal therapeutic exposure required for CMV prevention and treatment. Few reports directly address the incidence, consequences, and risk factors for hematologic toxicities related to ganciclovir therapy. Nevertheless, leukopenia, thrombocytopenia, and anemia occur in 5-50% of patients. Current strategies, focused on ganciclovir dose reduction, may increase the risk of CMV reactivation and drug-resistant disease. The current article reviews the incidence, risk factors, and consequences of ganciclovir-associated hematologic adverse events in transplant recipients. Management strategies, including ganciclovir dose reduction, and the addition of CMV hyperimmune globulin are discussed. Exposing this relatively frequently occurring, but uncommonly discussed, toxicity should lead to better avoidance and treatment strategies, without placing patients at increased risk of CMV disease.

Use of Valganciclovir for Prevention and Treatment of Cytomegalovirus Disease

The development of a valine ester for ganciclovir has allowed much-improved absorption and bioavailability of oral ganciclovir and has had a significant impact on the prevention and management of cytomegalovirus infection in the immunocompromised host. Valganciclovir offers ~60% bioavailability after oral ingestion. The administration of 900 mg of valganciclovir orally is comparable to a dose of intravenous ganciclovir of 5 mg/kg, although the peak level of ganciclovir is higher than that of oral valganciclovir [ 1 ] . Given this comparability, the use of valganciclovir for preemptive therapy and even for treatment of cytomegalovirus is becoming quite common, even with limited comparative trial data [2]. The article by Len et al [3] from the Spanish Network for Research on Infection in Transplantation (RESITRA) in this issue of Clinical Infectious Diseases reports an observational study that offers some insight into the efficacy of valganciclovir for the prevention and treatment of cytomegalovirus disease. Although this study d sign is not as rigorous as a randomized trial, and although it was subject to some investigator and clinician bias in patient selection, the data are nevertheless compelli g and demonstrate that valganciclovir can be used for preemptive therapy for cy omegalovirus infection in organ transplant recipients [3]. The definitions of success and failure in this study are rigorous, and the number of patients studied was large. Once viral replication was established, it is quite clear that valganciclovir is effective for viral suppression, to prevent progression to cytomegalovirus syndrome or organ disease. The vast majority of the data in this study derive from kidney and liver transplant recipients (and, to a lesser extent, from heart transplant patients); therefore, generalizability of the results to lung or kidney-pancreas transplant recipients is probably not justified. Furthermore, the data provide a limited experience regarding the cytomegalovirus antibody-negative recipients of transplants from antibody-positive donors, who represent the highestrisk group for late-onset and relapse. Thus, additional studies are required for these groups to provide information for such use. However, I think transplant programs can feel confident that the use of valganciclovir for preemptive therapy has merit for the vast majority of organ transplant recipients. In many programs, it is already the standard of care for either prophylaxis or preemptive therapy. Although the US Food and Drug Administration has cautioned against the use of valganciclovir for prevention of cytomegalovirus infection in liver transplant recipients [4], the data here support its use and also are consistent with what many liver transplantation programs are actually using as their protocol. The data from this Spanish group are not as compelling regarding the use of valganciclovir for treatment of invasive cytomegalovirus disease, which they label disease in tables 2 and 3 of their article [3]. The success rate of 75% is less than the success rate for intravenous ganciclovir (87.5%) or intravenous ganciclovir followed by valganciclovir (96.2%). Although the difference was not statistically significant, only 8 patients with focal were treated exclusively with valganciclovir. Although there were only 2 treatment failures, I believe that more experience will be necessary to conclude that valganciclovir therapy is appropriate initial management of tissue invasive disease. The authors do not indicate the lo-

Developing In Vivo Model for Studying Mechanisms of Osteoblast Suppression in Multiple Myeloma.

