January 7, 2013

Inactivation and Survival of Hepatitis C Virus on Inanimate Surface

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Journal of Infectious Diseases Advance Access published October 19, 2011

Juliane Doerrbecker,1 Martina Friesland,1 Sandra Ciesek,1,2 Thomas J. Erichsen,2 Pedro Mateu-Gelabert,5 Jorg Steinmann,3 Jochen Steinmann,4 Thomas Pietschmann,1 and Eike Steinmann1

1Division of Experimental Virology, Twincore, Centre for Experimental and Clinical, Infection Research, a Joint venture between the Medical School
Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), 2Department of Gastroenterology, Hepatology and Endocrinology, Hannover
Medical School, 3Institute of Medical Microbiology, University Hospital Essen, and 4MikroLab GmbH, Bremen, Germany; and 5National Development
Research Institutes, New York

"In summary, we show that infectious HCV can persist as a dried sample for up to 1 week. The most effective alcohol to inactivate the virus was 1-propanol, and commercially available disinfectants reduced HCV infectivity to undetectable levels, emphasizing strict hygiene measurements. These experimental developments should facilitate testing the virucidal activity against HCV of chemical biocides used for surface disinfection. In addition, these results will further improve the understanding of HCV cross-contaminations and its prevention in health-care settings and among inJection drug users."

Abstract

Background. Hepatitis C virus (HCV) cross-contamination from inanimate surfaces or obJects has been implicated in transmission of HCV in health-care settings and among inJection drug users. We established HCV-based carrier and drug transmission assays that simulate practical conditions to study inactivation and survival of HCV on inanimate surfaces.

Methods. Studies were performed with authentic cell culture derived viruses. HCV was dried on steel discs and biocides were tested for their virucidal efficacy against HCV. Infectivity was determined by a limiting dilution assay. HCV stability was analyzed in a carrier assay for several days or in a drug transmission assay using a spoon as cooker.

Results. HCV can be dried and recovered efficiently in the carrier assay. The most effective alcohol to inactivate the virus was 1-propanol, and commercially available disinfectants reduced infectivity of HCV to undetectable levels. Viral infectivity on inanimate surfaces was detectable in the presence of serum for up to 5 days, and temperatures of about 65-70°C were required to eliminate infectivity in the drug transmission assay.

Conclusions. These findings are important for assessment of HCV transmission risks and should facilitate the definition of stringent public health interventions to prevent HCV infections.

Hepatitis C virus (HCV) is an enveloped virus that, at present, chronically infects ~130 million people worldwide [1]. One hallmark of HCV is its high degree of sequence variability, which likely contributes to its ability to establish chronic infections. Different patient isolates are grouped into 7 genotypes and more than 100 subtypes within the genus Hepacivirinae of the family Flaviviridae [2]. Persistent infection is associated with a variable degree of liver damage often progressing in severity over the course of decades. Accordingly, a large number of patients are at risk of severe sequelae including life-threatening conditions like cirrhosis and hepatocellular carcinoma [3]. The best available treatment, a combination of polyethylene glycol (PEG)-conJugated interferon alpha (IFN-α) and ribavirin, is not effective in every patient and can be associated with severe adverse effects [4]. A prophylactic or therapeutic vaccine is so far not available.

Hepatitis C is a blood-borne viral infection transmitted mainly through intravenous drug use, blood transfusions, accidental needle sticks, and other parental exposures, including nosocomial transmissions [5-9]. With the implementation of routine testing of blood products for HCV, transfusion-transmitted infections became rare [10]. However, outbreaks in health-care settings have been consistently reported primarily attributed to contaminated medications or equipment and breaches in aseptic techniques in the United States, Europe, and Japan [11-15]. Furthermore, cross-contamination continues to occur among inJection drug users (IDUs) by the sharing of drug preparation equipment [16-18]. The seroprevalence of HCV among IDUs in the United States is high, ranging between 30% and 85%, with current estimates suggesting more than over 60% of newly acquired infections occur in individuals who have inJected drugs [19, 20]. The adequate assessment of transmission risks and the evaluation of the mechanisms of transmission have been difficult due to the lack of cell culture systems and animal models permissive to HCV infection. This obstacle has been overcome with the development of an HCV cell culture system based on the Japanese fulminant hepatitis (JFH1) HCV isolate, which reproduces the complete viral replication cycle in vitro [21-23]. This infection system was recently applied to evaluate the environmental stability of HCV and its susceptibility to chemical biocides in liquid suspensions [24]. Furthermore, Paintsil et al [25] analyzed in 2010 the survival of HCV in contaminated syringes and the duration of potential infectiousness; however, both studies did not analyze viability and infectivity of dried HCV.

Therefore, simulating realistic practical conditions, we established an HCV-based carrier and drug transmission assay to test inactivation and stability of HCV on inanimate surfaces. These results allow the further exploration of viral transmission from contaminated surfaces, obJects, or devices and the potential for recommendations for effective measures interrupting this transmission.

