Background. Geranylgeranylacetone (GGA), which is an isoprenoid compound, has been used orally as an antiulcer drug inJapan. GGA induces antiviral gene expression by stimulating the formation of interferon-stimulated gene factor 3 in humanhepatoma cells. This study verified the anti-hepatitis C virus (HCV) activity of GGA in chronic hepatitis C-infected patients.Methods. The present prospective study included 20 consecutive anti-HCV antibody-positive, HCV-genotype 1b, and chronicgastritis patients who visited Nagasaki University Hospital between January 1999 and December 1999. GGA (150 mg per day,which is the dose generally used for chronic gastritis) was taken orally for four weeks. We evaluated HCV-RNA titers and otherclinical parameters at pretreatment, posttreatment, and at the endpoint of the study. Pretreatment was the beginning point ofGGA treatment. Posttreatment was the termination point of GGA treatment. The endpoint was the point four weeks after theposttreatment point.Results. All patients completed four weeks of GGA treatment and four weeks of observation. HCV-RNA titers at postpointwere not significantly diminished compared to those at pretreatment. However, HCV-RNA titers were significantly diminishedat endtreatment compared to pretreatment. Unfortunately, we did not observe a case with no titer of HCV-RNA. Alanineaminotransferase values and other parameters were not affected by GGA treatment.Conclusion. GGA has anti-HCV activities in chronic hepatitis C-infected patients. In the future, it will be necessary to examinethe clinical effectiveness of the combination of treatment with both GGA and interferon in HCV patients