Multiple myeloma (MM) is an incurable neoplastic disease characterized by an accumulation of plasma cells in bone marrow. Osteolytic bone lesions are the major source of morbidity in MM patients and are associated with bone pain and fractures and hypercalcemia. The bone lesions result from increased osteoclastic bone destruction in areas adjacent to the myeloma cells. New bone formation that normally happens at sites of previous bone resorption still occurs in early stages of the disease but is absent in advanced MM. Although the molecular basis for the increased osteoclastic activity has been intensely investigated, the basis for the decreased osteoblast activity is just beginning to be understood. Recently, inhibitors of WNT signaling pathway, Dickkorpf1 (DKK1) and secreted Frizzle-Related Protein-2 (sFRP2) have been identified as factors involved in osteoblast suppression in MM. In addition, IL-3 and IL-7 are increased in plasma of MM patients and suppress osteoblastogenesis in cell culture models. However, the role of those factors in the osteoblastic activity in MM patients is unclear. Studies in patients are confounded by cytotoxic therapy as well as bisphosphonates, which are standard therapy for MM patients. Therefore, preclinical in vivo models are required to delineate the mechanisms responsible for the profound osteoblast suppression in MM. We have developed a mouse model of myeloma bone disease in which genetically modified myeloma cells can be selectively ablated without the confounding effects of cytotoxic therapies and allows us to tract the growth of MM cells. The 5TGM1 cell line which is the most common version of murine MM, was stably transfected with the thymidine kinase (TK) gene from herpes simplex virus, which permits eradication of myeloma cells with ganciclovir, as well as GFP and luciferase genes to detect the presence of MM cells. One ug/ml ganciclovir treatment in culture results in 100% death of the transfected 5TGM1 cells in 4 days. Importantly, ganciclovir treatment of primary marrow cell cultures had no effect on growth and differentiation of osteoblast and hematopoietic progentitors. Co-culturing of primary marrow cells with 5TGM1 expressing TK has no bystander effect on osteoblast differentiation with ganciclovir treatment. Subcutaneously implanted 5TGM1 cells into SCID mice were eradicated by intraperitoneal injection of 20mg/kg ganciclovir/d for 2 weeks. The dose of ganciclovir did not affect osteoblast differentiation of primary marrow culture from the mice treated with ganciclovir. Then we injected the 5TGM1 cells into tibia of SCID nude mice (n=4 per group). After measuring the increase of serum IgG2b level, half of the mice were treated with ganciclovir for 2 weeks and the other with saline. Our preliminary data show that osteogenic cultures of bone marrow from the ganciclovir treated mice had significantly higher alkaline phosphatase activity than cultures derived from the saline treated group (p=0.03). In addition, the ganciclovir treated mice had tendency of higher trabecular bone volume than the saline-treated group (p=0.08). These results demonstrate that this model should be useful for studying mechanisms of osteoblast suppression in MM.

Effective Ablation of Pancreatic Cancer Cells in SCID Mice Using Systemic Adenoviral RIP-TK/GCV Gene Therapy

Studies have demonstrated that adenovirus subtype 5 mediated rat insulin promoter directed thymidine kinase (A-5-RIP-TK)/ganciclovir (GCV) gene therapy resulted in significant enhanced cytotoxicity to both PANC-1 and MIA PaCa2 pancreatic cancer cells in vitro. However, little is known about the effect in vivo. In this study we examine the in vivo safety and efficacy of intravenous A-5-RIP-TK/GCV gene therapy. 1 x 10(6) Mia PaCa2 cells were injected intraperitoneally (i.p.) into SCID mice to create a mouse model of human pancreatic cancer. A-5-RIP-TK gene construct was administered intravenously (i.v.), followed by i.p. GCV administration. Intravenous injection of A-5-RIP-lacZ reporter gene constructs was used for evaluation of Ad-RIP-gene expression in tumors and other tissues. Optimal adenoviral and GCV doses and treatment duration were determined. Tumor volume, serum insulin, and glucose levels were measured. Immunohistochemical staining of pancreata and tumors were performed to assess morphology and hormone expression and apoptotic rates were determined. All A-5-RIP-TK/GCV-treated mice had reduced tumor volume compared with controls, but maximal tumor volume reduction was observed with 10(8) vp followed by GCV treatment for 4 wk. A-5-RIP-TK/GCV gene therapy contributed to significant survival advantage in MIA PaCa2 bearing mice, and the greatest survival benefit was observed with 10(8) vp and was not affected by length of treatment of GCV. A-5-RIP-TK/GCV therapy increased PDX-1 expression and tumor cells apoptosis, and altered islet morphology. However, A-5-RIP-TK/GCV gene therapy caused diabetes associated with islet cell apoptosis, increased delta-cells and reduced pancreatic polypeptide (PP)-cell numbers. Systemically administered A-5-RIP-TK/GCV is an effective treatment of pancreatic cancer. A-5-RIP-TK/GCV cytotoxicity to malignant cells varies with adenoviral dose and length of GCV treatment. However, A-5-RIP-TK/GCV is associated with islet cell toxicity and diabetogenesis. The type of diabetes observed is distinct from Types 1 and 2 and is associated with islet cell apoptosis and reduced delta- and PP-cells.