DISCUSSION

For better understanding and prevention of HCV transmission in medical settings and in the environment, experimental system simulating practical conditions are highly relevant. In this study, we addressed HCV inactivation and stability profiles on inanimate surfaces to mimic viral cross-transmissions among IDUs and in health-care settings where HCV infections continue to occur. We demonstrated that HCV could be dried and recovered efficiently in a carrier assay that can therefore be used to validate chemical biocides in their virucidal efficacy against HCV. Importantly, it also confirms that reusing HCV contaminated cookers could lead to infection even if using sterile syringes. Furthermore, by simulating the procedure for heating drugs into solution, we showed that HCV could be eliminated at temperatures of 65-70°C. These data can be used for the design of public health recommendations and prevention of viral spread among IDUs. Until recently, experimental data about the environmental stability of HCV were not reported or performed with surrogate markers (antigens, RNA, enzyme activity) for the presence or absence of infectious particles. The HCV infection system used here is based on human hepatoma cells and viruses generated in vitro [21-23], and substantial progress has been made in HCV basic and translational research with this model [31]. However, limitations are that in vivo hepatocytes and patient-derived particles might be slightly different or that not all genotypes can be grown in cell culture.

In the environment, viruses are normally found on surfaces and/or embedded in body fluids like excrements, serum, blood, or other excretions, and the risk of viral transmission depends on the contact number, time, body parts, and how readily the virus is released from such surfaces. The carrier test method for HCV developed here allows predicting the activity of chemical biocides simulating practical conditions. Dried HCV was exposed to a test product for a defined contact time. At the end of the contact time, the virus-biocide mixture was recovered from the surface of the carrier and titrated to determine the degree of loss in virus infectivity. We could previously show in a quantitative suspension assay that 1-propanol is the most effective alcohol in activating HCV [24]. However, whereas in a suspension test a concentration of 20% 1-propanol was sufficient to eliminate Jc1 with a viral titer of 106 TCID50/mL, higher concentration of the alcohol are needed to inactivate dried HCV due to a stronger challenge for the disinfectant [29]. Importantly, we could demonstrate that commercially available surface disinfectants have a high virucidal efficacy at concentrations recommended by the manufacturers as previously shown for hand antiseptics [24]. While dried virus in the presence of serum could survive for up to 5 days at room temperature, we could show that HCV in suspension could survive for even 3 weeks [24], and in syringes infectivity was detected for up to 63 days [25]. Kamili and colleagues [32] demonstrated in a chimpanzee animal model that dried HCV derived from patient sera could survive for at least 16 hours but was not detectable after storage of 4 or 7 days. Differences in the viral dose, storage conditions, or determination of infectivity in vitro or in vivo [33, 34] might account for the different survival times between these studies. We used here a highly sensitive detection assay and were able to determine precise survival times of the virus on dried surfaces in the presence or absence of serum. The transmission patterns for hepatitis B virus (HBV) are very similar to HCV, and high stability in the environment has been reported for this hepatotropic virus as well [35]. In line with our results, infectivity after drying of HBV-positive human plasma could be detected for at least 1 week while no longer incubation times were analyzed [35]. In summary, these reports showed that HCV could remain viable for a prolonged time in the environment indicating that blood-contaminated surfaces can serve as HCV reservoirs. Consequently, effective disinfection of surfaces is crucial in the prevention of HCV transmission.

Transmission of HCV remains high among IDUs in recent years, with incidence rates ranging from 16% to 42% per year [36]. Furthermore, the risk of HCV transmission estimated per exposure to a contaminated syringe is 5-fold to 20-fold higher than that of HIV [37-39]. Recently, Paintsil et al [25] contributed to the understanding of biological mechanisms of HCV transmission by studying contaminated syringes with HCV cell culture derived virus [40]. They found that HCV survival was dependent on syringes type, time, and temperature. Infectivity could be detected for up to 63 days in high void volume tuberculin syringes. These results suggest that this long survival contributes to the high prevalence of HCV in comparison to HIV among IDUs in spite of successful syringe exchange programs. Besides syringes, the sharing of drug cookers and cotton for filtration was also significantly associated with HCV infection independent of sharing needles and syringes [18, 30]. We show here that HCV on a spoon as cooker can survive temperatures up to 65°C, which corresponds to a heating time of 80-95 seconds in this assay setup, indicating that virus survival on cookers could also be a potential source of infectious HCV aside from syringes.

In summary, we show that infectious HCV can persist as a dried sample for up to 1 week. The most effective alcohol to inactivate the virus was 1-propanol, and commercially available disinfectants reduced HCV infectivity to undetectable levels, emphasizing strict hygiene measurements. These experimental developments should facilitate testing the virucidal activity against HCV of chemical biocides used for surface disinfection. In addition, these results will further improve the understanding of HCV cross-contaminations and its prevention in health-care settings and among inJection drug users.

RESULTS

Development of a HCV-Based Carrier Test

In general, the carrier test method is designed to evaluate the ability of chemical biocides to inactivate vegetative bacteria, viruses, fungi, mycobacteria and bacterial spores on inanimate surfaces [29]. Here, the experimental procedure of the carrier assay was used for the first time to test the virucidal activity of biocides against dried HCV. First, stainless steel discs were inoculated with a virus preparation of the HCV genotype 2a chimera Jc1 [26] and dried under a laminar flow (Figure 1A). After drying, the virus-contaminated discs were transferred into plastic vial holders, which were previously filled with glass beads to increase virus recovery by mechanical abrasion. Next, the tested biocides were distributed onto the dried virus and incubated for 1 or 5 minutes. In order to neutralize the test substance, culture medium was immediately added at the end of the exposure time. The vials were directly vortexed to recover residual infectivity, before the eluate was diluted to determine viral infectivity using a limiting dilution assay.