Hamasaki Keisuke

Honda Takuya

Ichikawa Tatsuki

Kato Yuji

Miyaaki Hisamitsu

Muraoka Toru

Nakao Kazuhiko

Shibata Hidetaka

Takeshima Fuminao

Takeshita Shigeyuki

Taura Naota

Yamaguchi Tohei

Nagasaki University's Academic Output SITE: NAOSITE

IntroductionCurrently, chronic hepatitis C virus (HCV) infections arethe major cause of hepatocellular carcinoma (HCC) world-wide (1). Therefore, an anti-HCV strategy is important forthe prevention of carcinogenesis. The treatment of HCVwith a combination of pegylated interferon (IFN) andribavirin is effective in 80% of HCV genotype 2 or 3 casesbut is less than 50% effective in genotype 1 cases. Newanti-HCV agents designed to inhibit the life cycle of HCVhave been developed and are used in combination withIFN-α to ameliorate the salvage rate of HCV infection (2).However, this combination therapy cannot completely elimi-nate chronic HCV infections. Therefore, long-term man-agement and safety drugs for chronic hepatitis C (CHC) pa-tients are required.Geranylgeranylacetone (GGA) is an isoprenoid compound,which includes retinoids. GGA was developed in Japan andhas been used orally as an antiulcer drug (3). GGA protectsthe gastric mucosa from various types of stress without af-fecting gastric acid secretion (4,5). Moreover, GGA sup-presses cell growth and induces differentiation or apoptosisActa Med. Nagasaki 57: 1－4Address correspondence: Tatsuki Ichikawa, Department of Gastroenterology and Hepatology, Graduate School of BiomedicalSciences, Nagasaki University 1-7-1 Sakamoto, Nagasaki 852-8501, JapanPhone: +81-95-819-7482, Fax: +81-95-819-7481, E-mail address: ichikawa@net.nagasaki-u.ac.jp, All authors: There are no conflicts of interestReceived September 15, 2011; Accepted January 6, 2012MS#AMN 07096Anti-hepatitis C virus activity of geranylgeranylacetone treatmentin hepatitis C-infected patientsTohei YAMAGUCHI, Tatsuki ICHIKAWA, Shigeyuki TAKESHITA, Naota TAURA, Hisamitsu MIYAAKI, Toru MURAOKA,Hidetaka SHIBATA, Takuya HONDA, Keisuke HAMASAKI, Yuji KATO, Fuminao TAKESHIMA, Kazuhiko NAKAODepartment of Gastroenterology and Hepatology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, JapanBackground. Geranylgeranylacetone (GGA), which is an isoprenoid compound, has been used orally as an antiulcer drug inJapan. GGA induces antiviral gene expression by stimulating the formation of interferon-stimulated gene factor 3 in humanhepatoma cells. This study verified the anti-hepatitis C virus (HCV) activity of GGA in chronic hepatitis C-infected patients.Methods. The present prospective study included 20 consecutive anti-HCV antibody-positive, HCV-genotype 1b, and chronicgastritis patients who visited Nagasaki University Hospital between January 1999 and December 1999. GGA (150 mg per day,which is the dose generally used for chronic gastritis) was taken orally for four weeks. We evaluated HCV-RNA titers and otherclinical parameters at pretreatment, posttreatment, and at the endpoint of the study. Pretreatment was the beginning point ofGGA treatment. Posttreatment was the termination point of GGA treatment. The endpoint was the point four weeks after theposttreatment point.Results. All patients completed four weeks of GGA treatment and four weeks of observation. HCV-RNA titers at postpointwere not significantly diminished compared to those at pretreatment. However, HCV-RNA titers were significantly diminishedat endtreatment compared to pretreatment. Unfortunately, we did not observe a case with no titer of HCV-RNA. Alanineaminotransferase values and other parameters were not affected by GGA treatment.Conclusion. GGA has anti-HCV activities in chronic hepatitis C-infected patients. In the future, it will be necessary to examinethe clinical effectiveness of the combination of treatment with both GGA and interferon in HCV patients.ACTA MEDICA NAGASAKIENSIA 57: 1－4, 2012Keywords: Hepatitis C virus, geranylgeranylacetone, chronic hepatitis CTohei Yamaguchi et al.: GGA has anti-HCV activityin several human leukemia cells (6,7). 3,7,11,15-Tetramethyl-2,4,6,-10,14-hexadecapentaenoic acid is another isoprenoidcompound that was designated as an acyclic retinoid be-cause it has the ability to interact with nuclear retinoid re-ceptors (8) that cause apoptosis in certain human hepatomacells (9). GGA acts as a potent inducer of antiviral gene ex-pression, and it induces the expression by stimulating theformation of IFN-stimulated gene factor 3 (ISGF3) in humanhepatoma cells (10). GGA induces the expression of antiviralproteins such as 2′5′-oligoadenylate synthetase (2′5′-OAS)and double-stranded RNA-dependent protein kinase (PKR)in hepatoma cell lines. GGA stimulates 2′5′-OAS and PKRgene expression at the transcriptional level through the for-mation of ISGF-3, which regulates the transcription of bothgenes. GGA induces the expression of signal transducersand activators of transcription 1, 2 (STAT-1, STAT-2) andp48 proteins, components of ISGF3, together with thephosphorylation of