Control of graft-versus-host disease with maintenance of the graft-versus-leukemia effect in a murine allogeneic transplant model using retrovirally transduced murine suicidal lymphocytes

Limited clinical trials have validated the hypothesis of controlling graft-versus-host disease (GVHD) arising from stem cell transplant utilizing suicidal T-lymphocytes that have been transduced to express the HSV-TK gene. However, clinical utility has been limited by diminished T-cell function arising from the production process. To evaluate strategies for harnessing the graft-versus-leukemia (GVL) effect while improving the safety and function of suicidal lymphocytes, we have developed techniques to produce fully functional, retrovirally transduced, HSV-TK-positive murine T cells (TK+TC). Utilizing a murine major histocompatibility complex-matched transplant model, we evaluated the ability of TK+TC to generate a GVL effect and the ability to control GVHD in experiments where we varied the dose of TK+TC, ganciclovir (GCV) dose, the start of GCV administration (day 4, 7, 10, 13, 15, or 19) posttransplantation, and the GCV administration route (osmotic pump versus intraperitoneal). At TK+TC doses in excess of the standard lethal dose (SLD) of unmanipulated T-cells, GCV administration completely (2 x SLD) and partially (4 x SLD) controlled GVHD. Additionally, GVHD remained reversible despite delaying administration of GCV for a week after GVHD developed. Importantly, GVHD was controlled with a 1-log but not 2-log reduction in GCV dose, and this "partial suicide" preserved more circulating TK+TC compared with standard-dose GCV. Survival of leukemia-positive mice receiving TK+TC and GCV was significantly increased compared with control cohorts not receiving GCV or transplanted with unmanipulated T cells, thereby demonstrating a GVL effect. Retrovirally transduced suicidal lymphocytes generate a potent GVL effect while simultaneously enabling control of GVHD, which results in improved leukemia and GVHD-free survival.

Exogenous wt-p53 enhances the antitumor effect of HSV-TK/GCV on C6 glioma cells

To study on the antitumor effect of combining wt-p53 gene with suicide gene therapy (HSV-tk+GCV) for malignant gliomas. AdCMV-p53 was transfected into C6 glioma cells at MOI of (Multiplicity of infection) 0(G100), 10(TPG1), 100(TPG2), then AdCMV-tk was transducted to C6 glioma cells of G100, TPG1 and TPG2, respectively, at MOI of 100. The C6 glioma cells tranfected with both AdCMV-p53 and AdCMV-tk were exposed to various concentration of GCV. The cell survival rate was measured by MTT assay in vitro. Rat glioma model was established by injecting 5 x 10(5) C6 glioma cells into right caudate nucleus of SD rats. AdCMV-p53 and AdCMV-tk were injected into glioma on day 5 and 6, respectively. On day 7, ganciclovir (GCV) was administrated intraperitoneally at 15 mg/kg/day for 14 days. The survival time of all rats was observed. The growth of intracerebral tumors was monitored dynamically by enhanced MRI. Cell apoptosis was evaluated by TUNEL method. Expression of HSV-tk gene was identified by in situ hybridization and expression of exogenous p53 gene was detected with Western blotting. In vitro, wt-p53 significantly enhanced antitumor effect of HSV-tk/GCV. The concentration of GCV for ID50 of TPG2 cells (0.001 microg/ml GCV) was 10 times lower than that for the cells of tk-GCV group (MOI = 100), while the concentration of GCV for ID100 of TPG2 (0.01 microg/ml GCV) and TPG1(0.1 microg/ml GCV) was 100 and 10 times lower than that for the cells of tk-GCV group (MOI = 100), respectively. Apoptosis of C6 glioma cells also could be induced by transfection with wt-p53 gene slightly. For in vivo study, the survival time of tumor-bearing rats treated with HSV-TK/GCV or wt-p53 combined with HSV-TK/GCV was significantly prolonged and the intracerebral tumors were regressed and disappeared earlier in the combined gene therapy group than those in the HSV-TK/GCV therapy group as shown in enhanced MRI. However, only half dose of GCV for the rats treated with both wt-p53 and HSV-TK/GCV was needed to obtain the same efficacy as those rats treated with HSV-TK/GCV alone. These results indicate that the transfection of wt-p53 potentiates the effect of HSV-TK/GCV therapy. The combination of HSV-tk/GCV system with wt-p53 gene transduction is optimal for clinical therapeutic trials of suicide gene therapy for malignant gliomas.