It has been described that depending on which virus type is dried on the carrier the amount of infectivity recovered might vary [29]. Therefore, to determine the recovery efficiency for HCV, we titrated Jc1 incubated 1 hour in suspension and a virus inoculum that was dried for the same time on a carrier disc. As depicted in Figure 1B, the infectivity of HCV recovered from the carrier surface by our procedure was about 10-fold lower compared with the HCV stored in a liquid environment. Thus, ~10% of the viral infectivity was recovered in the carrier assay.

Virucidal Efficacy of 1-Propanol, 2-Propanol, and Ethanol Against Dried HCV
Surface disinfectants used in health care and other medical settings often contain 1-propanol, 2-propanol or ethanol as active ingredients for decontamination of surfaces. To assess the virucidal efficacy of these alcohols at concentrations ranging from 10% to 60% on contaminated surfaces, we incubated each alcohol for 1 minute (Figure 2A) and 5 minutes (Figure 2B) on dried HCV. The most effective alcohol to inactivate HCV was 1-propanol, reducing viral titers to background levels at a concentration of 30% with both incubation times (Figure 2). For 2-propanol, a concentration of 30% decreased infectivity about 10-fold, and complete inactivation was observed at an alcohol content of 50% with a 1-minute exposure time and 40% with 5 minutes incubation, respectively. Ethanol showed the lowest virucidal efficacy with a required concentration of 50% to reduce viral titers to undetectable levels in the 5-minute exposure (Figure 2B).

Effect of Commercially Available Surface Disinfectants Against Dried HCV
To directly determine the efficacy of commercially available surface disinfectants, we chose 6 different chemical biocides with different virucidal substances as ingredients. Products A and B were both based on ethanol and 2-propanol or 1-propanol, respectively. Product C contained glutaraldehyde as active ingredient. Product D and E were on the basis of quaternary ammonium compounds, whereas for product F peroxide compounds were used as virucidal substance. The alcohol-based biocides were tested as recommended with an incubation time of 5 minutes in the concentrations of 10%, 50%, and 100%. As depicted in Figure 3A, a concentration of 50% for product A reduced viral titers about 50-fold. In an undiluted preparation no infectivity could be detected; however, at a 100% concentration also cytotoxicity was visible. Product B containing ethanol and 1-propanol demonstrated a higher virucidal efficacy than product A reducing viral titers to background levels already at a concentration of 50%, thus confirming the previous results that 1-propanol is superior over 2-propanol as biocide for HCV. The other commercially available disinfectants were tested at concentrations of 0.025%, 0.25%, and 0.5% in the carrier test (Figure 3B). A complete inactivation could be achieved by all products at the highest concentration with only slight cytotoxicity for products C, D, and E. These results show that ingredients like glutaraldehyde, quaternary ammonium, and peroxide compounds have a high virucidal efficacy against HCV.

Survival of Dried HCV on Inanimate Surfaces

Recently, it could be shown that HCV can be stable for several weeks in a liquid environment or in syringes [24, 25]. To evaluate the stability of nonliquid HCV, Jc1 virus was dried on carrier discs and incubated for several days at room temperature. As HCV infection is typically transmitted via blood, the effect of healthy serum on the stability of dried HCV was analyzed in parallel. Infectivity of dried virus in the presence of serum was reduced 10-fold after 2 days and reached undetectable levels after 6 days. Furthermore, the addition of serum resulted in reduced viral titers compared with the virus without serum (Figure 4A). In the latter case, we still could measure infectious HCV with a titer of about 30 TCID50/mL after 7 days of incubation demonstrating a stability of dried HCV for more than a week on the carrier surface. In the next set of experiments, we analyzed if the addition of serum before the drying procedure influences the ability of the different biocides to inactivate HCV as reported for other viruses. The different alcohols or commercial disinfectants that were used in a concentration completely inactivated HCV as shown before (compare Figures 2 and 3). All tested biocides were able to inactivate HCV infectivity to undetectable levels in the presence or absence of serum (Figure 4B), indicating that serum cannot confer viral resistance to the tested biocides.

Heat Stability of HCV in a Drug Transmission Assay

Epidemiologic studies indicate that the sharing of the drug preparation equipment among IDUs is an important risk factor for HCV transmission [18, 30]. Spoons and/or cookers are used to heat diluted heroin into solution. Cookers are mostly used in the United States, whereas spoons are mostly used in Europe. During the drug preparation, spoons are often reused and shared between users. The drug dilution from the spoon is drawn into a syringe, and blood contaminated with HCV can be exposed to the drug dilution by insertion of an HCV-contaminated syringe into spoons that are shared. Therefore, blood on spoons/cookers could be source for contamination with infectious HCV, and the ability of the virus to survive on such surfaces can have a strong impact on cross-transmissions. To evaluate the transmission risk via this route, we contaminated a spoon with Jc1 reporter virus (Figure 5A). With the use of a tea candle, increasing temperatures were simulated with the cooker device. At indicated time intervals, aliquots were taken and used to determine infectivity by luciferase reporter assay. Viral infectivity started to decrease at a temperatures of ~50°C and was below the detection limit at about 65-70°C in 9 independent measurement series (Figure 5B). The time required to reach certain temperatures depends highly on the experimental setup, but in our case ~80-95 seconds were necessary when small bubbles start to appear on the spoon. The half-life of HCV at different temperatures did not differ significantly between reporter virus and authentic wild-type HCV Jc1 (data not shown). Next, we tested the impact of water and serum in this drug transmission assay. As depicted in Figure 5C, the addition of water or serum to the virus solution did not influence HCV stability. Again, ~65°C was the temperature required to inactivate viral infectivity to background levels.