STAT1 (10). However, the anti-HCVactivity of GGA has not been observed in vivo and in vitro.At present, new treatments for CHC patients are neces-sary, and GGA has an IFN-like action in hepatoma cells(10). Therefore, we attempted to verify the anti-HCV activ-ity of GGA in CHC patients.MethodsPatientsThe present prospective study included 20 consecutiveanti-HCV antibody-positive, HCV-genotype 1b, and chronicgastritis patients who visited the Nagasaki University Hospitalbetween January 1999 and December 1999. Patients wereenrolled in the study after informed consent was obtained.The patients had not been previously treated with IFN ther-apy and were diagnosed with CHC on the basis of clinicaldata. The patients were evaluated with a HCV-RNA polymerasechain reaction (PCR) method (Amplicor method). TheHCV-RNA high group (100,000 IU/mL or more in theserum) was identified by quantitative PCR. The criteria forHCC were assessed by abdominal imaging methods and byHCC history. The patients who were not previously diag-nosed with diabetes mellitus (DM) were evaluated by the75-g oral glucose tolerance test (OGTT). All subjects un-derwent OGTT with 75 g of glucose according to the rec-ommendations of the National Diabetes Data Group of theNational Institute of Health. Blood samples were taken at 0,30, 60, 90, 120, and 180 min after administration in order tomeasure the plasma glucose (PG) and insulin concentrations.In this study, the DM group consisted of patients with clini-cally diagnosed DM or 110 mg/dL fasting PG and/or 140mg/dL or high PG at 120 min.White blood cell counts, red blood cell counts, plateletcounts, hemoglobin A1c levels, alanine aminotransferase(ALT) levels, aspartate aminotransferase (AST) levels, andγ-glutamyl transpeptidase (GTP) levels were determinedby hematometry and standard laboratory techniques. Clinicalcharacteristics are shown in the Table.gMethodsThe dose of 150 mg of GGA per day, which is generallyused to treat chronic gastritis in Japan, was taken orally forfour weeks, and it was assumed that patients took one dosea day. Pretreatment was the beginning point of GGA treat-ment. We evaluated HCV-RNA titers and other clinical pa-rameters at pretreatment, posttreatment, and study end-point. Posttreatment was the termination point of GGAtreatment. Endpoint was the point four weeks after the2Age (years) 56 (16)Sex (F/M) 10/10BMI 21.0 (3.02)Genotype 1b 20HCV high titer 14HCV-RNA titer 489 (378)HCC +/- 0/20WBC count 6004 (1585)RBC count 447 (60)Plt count 18.9 (7.9)Alb level 4.46 (3.0)AST level 49.5 (21.2)ALT level 71 (28)γ-GTP level 50.5 (32)DM +/- 0/20HbA1c level 5.05 (0.8)FPG level 96 (13)Table. Clinical characteristics at pre-GGA treatmentCharacteristic mean (SD) or numberData are shown as means (standard deviation) and numbers.BMI, body mass index; HCV, Hepatitis C virus; HCC, hepatocellular car-cinoma; WBC, white blood cells; RBC, red blood cells; Plt, platelets; Alb,albumin; AST, aspartate aminotransferase; ALT, alanine aminotransferase;γ-GTP,γ-glutamyl transpeptidase; DM, diabetes mellitus; HbA1c, hemo-globin A1c; FPG, fasting plasma glucose.Normal values in laboratory tests: ALT (IU/L), 5-40; AST (IU/L), 10-40;γ-GTP (IU/L), <70 in men, <30 in women; Alb (g/dL), 4.0-5.0; WBC(cells/μL), 3500-9000; RBC (× 104 cells/μL), 450-580 in men, 380-480in women; Plt (× 104 platelets/μL), 14-33; ferritin (ng/mL), 39.4-340 inmen, 3.6-114 in women; FPG (mg/dL), 70-110; HbA1c (%), 4.3-5.8; BMI,body weight (kg)/height2 (m).Tohei Yamaguchi et al.: GGA has anti-HCV activityposttreatment of GGA. During this study, all patients werenot treated with Stronger Neo-Minophagen C (MinophagenPharmaceutical Co., Ltd., Tokyo, Japan) because of its anti-hepatitis effects or with IFN because of its anti-HCV effects.Statistical analysisData were processed on a personal computer and ana-lyzed using StatView 5.0 (SAS Institute, Inc., Cary, NC).The differences in the values of each laboratory parameterwere analyzed with a t-test. P values less than 0.05 wereconsidered statistically significant.ResultsGGA decreased the HCV-RNA titers in patients but did notaffect the values of ALTAll patients completed four weeks of GGA treatment andfour weeks of observation. Adverse effects were not observedin any patient. The titers of HCV-RNA (Fig. 1A) changedafter the patients completed GGA treatment. Comparedwith HCV-RNA titers at pretreatment, titers at endpoint didnot diminish significantly. However, compared to HCV-RNA titers at pretreatment, the titers were significantly di-minished at posttreatment. Unfortunately, we did not ob-serve a case with no titer of HCV-RNA. Values of ALT(Fig. 1B) and other parameters were not changed by GGAtreatment. The diminished HCV-RNA titers at the posttreatmentpoint were increased at the endpoint, which was four weeksafter the posttreatment point.In Fig. 2, we present the case of a patient who had themost diminished HCV-RNA titers among the 20 GGA-treated patients (Fig. 2). This case had mild