Human herpes virus-6 encephalitis in a paediatric bone marrow recipient: successful treatment with pharmacokinetic monitoring and high doses of ganciclovir

Human herpes virus-6 encephalitis in a paediatric bone marrow recipient: successful treatment with pharmacokinetic monitoring and high doses of ganciclovir

A guanylate kinase/HSV-1 thymidine kinase fusion protein enhances prodrug-mediated cell killing

Herpes simplex virus thymidine kinase (HSVTK) with the guanosine analog ganciclovir (GCV) is currently the most widely used suicide gene/prodrug system for gene therapy of cancer. Despite the broad application of the HSVTK/GCV approach, phosphorylation of GCV to its active state is inefficient such that high, myelosuppressive doses of GCV are needed to observe an antitumor effect. One strategy used to overcome the poor substrate specificity of HSVTK towards GCV (Km = 45 microM) has been to create novel forms of TK with altered substrate preferences. Such mutant TKs have shown benefit and are currently in clinical use. We describe here a second strategy to increase the amount of intracellular triphosphorylated GCV by involving the second enzyme in the GCV activation pathway, guanylate kinase (GMK). As a means to overcome the bottleneck of prodrug activation from the monophosphate to the diphosphate, we sought to combine both the critical HSVTK and GMK activities together. In this report we describe the construction of a fusion or chimeric protein of HSVTK and guanylate kinase, show data that demonstrate it confers a approximately 175-fold decrease in IC50 compared to HSVTK alone in response to ganciclovir treatment in stably transfected C6 glioma cells and finally, we present biochemical evidence of a kinetic basis for this improved cell killing.

Valganciclovir: What is the Status in Solid Organ Transplantation?

Prophylactic drug therapy for cytomegalovirus disease in solid organ transplant recipients is effective and simple to implement, but it is associated with patient nonadherence and viral resistance. Recent data show that the efficacy and safety of oral ganciclovir and oral valganciclovir are similar. However, three large daily doses of oral ganciclovir are required, which is inconvenient, and viral resistance can develop to the drug. The single daily dose and lack of viral resistance are advantages of valganciclovir. This has become the primary agent for the prevention of cytomegalovirus disease. Current trials are underway to determine its effectiveness for treatment of cytomegalovirus disease, the optimal length of prophylaxis, and the safety and efficacy of a syrup formulation in children.

Question of using valganciclovir for cytomegalovirus (CMV) infection prophylaxis in post–liver transplant recipients