MATERIALS AND METHODS

Plasmids and Viruses

The plasmid pFK-Jc1 has been described recently [26]. Construct Luc-Jc1 encodes a chimeric HCV polyprotein that consists of codons 1-846 derived from J6/CF [27] combined with codons 847-3033 of JFH1. In this genome the HCV polyprotein-coding region is located in the second cistron and is expressed via an internal ribosomal entry side element derived from the encephalomyocarditis virus. The first cistron contains the firefly luciferase reporter gene fused to the JFH1-derived 5'NTR and coding region of the N-terminal 16 amino acids of JFH1 core [28].

Chemical Biocides

The alcohol substances 1-propanol, 2-propanol and ethanol were purchased from Carl Roth, Karlsruhe, Germany. Six commercially available biocides for surface disinfection were chosen to study the efficacy against dried HCV: Product A (based on ethanol, 2-propanol), product B (based on ethanol, 1-propanol), product C (based on glutaraldehyde), product D and E (based on quaternary ammonium compounds), and product F (based on peroxide compounds).

Cell Culture

Huh7.5 cells were cultured in Dulbecco modified Eagle medium (DMEM, Invitrogen) with 10% fetal bovine serum, 1x nonessential amino acids (Invitrogen), 100 μg/mL streptomycin (Invitrogen) and 100 IU/mL penicillin (Invitrogen).

In Vitro Transcription, Electroporation, and Production of Cell Culture-Derived HCV

Infectious HCV particles were produced as described elsewhere [28]. Briefly, Jc1 or Luc-Jc1 plasmid DNA was linearized and transcribed into RNA, which was then electroporated into Huh7.5 cells. Virus-containing culture fluids were harvested after 48 or 72 hours filtered through a 0.45 μm pore size filter. For determination of viral infectivity cell-free supernatants were used to infect naive Huh7.5 target cells.

Determination of HCV Infectivity

Titers of infectious virus were determined by using a limiting dilution assay on Huh7.5 cells with a few minor modifications and tissue culture infectious dose 50 (TCID50) was determined as described elsewhere [23]. For determination of Luc-Jc1 reporter activity, infected cells were washed with phosphate-buffered saline (PBS) and lysed in luciferase lysis buffer (1 % Triton X-100, 25 mmol/L glycylglycine, 15 mmol/L MgSO4, 4 mmol/L EGTA, and 1 mmol/L DTT, pH 7.8). Firefly luciferase activity was measured as described previously [28].

Preparation of the Carrier

Stainless steel discs with grade 2B finish on both sides (20 mm diameter, GK Formblech GmbH) were incubated in a 5% (vol/vol) Decon 90-solution (Decon Laboratories Ltd) for 1 hour. Afterward the discs were rinsed off twice with freshly distilled water for 10 seconds, ensuring that the carriers did not dry to any extent, and were then placed in 70% ethanol (vol/vol) for 15 minutes. Finally, the carriers were dried by evaporation in sterile petri dishes under a biological safety cabinet.

Experimental Procedure of HCV Carrier Assay

In total, 50 μL of the virus inoculum were pipetted in the center of each pretreated carrier and dried in a desiccator or under a laminar flow for about 1-3 hours at room temperature. After drying, the virus contaminated discs were transferred with forceps into 25 mL plastic vial holders (Sarstedt AG & Co KG), which were previously filled with 0.5 g of sterile glass beads (0.25-0.50 mm diameter, Carl Roth GmbH) to increase virus recovery by mechanical abrasion. Then, 100 μL of the test substance were pipetted on the dried virus inoculum and incubated for 1 or 5 minutes. Control carriers received 100 μL of water instead of the chemical biocide. In order to neutralize the test substance, 900 μL of culture medium were immediately added at the end of the chosen exposure time. The vials were directly vortexed for 1 minute to recover the residual virus, before the eluate was diluted to measure viral infectivity. To determine cytotoxicity of the biocides, 1 part of PBS was mixed with 9 parts of the biocide and used to inoculate, permissive Huh7.5 cells. Cytotoxicity was determined by examining permissive cells by microscopy for any significant changes in the cell monolayer and calculated analogously to virus titer (TCID50/mL).

For testing HCV stability and inactivation in the presence of serum, whole blood samples of healthy donors were centrifuged for 5 minutes at 5000 rpm to obtain serum. The effect of serum on HCV stability was tested by mixing serum and virus suspension in a ratio 1:1 in a total volume of 0.1 mL before the drying procedure.