fluctuations ofALT levels before GGA treatment. The HCV-RNA titerwas 420 K copies/mL and 380 K copies/mL at 12 weeksbefore treatment and at the pretreatment point, respectively.After GGA treatment, HCV-RNA titers were decreased to2 K copies/mL and 4 K copies/mL at the endpoint and atthe posttreatment point, respectively. In this case, the ALTvalues were also diminished in a similar manner as HCV-RNA. After the observation period, +12 weeks, HCV-RNAtiters and ALT values were increased compared to those atthe pretreatment point.DiscussionGGA demonstrated anti-HCV activity in this study. Theanti-HCV effect that was due to GGA did not result in adisappearance of HCV-RNA titers in CHC patients. An ad-verse effect was not observed with GGA treatment.GGA is a non-toxic heat shock protein (HSP) 70 inducer(11). Various GGA activities outside of the stomach arealso related to HSP induction (12,13,14). GGA induces3Figure 1. Titers of hepatitis C virus (HCV)-RNA at the endpointwere decreased compared to the levels at pretreatment (A), butalanine aminotransferase (ALT) levels were not changed (B).Panel A shows serum HCV-RNA titers, and panel B shows serumALT levels at each of the indicated points. The bar indicates themean value. Statistical significance was accepted with p-values lessthan 0.05. "Pre" indicates the point of pre-geranylgeranylacetone(GGA) treatment. "Post" indicates the termination point of GGAtreatment. "End" indicates the point four weeks after posttreatmentof GGA. Compared to HCV-RNA titers at pretreatment, GGAtreatment decreased the titer at pretreatment but not at posttreatment.Figure 2. The clinical course of a geranylgeranylacetone (GGA)-treated chronic hepatitis C (CHC) patient.Here, we present a case of a 53-year-old man who was an out-patient of our hospital for 5 years. He was diagnosed with chronichepatitis on the basis of clinical data. The patient had not beenpreviously treated with interferon (IFN). The y-axis indicates alanineaminotransferase (ALT) levels, and the x-axis indicates the timecourse. The duration of the GGA treatment periods is shown inthe gray field. The zero point on the x-axis is the GGA treatment-starting day. HCV-RNA titers are 420, 380, 2, and 4 K copies/mLat -12 weeks, 0 weeks (pretreatment), +4 weeks (posttreatment),and +8 weeks (end of follow-up period), respectively.Tohei Yamaguchi et al.: GGA has anti-HCV activitythioredoxin, as well as HSP-70, in hepatocytes and othercells (15). The antiviral activity of thioredoxin is inducedby AP-1 and NF-κB but not by HSP-70 (16). GGA, whichhas potent antiviral activities through the enhancement ofantiviral factors, can clinically provide protection from in-fluenza viral infections (17). Previously, we reported thatGGA induction of antiviral proteins was dependent uponSTAT-1 tyrosine phosphorylation in HuH-7 and HepG2with which HCV was not infected (10). However, HCVproducts inhibit the Jak-STAT pathway in HCV-infectedhepatocytes (18). The mechanism of inhibition of the Jak-STAT pathway is multifactorial and includes the expres-sion of suppressor of cytokine signaling 3 (SOCS-3) (19),protein phosphatase 2A induction (20), STAT-3 expression(21), and IL-8 expression (22). A clarification of GGA-induced anti-HCV activity is necessary for further exami-nation of the in vitro and in vivo effects.The peak venous blood concentration after taking 150mg of GGA orally is 5-7μmol/L (23), but 50μmol/L is thebest dose for induction of PKR and 2′5′-OAS in hepatomacell lines (10). In this study, we employed the usual dosageof GGA used to treat chronic gastritis in Japan, which is150 mg per day. In a previous study, it was reported thatportal blood concentration after taking 150 mg of GGAorally was several-fold that of the venous blood concentra-tion (23). The usual dosage of GGA also may have a possi-ble antiviral gene expression effect in the liver.In conclusion, GGA, a drug that can be safely adminis-tered orally, has anti-HCV activity. Unfortunately, we didnot observe a case that exhibited disappearance of HCV-RNA titers. GGA treatment is insufficient for clearance ofHCV, and, therefore, it will be necessary to examine theclinical effectiveness of the combination treatment withGGA and IFN in HCV patients in the future.References1. Fattovich G, Stroffolini T, Zagni I, Donato F. Hepatocellular carci-noma in cirrhosis: incidence and risk factors.: Gastroenterology.; 127:S35-50.2. Pawlotsky JM, Chevaliez S, McHutchison JG. The hepatitis C viruslife cycle as a target for new antiviral therapies.: Gastroenterology.;132: 1979-98.3. Murakami M, Oketani K, Fujisaki H, Wakabayashi T, Ohgo T. 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Anti-hepatitis C virus activity of geranylgeranylacetone treatment in hepatitis C-infected patients

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Anti-hepatitis C virus activity of geranylgeranylacetone treatment in hepatitis C-infected patients

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