We read the article by Park et al. with great interest.1 The authors retrospectively studied the efficacy of low-dose oral valganciclovir (n = 49) compared with the standard dose of ganciclovir (n = 60) for the prevention of cytomegalovirus (CMV) disease in adult liver transplant recipients. The authors found 2 patients in each group with CMV disease within 1 year of follow-up. Both patients in the ganciclovir group were high risk for CMV (donor positive, recipient negative), while in valganciclovir group, 1 patient was high-risk and 1 was low risk.1 Absorption of valganciclovir has been found to be 10 times higher than oral ganciclovir, with bioavailability of nearly 60% in healthy volunteers and in patients who are human immunodeficiency virus positive with CMV. Based on these findings, we agree that the greater absorption and antiviral activity of valganciclovir offers an attractive option for prophylaxis against CMV after solid-organ transplantation.2-4 However, when we analyzed 203 consecutive post–liver transplant recipients treated with prophylactic valganciclovir between July 2001 and May 2003, we found an overall rate of 17% symptomatic CMV, when either the donor or the recipient was exposed to CMV. It was 25.9% in the high-risk group, (donor positive, recipient negative, based on CMV serology). Some of the patients developed CMV even while on valganciclovir prophylaxis.5, 6 Since our publication, we have screened more high-risk recipients for CMV during and after valganciclovir prophylaxis and have found a higher rate than the rate published earlier. In concurrence with our findings, a double-blind, prospective trial was conducted by Roche laboratories (manufacturers of valganciclovir and ganciclovir) in 372 posttransplantation patients with high risk for CMV disease (heart, n = 56; liver, n = 177; kidney, n = 120; and kidney/pancreas, n = 11) comparing valganciclovir and ganciclovir prophylaxis. The overall results at 6 months suggested that the rate of CMV disease (including invasive CMV) was 12.1% with valganciclovir prophylaxis compared to 15.2% with ganciclovir. However, in liver transplant recipients, the rate of invasive CMV disease was 12% in the valganciclovir group compared to 3% in oral ganciclovir group. Based on these findings, valganciclovir was not recommended for CMV prophylaxis in high-risk liver transplant recipients.7 One fails to understand why liver transplant recipients had higher rates of CMV compared to kidney or heart transplant recipients under valganciclovir prophylaxis. Both mycophenolate mofetil and valganciclovir require esterase to convert them into active forms (mycophenolic acid and ganciclovir, respectively). Post–liver transplant recipients often have initial hepatic and/or bowel dysfunction, and therefore the enzyme may be quantitatively deficient or qualitatively ineffective. Improved absorption of valganciclovir over time has been reported in liver transplant recipients. Also, valganciclovir could be competing with mycophenolate mofetil for esterase to be converted to active ganciclovir. Previous pharmacokinetic studies on valganciclovir were performed 21 to 180 days after liver transplant.2-4 There is a lack of pharmacokinetic data in the early post–transplant period. In the present report, it is possible that the authors might have found a lower rate of CMV because not all patients were screened for CMV disease after discontinuation of prophylaxis. In addition, because of the retrospective nature of the study, some patients might have been missed, as some patients might not have presented to their clinic or might not have been documented adequately. It is particularly important to note that the overall rate of CMV disease was only 4% in the entire group,1 much lower than that observed by others. We feel that the findings of this retrospective study, which indicates that valganciclovir provides adequate prophylaxis for liver transplant recipients, should be treated with caution in clinical practice until more prospective clinical trials are conducted. It is important to have a high degree of suspicion for CMV infection during and, particularly, after discontinuation of valganciclovir prophylaxis. Ashok Jain*, Ravi Mohanka*, Mark Orloff*, Peter Abt*, Charlotte Ryan , Adel Bozorgzadeh*, * Department of Surgery, Division of Transplantation, University of Rochester Medical Center, Rochester, NY, Department of Pathology, University of Rochester Medical Center, Rochester, NY.

Tat8–TK/GCV Suicide Gene Therapy Induces Pancreatic Tumor Regression In Vivo

Suicide gene therapy using the herpes simplex virus thymidine kinase (TK) gene in combination with ganciclovir (GCV) has been shown to produce therapeutic, but limited, efficacy because of poor gene transfer efficiency and reduced bystander effect. Here we report that fusion of TK to an eight-amino acid peptide from the basic domain of the human immunodeficiency virus (HIV) Tat protein significantly increases the cytotoxic efficacy of the TK/GCV system in pancreatic cancer cells. We demonstrate that Tat8-TK protein is released from the intracellular compartment of Tat8-TK-expressing cells to the extracellular medium after GCV treatment. Interestingly, we show that this conditioned medium is then able to mediate cytotoxicity of wildtype cultures, suggesting the internalization of the Tat8-TK protein. Moreover, a strong antitumoral effect of Tat8-TK/GCV treatment could be achieved by two different in vivo approaches. Tumors injected with NIH 3T3/Tat8-TK cells attached to microcarriers (MC+Tat8-TK) and treated with GCV led to a 35.6% reduction in the initial tumor volume and to 50% tumor eradication. Furthermore, electrogene transfer of TK or Tat8-TK followed by administration of high doses of GCV led to an overall statistically significant reduction in tumor growth. However, the reduction in initial tumor volume was statistically significant only for the Tat8-TK group (59.5% reduction). Moreover, in this group 50% complete tumor eradication was achieved. When moderate doses of GCV were administered, the overall reduction in tumor growth was statistically significant only in the Tat8-TK group. Therefore, our results suggest that fusion of TK to the Tat8 peptide enhances TK/GCV suicide gene therapy.