Experimental Procedure for HCV Drug Transmission Assay

To test the effect of different temperatures on HCV infectivity in a drug preparation simulation, viral suspensions of 800 μL were used as inoculum of a standard household spoon (stainless steel). A heating procedure was started with a tea candle with a distance of ~4 cm between the spoon and the top of the flame. Temperatures of the suspensions were measured at specific time intervals using a thermometer for small liquids (YEW pocket thermometer 2542). At given temperatures, 70 μL of the viral suspension was sampled. To Judge the influence of human serum on virus stability in the drug transmission assay, virus suspension was diluted in a ratio of 1:8 with serum or water. Viral infectivity was determined by a luciferase reporter assay as described elsewhere [28].

Source

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Hepatology (Early publication, ahead of print] Accepted manuscript online: 23 NOV 2012
Norah A. Terrault1 Jennifer L. Dodge1, Edward L. Murphy1,2, John E. Tavis3, Alexi Kiss3, T.R. Levin4, Robert Gish5, Michael Busch1,2, Arthur L. Reingold6, Miriam J. Alter7 University of California San Francisco1, Blood Systems Research Institute2, St. Louis University3, Kaiser Permanente Division of Research4, California Pacific Medical Center5, University of California Berkeley6; Centers for Disease Control and Prevention7*

the authors summarize: "In summary, we conclude that HCV transmission by sex from chronically infected persons to their heterosexual partners in a long-term monogamous relationship likely occurs but is a rare event. Our results provide a basis for specific counseling messages that clinicians can use with their patients. These messages should be qualified given the limitations of the sample size, but they support the current national recommendations that couples not change their sexual practices if they are in a monogamous heterosexual relationship." from Jules of NATAP: there are some buts here......

from Jules of NATAP: this study is composed of a study population that is selected (heterosexual, monogamous) and thus a study participant group that by study definition eliminates IDUs if both partners had a history of IDU, HIV & HBV, thus also likely eliminating or limiting participation of individuals that may be more likely to have STDs, sex workers; findings from prior studies have suggested that the presence of HIV & STDs increase risk for sexual transmission, as well HIV appears to increase risk of sexual transmission perhaps due to higher HCV viral load, or perhaps due to risky sexual behaviors (rough sex, anal sex that may rupture mucosal environment & also perhaps due to the presence of other sexually transmitted diseases), so the results of this study are limited to the study group they studied (heterosexual momogomous couples in a relationship for at least 36 months): "Criteria for study participation by each couple included a heterosexual relationship for a minimum of 36 months, monogamy for the duration of the relationship reported by both partners, and a minimum of 3 sexual contacts by the couple in the preceding 6 months. Couples were excluded if either partner had known HIV or hepatitis B virus infection, prior organ transplantation, or was currently using antiviral or immunosuppressive therapy; or if both partners reported a history of injection drug use (IDU)."

"The 500 couples were predominantly non-Hispanic White race, educated, employed, and native-born (Table 1). The median duration of the couples' sexual relationships was 15 years (range 2 to 52)." from Jules: in Table 1 demographics you can see 72% white, 73% had college education, 21% had income over $100k, and 35% income $50-100k. I don't see any mention in the study of the presence of STds among the study population. The authors also say: "employing optimal methods to detect HCV RNA, the minority of samples were positive for HCV RNA and all positive samples were of low titer (≤102 IU/mL) (19-20). A low titer of virus in genital secretions may be one reason that HCV is transmitted less efficiently than hepatitis B virus or HIV (21-22). Additionally, transmission of infection by sex may require a specific genital tract environment such as disrupted mucosal integrity or the presence of viral or bacterial coinfections. These factors may explain the recent reports of HCV transmission by sex in HIV-infected men who have sex with men (23)"

"The 500 couples were predominantly non-Hispanic White race, educated, employed, and native-born (Table 1). The median duration of the couples' sexual relationships was 15 years (range 2 to 52). The most frequently reported risk factors for HCV infection among index persons were IDU (53.8%) and blood transfusion before 1992 (31.6%); these risks were infrequently reported by partners."

"Based upon the frequencies of sexual contact and length of relationships reported, a cumulative 8,377 person-years of risk for acquiring HCV by sexual activity was calculated. With 3 viremic confirmed concordant couples and 3 possible concordant couples, the estimated incidence of HCV infection among partners ranged from 3.6 per 10,000 person-years (95% CI: 0.0, 7.7) (minimum estimate) to 7.2 per 10,000 person-years (95% CI: 1.3, 13.0) (maximum estimate). The estimated risk per sexual contact ranged from 1 per 380,000 (95% CI 1/600,000, 1/280,000) to 1 per 190,000 (95% CI 1/1.03 mil, 1/100,000)."

"Concordantly-infected couples were no more likely to share blood-contaminated objects, such as nail clippers, razors, and toothbrushes, than couples in which one partner remained uninfected (0.0% vs. 10.1%, p=1.00), but were more likely to have vaginal intercourse during menses (100.0% vs. 65.6%, p= 0.55) and anal intercourse (66.7% vs. 30.2%, p=0.22), and less likely to use condoms (0.0% vs. 30.4%, p=0.56). These differences, however, were not statistically significant."

"Sexual transmission of HCV presumably occurs when infected serum-derived body fluids are exchanged across mucosal surfaces. Potential factors that may influence this exchange include the titer of virus, the integrity of the mucosal surfaces, and the presence of other genital infections (viral or bacterial). Studies to detect HCV RNA in semen (seminal fluid and cells), vaginal secretions, cervical smears, and saliva have yielded mixed results (14-20). Failure to detect HCV RNA in body secretions from chronically infected persons may be due to technical factors including specimen collection and storage, and the inability to exclude cellular components and to overcome the presence of polymerase inhibitors in body fluids. Even in studies employing optimal methods to detect HCV RNA, the minority of samples were positive for HCV RNA and all positive samples were of low titer (≤102 IU/mL) (19-20). A low titer of virus in genital secretions may be one reason that HCV is transmitted less efficiently than hepatitis B virus or HIV (21-22). Additionally, transmission of infection by sex may require a specific genital tract environment such as disrupted mucosal integrity or the presence of viral or bacterial coinfections. These factors may explain the recent reports of HCV transmission by sex in HIV-infected men who have sex with men (23)."

Abstract

BACKGROUND: The efficiency of hepatitis C virus (HCV) transmission by sexual activity remains controversial. We conducted a cross-sectional study of HCV-positive persons and their partners to estimate the risk for HCV infection among monogamous heterosexual couples.

METHODS: 500 anti-HCV-positive, HIV-negative index persons and their long-term heterosexual partners were studied. Couples were interviewed separately for lifetime risk factors for HCV infection, within-couple sexual practices and sharing of personal grooming items. Blood samples were tested for anti-HCV, HCV RNA, and HCV genotype and serotype. Sequencing and phylogenetic analysis determined the relatedness of virus isolates among genotype-concordant couples.

RESULTS: HCV-positive index persons were mostly Non-Hispanic Whites, with median age 49 years (range 26-79) and median 15 years (range 2-52) of sexual activity with their partners. Overall, HCV prevalence among partners was 4% (n=20), and 9 couples had concordant genotype/serotype. Viral isolates in 3 couples (0.6%) were highly related, consistent with transmission of virus within the couple. Based upon 8377 person-years of follow-up, the maximum incidence rate of HCV transmission by sex was 0.07% per year (95% CI: 0.01, 0.13) or ~1 per 190,000 sexual contacts. No specific sexual practices were related to HCV-positivity among couples.

CONCLUSIONS: The results of this study provide quantifiable risk information for counseling long-term monogamous heterosexual couples in which one partner has chronic HCV infection. In addition to the extremely low estimated risk for HCV infection in sexual partners, the lack of association with specific sexual practices provides unambiguous and reassuring counseling messages.

INTRODUCTION

Chronic hepatitis C virus (HCV) infection affects 3 to 4 million people in the United States, most of whom are sexually active adults (1). The primary means of transmission of HCV is by direct percutaneous exposures to infectious blood and there are clearly defined counseling messages for infected persons to prevent spread from such exposures (2). The accumulated epidemiological evidence indicates that HCV can be transmitted by sex with an infected partner, presumably by mucosal exposure to infectious blood or serum-derived fluids. However, sexual activity is much less efficient for transmitting HCV than for other blood borne, sexually transmitted viruses, such as hepatitis B virus and human immunodeficiency virus (HIV) (3).

The association between sexual activity and HCV infection was first demonstrated by case-control studies of persons with acute hepatitis C (4). The few prospective cohort studies of monogamous heterosexual couples have reported incidence rates of HCV infection of 0 - 0.6% per year in seronegative partners of persons with chronic HCV infection (5-7), In cross-sectional studies, HCV prevalences among partners vary widely (0-27%) but are <5% in studies excluding partners with known percutaneous exposures (3). For HCV-infected persons in the United States, the risks quantified by previous incidence studies may not apply as they were performed in countries where the epidemiology of HCV infection differs from that in the United States due to potentially confounding by unmeasured non-sexual risk factors. Although several seroprevalence studies of monogamous heterosexual couples have been reported from the United States (8, 9), their sample sizes were insufficient to evaluate overall risk or that related to specific sexual practices, and detailed virologic analyses of antibody-concordant couples were lacking, leading to an overestimation of transmission risk.

While it is generally agreed that the risk for transmitting HCV to sex partners is very low, the lack of quantifiable data has been a limitation to clinicians counseling their patients. Thus, the major objectives of this study were to quantify the risk for sexual transmission of HCV infection from chronically infected persons to their long-term heterosexual partners and identify specific sexual practices associated with that risk.

STUDY METHODS

Study Population

The recruitment phase of the study was conducted in Northern California sites between January 2000 and May 2003. Recruitment began by first identifying a known HCV-positive person (referred to as the index person) from multiple sources, including liver clinics at the University of California at San Francisco, members of Kaiser Permanente Medical Care Plan in Northern California, California Pacific Medical Center and affiliated clinics, other community-based practices in the greater San Francisco Bay Area, and blood donors from Blood Centers of the Pacific/Blood Systems Research Institute. Researchers contacted index persons for study enrollment, and if eligible based on prescreening, contacted their sexual partner. Criteria for study participation by each couple included a heterosexual relationship for a minimum of 36 months, monogamy for the duration of the relationship reported by both partners, and a minimum of 3 sexual contacts by the couple in the preceding 6 months. Couples were excluded if either partner had known HIV or hepatitis B virus infection, prior organ transplantation, or was currently using antiviral or immunosuppressive therapy; or if both partners reported a history of injection drug use (IDU).