High-dose (5000-μg) Intravitreal Ganciclovir Combined with Highly Active Antiretroviral Therapy for Cytomegalovirus Retinitis in HIV-Infected Patients in Venezuela

To describe the use of high doses of intravitreal ganciclovir combined with highly active antiretroviral therapy (HAART) for the treatment of cytomegalovirus (CMV) retinitis in human immunodeficiency virus (HIV)-infected patients. Thirteen HIV-infected patients (18 eyes) with active CMV retinitis (83.3% in zone 1 and 38.4% resistant) participated in this prospective interventional case series. Patients were treated with high dose intravitreal ganciclovir (5.0 mg/0.1 mL once a week) in combination with HAART therapy. Intravitreal injections were discontinued once CMV retinitis healed if there was a significant increase in CD4+ count (any increase of > or 50 cells/microL to levels over 100 cells/microL sustained for at least 3 months). Mean follow-up was 15.6 months. Main outcome measures included assessment of visual acuity and retinal inflammation (CMV retinitis activity). A matched historical control group of 20 eyes (15 patients) with CMV retinitis treated with systemic ganciclovir (intravenous [induction] and oral [maintenance]) was included. Complete regression of the retinitis was obtained with high doses of intravitreal ganciclovir in 88.8% of eyes (two patients died during follow-up) at a mean of 4.5 weeks (2 to 8 weeks). Visual acuity improved two or more lines in 61.1% of eyes. No ganciclovir retinal toxicity was identified. Three eyes presented CMV retinitis reactivation at a mean of 25.6 days after their last injection. Complications (33.3%) included retinal detachment (RD; 3 eyes), immune recovery uveitis (IRU; 2 eyes), and endophthalmitis (1 eye). In our control group complete regression of the retinitis was obtained in 100% of eyes at a mean of 4 weeks (3 to 7 weeks). However, 12 eyes (60%) presented with CMV retinitis relapse at a mean of 29 days (21 to 32 days) after initiating oral ganciclovir (maintenance). Complications included RD (7 eyes, 35%) and IRU (3 eyes, 15%). Severe neutropenia occurred in 2 patients (13%). High doses of intravitreal ganciclovir (5.0 mg) once a week in combination with HAART therapy is effective to control CMV retinitis, and may be discontinued after CMV retinitis has healed if immune reconstitution with a significant increase in CD4+ count has occurred.

Cytomegalovirus in Renal Transplant Recipients: Our Experience and Review

We have done a retrospective study to see CMV disease in our transplant patients. Seventy two transplants were done during last three and a half years. All were included in the study and those with a suspicion of CMV disease were monitored. Seven patients (9.72%) had CMV disease. All patients presented with fever (100%). Other features included – graft dysfunction (4 patients - 57.1%), leucopoenia (4 patients 57.1%), pneumonia (3 patients - 42.85%), and hepatitis (2 patients - 28.57%). Time of onset in the post transplant period was - first six months - 3 patients (42.85%), 6 months to 2 years-2 patients (28.57%), >2 years - 2 patients (28.57%). In all cases IgM CMV was done as first line test. IgM CMV was positive in 4 out of 7 (57.1%) cases. One patient had a significant elevation in IgG CMV titers (14.28%). Two cases were IgM CMV negative. NASBA was done in those two cases on firm clinical suspicion, which was positive. All cases were started on I/V Ganciclovir according to creatinine clearance. All patients improved except one (14.28%) who died of multiorgan failure. Once the patient was asymptomatic, maintenance dose of ganciclovir was given, i.e., 500 mg TDS for 3 months, though the advised dose for maintenance therapy is 1000 mg TDS. To conclude, we observed the following facts: Pyrexia is the most common presentation (100%). CMV IgM is not 100% sensitive test and NASBA can pick up those cases that are IgM negative. CMV disease can occur at any time post transplant rather than as usual presentation within 6 weeks. So high degree of suspicion is required even late in course. The outcome is better if treatment is started early. Our patients did well on half of the dose for maintenance therapy, which reduces the financial burden on the patients significantly.