Partners of each couple were interviewed independently by phone (76%) or in-person (24%) by trained interviewers, with no difference in completing the questionnaire by interview type. Detailed information was obtained on the sexual history with the study partner (Appendix 1), non-sexual household exposures (sharing of personal items including nail-grooming tools, razors, and toothbrushes), and all other risk factors for HCV acquisition. The risk period for sexual transmission was defined using a uniform method to capture sexual activities over the entire duration of the couple's relationship. Sexual histories were collected in discrete time intervals defined by events in each participant sexual history and beginning from the time of first sexual contact with the current partner up to the time of interview. Each participant identified life events such as pregnancy, childbirth, medical illness, and absences that significantly changed sexual activities with their study partner and the corresponding year and age for each life event. Sexual practices, including type and frequency of sexual contact and use of protective barriers, were obtained during each of these defined intervals. When responses to questions about sexual or personal grooming practices were discordant between partners, responses were recoded for presence rather than absence of the practice.

RESULTS

Eligible and Enrolled Couples

Of the 2077 couples screened for study inclusion, 672 (32%) were eligible. Reasons for study exclusion occurring in ³5% of the 1405 ineligible couples included lack of sexual activity (31%), prior organ transplant (12%), refused study participation (11%), doctor refused (8%), HIV or hepatitis B virus coinfection (8%), partnership less than 3 years or non-monogamous (6%), and history of IDU in both partners (6%). Of the 672 eligible couples, 500 (74%) enrolled and completed all the study requirements at which time study enrollment was halted. The primary reasons for failure to participate among the remaining 172 eligible couples were non-response (54%) or refusal (29%). Of the 500 enrolled couples, 43% were referred from tertiary referral practices, 34% from community sources, and 21% were blood donors.

Characteristics of Participating Couples

The 500 couples were predominantly non-Hispanic White race, educated, employed, and native-born (Table 1). The median duration of the couples' sexual relationships was 15 years (range 2 to 52). The most frequently reported risk factors for HCV infection among index persons were IDU (53.8%) and blood transfusion before 1992 (31.6%); these risks were infrequently reported by partners. Twenty or more lifetime sex partners prior to the current relationship were reported by 46.2% of index persons and 26.8% of partners.

The median number of sexual contacts per month was highest for vaginal intercourse during the first year of the relationship (12 contacts per month) (Table 2). The frequency of sexual contacts decreased over time for all types of sexual activity. Vaginal intercourse during menses and anal intercourse (³1 occasion) were reported by 65.2% and 30.4% of couples, respectively. Condom use during vaginal intercourse was reported by 29.9% of couples and condom use decreased over time for vaginal and anal intercourse.

HCV Sequencing and Phylogenetic Analyses of HCV Strains

Among the 500 partners of anti-HCV positive index persons, 20 were confirmed anti-HCV positive and 13 of the 20 partners were HCV RNA positive. HCV genotyping/subtyping and HCV serotyping confirmed 9 couples to be concordant, 8 to be discordant and 3 couples to be of indeterminant status (Table 3).

Of the 9 genotype concordant couples, both partners of 6 couples were viremic, allowing phylogenetic analyses; 3 had strong evidence that the partners were infected with the same HCV isolate and 3 were consistent with infection by different HCV strains (Table 4). Couple 15 had HCV 1a strains that were more similar to each other than 99% of random pairings of HCV sequences of subtype 1a. Both partners of Couple 17 were infected with both HCV 1a and 1b strains, and their 1b strains were more similar to each other than 99% of random pairings of HCV 1b sequences, however, their 1a strains were no more closely related than to random HCV isolates in the population. Both partners of Couple 14 were infected with HCV strains 2b and 1a.

The 2b strains were highly similar, with only 1.8% difference in basepairs over a 944 basepair region analyzed, whereas, their 1a strains were no more closely related than random pairs of 1a sequences in the population. The HCV isolates in couples 9, 11 and 13 were no more similar to each other than random HCV isolates of the same subtype in the population.

Among the partners with highly-related strains (Couples 14, 15 and 17), the estimated minimum divergence time was 6.5 years for couple 14, whose sexual relationship duration was 18 years; 14.6 years for couple 15, whose sexual relationship duration was 28 years; and 6.2 years for couple 17, whose sexual relationship duration was 10.0 years. The risk factor profiles of Couple 14 revealed that the female partner had a history of IDU and the male no identifiable risk factors for HCV infection other than contact with his female partner. In Couple 17, the female partner had a history of IDU and both partners reported more than 20 prior sexual partners, a history of sexual transmitted diseases and snorting of drugs. In Couple 15, the male partner had a history of IDU, of being stuck by a sharp bloody object while working in a hospital, and more than 20 prior sexual partners; both partners reported snorting drugs and sharing snorting equipment with each other.

Prevalence and Incidence of HCV Infection in Partners

While the overall prevalence of HCV infection among the partners of anti-HCV-positive index persons was 20/500 (4%), the prevalence of HCV infection among partners potentially attributable to sexual contact was 3/500 (0.6%, 95% CI: 0.0%, 1.3%) assuming all HCV RNA negative partners were discordant (minimum estimate) and 6/500 (1.2%, 95% CI 0.2%, 2.2%) assuming all HCV RNA negative, antibody concordant couples were concordant (maximum estimate), respectively.