Attenuated Preemptive Therapy Against Cytomegalovirus (CMV) Reactivation/Disease Consisting of a Higher Cutoff Point and a Lower Initial Dose of Ganciclovir after Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) with an HLA-Identical Sibling Donor.

CMV continues to be a major cause of morbidity following allogeneic HSCT. Although preemptive ganciclovir therapy is widely used to prevent CMV disease, the optimal initial dose and timing of ganciclovir administration have not yet been determined. Unnecessary routine ganciclovir therapy is not acceptable, since it can cause considerable neutropenia and may impair the de novo recovery of CMV-specific T cell immunity. We performed a prospective study to evaluate the safety and effectiveness of a novel preemptive protocol with a higher cutoff point to initiate therapy and a lower initial dose of ganciclovir. The primary endpoint of this study was defined as the occurrence of CMV disease. CMV antigenemia was measured weekly after engraftment. The decision to start ganciclovir was based on the detection of ≥10 positive cells per 50,000 cells by the antigenemia assay, or by the clinical diagnosis of CMV disease with apparent infection-related symptoms. Intravenous ganciclovir was started at 5 mg/kg/day three times per week, and was continued until the positive cell count had decreased to < 10 per 50,000 cells. If the number of positive cells increased or CMV-related symptoms worsened, the dose of ganciclovir was increased. Forty-six consecutive patients undergoing allogeneic HSCT from an HLA-identical sibling at National Cancer Center Hospital between April 2003 and March 2004 were enrolled. The median age of the patients was 51 years (range, 17 to 68 years). There were 40 cases of hematological malignancy and 6 solid tumor. The risk status of CMV was recipient (R) +/donor (D)+ in 39, R+/D- in 4, R−/D+ in 1, and R−/D− in 2. Fludarabine- or cladribine-based reduced-intensity regimens were used in 35 patients and conventional myeloablative in 11. All patients were given acyclovir 750 mg/day orally from day −7 to the discontinuation of GVHD prophylaxis. The median follow-up was 275 days (26 – 481). Twenty-six patients (57%) developed positive antigenemia at a median of 42 days (12 – 151) after transplantation. In 10 patients (22%), ≥10 positive cells per 50,000 cells were detected by the antigenemia assay, and these patients, except two with renal dysfunction, received low-dose ganciclovir. The dose of ganciclovir was increased in 5 patients. Three patients developed overt CMV gastroenteritis which was successfully treated with a regular therapeutic dose of ganciclovir at 10 mg/kg/day. No case of late CMV disease was observed in this study. Overall, the total dose of ganciclovir was significantly less than that in a previous protocol using a conventional double dose (5 mg/kg twice daily) of ganciclovir (111 mg/kg vs. 225 mg/kg on average, P=0.006), reducing the cost by as much as $ 1,500 per patient. The incidence of neutropenia after ganciclovir was significantly less than that in the previous approach (0% vs. 11% (4/36), P=0.038). In a multivariate analysis, factors associated with ≥10 positive antigenemia included grade II – IV acute GVHD (P < 0.0001), the use of systemic steroid of ≥1.0 mg/kg/day (P < 0.0001), and a reduced- intensity conditioning regimen (P =0.036). In conclusion, our attenuated preemptive treatment protocol appeared to be safe, better tolerated, less expensive, and effective at controlling CMV infection.

The CARI guidelines. CMV disease and kidney transplant: treatment of cytomegalovirus disease in renal transplant recipients.

The CARI guidelines. CMV disease and kidney transplant: treatment of cytomegalovirus disease in renal transplant recipients.

Early immune reconstitution and abrogation of GvHD after infusion of HSV-TK engineered donor lymphocytes after haplo-identical hemopoietic stem cell transplantation

6515 Background: Haplo-identical stem cell transplantation (haplo-SCT) is a therapeutic option for patients with high risk hematologic malignancies lacking an HLA matched donor. The full exploitation of haplo-SCT is limited by delayed immune recovery, resulting in prolonged risk of life-threatening infections. Infusion of small numbers of donor T cells, aimed at providing immune reconstitution, results in severe forms of graft-versus-host-disease (GvHD). We previously showed that infusion of donor lymphocytes (DLI) expressing the herpes simplex virus thymidine kinase (Tk) is an efficient tool for controlling GvHD and preserving anti-tumor activity. Methods: We designed a dose-finding clinical protocol of Tk-DLI for immune reconstitution and relapse prevention after haplo-SCT. Eight pts with high risk hematologic malignancies who underwent haplo-SCT received escalating doses (10e6–10e7/kg) of Tk-DLI from day +42. Results: Transduced cells were documented ex vivo in all evaluable pts. While no CD3+ cells were observed after infusion of 10e6/kg, the majority of pts who received 10e7/kg Tk-DLI reached 50 CD3+/μl by day 50, increasing steadily thereafter. Analysis on ex vivo PBLs showed Th1/Tc1 cells with a wide TCR repertoire able to proliferate, lyse and produce IFN-γ after stimulation with viral and allogeneic antigens. Immune reconstitution resulted in complete control of viral infections and complete remission of leukemia in the majority of patients. In 1 pt a grade II biopsy-proven acute GvHD, involving skin and liver was observed. Multiple doses of ganciclovir (10 mg/kg/die), in the absence of immunosuppressive drugs, quickly resulted in the complete resolution of all signs of GvHD. Ganciclovir-mediated abrogation of alloreactive T cells was confirmed by ex vivo ELISPOT analysis of host-reactivity. Conclusions: Tk-DLI at a dose of 10e7/kg is a promising tool for preventing disease relapse and promoting immune reconstitution after haplo-SCT while providing an effective and selective treatment for GvHD. A multicentric study aimed confirming these results on a larger cohort of patients recently started. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Molmed s.p.a.

Early immune reconstitution and abrogation of GvHD after infusion of HSV-TK engineered donor lymphocytes after haplo-identical hemopoietic stem cell transplantation

6515 Background: Haplo-identical stem cell transplantation (haplo-SCT) is a therapeutic option for patients with high risk hematologic malignancies lacking an HLA matched donor. The full exploitation of haplo-SCT is limited by delayed immune recovery, resulting in prolonged risk of life-threatening infections. Infusion of small numbers of donor T cells, aimed at providing immune reconstitution, results in severe forms of graft-versus-host-disease (GvHD). We previously showed that infusion of donor lymphocytes (DLI) expressing the herpes simplex virus thymidine kinase (Tk) is an efficient tool for controlling GvHD and preserving anti-tumor activity. Methods: We designed a dose-finding clinical protocol of Tk-DLI for immune reconstitution and relapse prevention after haplo-SCT. Eight pts with high risk hematologic malignancies who underwent haplo-SCT received escalating doses (10e6–10e7/kg) of Tk-DLI from day +42. Results: Transduced cells were documented ex vivo in all evaluable pts. While no CD3+ cells were observed after infusion of 10e6/kg, the majority of pts who received 10e7/kg Tk-DLI reached 50 CD3+/μl by day 50, increasing steadily thereafter. Analysis on ex vivo PBLs showed Th1/Tc1 cells with a wide TCR repertoire able to proliferate, lyse and produce IFN-γ after stimulation with viral and allogeneic antigens. Immune reconstitution resulted in complete control of viral infections and complete remission of leukemia in the majority of patients. In 1 pt a grade II biopsy-proven acute GvHD, involving skin and liver was observed. Multiple doses of ganciclovir (10 mg/kg/die), in the absence of immunosuppressive drugs, quickly resulted in the complete resolution of all signs of GvHD. Ganciclovir-mediated abrogation of alloreactive T cells was confirmed by ex vivo ELISPOT analysis of host-reactivity. Conclusions: Tk-DLI at a dose of 10e7/kg is a promising tool for preventing disease relapse and promoting immune reconstitution after haplo-SCT while providing an effective and selective treatment for GvHD. A multicentric study aimed confirming these results on a larger cohort of patients recently started. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Molmed s.p.a.