Based upon the frequencies of sexual contact and length of relationships reported, a cumulative 8,377 person-years of risk for acquiring HCV by sexual activity was calculated. With 3 viremic confirmed concordant couples and 3 possible concordant couples, the estimated incidence of HCV infection among partners ranged from 3.6 per 10,000 person-years (95% CI: 0.0, 7.7) (minimum estimate) to 7.2 per 10,000 person-years (95% CI: 1.3, 13.0) (maximum estimate). The estimated risk per sexual contact ranged from 1 per 380,000 (95% CI 1/600,000, 1/280,000) to 1 per 190,000 (95% CI 1/1.03 mil, 1/100,000).

Concordantly-infected couples were no more likely to share blood-contaminated objects, such as nail clippers, razors, and toothbrushes, than couples in which one partner remained uninfected (0.0% vs. 10.1%, p=1.00), but were more likely to have vaginal intercourse during menses (100.0% vs. 65.6%, p= 0.55) and anal intercourse (66.7% vs. 30.2%, p=0.22), and less likely to use condoms (0.0% vs. 30.4%, p=0.56). These differences, however, were not statistically significant.

DISCUSSION:

Sexual transmission of HCV among monogamous heterosexual couples is an extremely infrequent event. The maximum prevalence of HCV infection among sexual partners of persons with chronic HCV infection was only 1.2%, and the maximum incidence of HCV transmission by sex was 0.07% per year or ~1 per 190,000 sexual contacts. Condom use was infrequent among the study participants and decreased over the duration of the sexual relationship, indicating that the very low rate of sexual transmission in our study population was not due to use of barrier methods during sexual activity.

This estimate includes couples who were antibody concordant by serotyping assays but without confirmation of HCV strain relatedness by phylogenetic analysis because at least one of the partners was HCV RNA negative. By including these couples, we minimized selection bias, but since couples with the same genotype/serotypes may not be infected with the same strain of HCV, we provided maximum (including aviremic serotype concordant couples) and minimum (based on viremic couples only) estimates of HCV prevalence and incidence, The minimum estimate of prevalence of HCV infection among viremic couples was 0.6% (95% CI: 0.0%, 1.3%) and the incidence was 0.04% per year.

Sexual transmission of HCV presumably occurs when infected serum-derived body fluids are exchanged across mucosal surfaces. Potential factors that may influence this exchange include the titer of virus, the integrity of the mucosal surfaces, and the presence of other genital infections (viral or bacterial). Studies to detect HCV RNA in semen (seminal fluid and cells), vaginal secretions, cervical smears, and saliva have yielded mixed results (14-20). Failure to detect HCV RNA in body secretions from chronically infected persons may be due to technical factors including specimen collection and storage, and the inability to exclude cellular components and to overcome the presence of polymerase inhibitors in body fluids. Even in studies employing optimal methods to detect HCV RNA, the minority of samples were positive for HCV RNA and all positive samples were of low titer (≤102 IU/mL) (19-20). A low titer of virus in genital secretions may be one reason that HCV is transmitted less efficiently than hepatitis B virus or HIV (21-22). Additionally, transmission of infection by sex may require a specific genital tract environment such as disrupted mucosal integrity or the presence of viral or bacterial coinfections. These factors may explain the recent reports of HCV transmission by sex in HIV-infected men who have sex with men (23).

Epidemiologically, specific factors that facilitate sexual transmission of HCV have not been identified, although most studies were not large enough to do so. Our study is the largest conducted in the United States and the first to include a rigorous assessment of sexual practices, none of which were associated with concordant HCV-positivity in couples. Although a considerably larger sample size might yield different results, the very low estimated overall transmission risk indicates that any risk for infection from engaging in specific "high-risk" practices would be very low. Thus, this study supports the current recommendations that persons with HCV infection in long-term monogamous relationships need not change their sexual practices (2). Prospective studies from other countries of monogamous couples provide additional support for this recommendation (5, 6). An Italian study of 775 HCV-negative partners followed for 10 years average, identified new HCV infection in 3 partners but none of these partners had viral strains related to those in the HCV-infected partner, indicating an outside source of infection rather than possible sexual transmission (6). However, this study excluded 33 partners who were infected at baseline, introducing a potential bias into the study. It is possible that that risk period of HCV acquisition by sexual contact by be early in the relationship and exclusion of infected partners in long-term relationships, excludes those partners at greatest risk. In contrast to the Italian study, we chose to include all anti-HCV positive partners and rely about the phylogenetic analysis and detailed risk histories to estimate likelihood of sexual transmission. The ideal prospective study to assess risk of HCV transmission among monogamous couples would target HCV-negative partners initiating a sexual relationship with an HCV-infected individual, but such a study would be extremely difficult to execute.

Interestingly, in two couples (Couples 14 and 17), each of the partners had evidence of HCV superinfection with only one of the strains phylogenetically similar in both partners. In Couple 14, it seems likely that the related strain was transmitted from the partner with a history of IDU to the partner who reported no risk factors for HCV infection other than contact with the infected partner. However, the origin of the unrelated HCV strain in the partner with no other HCV-related risk factors is unexplained. In Couple 17, the index person and partner both had different risk factors for HCV, which could explain why each member of the couple was infected with different HCV strains while they also shared a similar strain that was likely transmitted from one partner to the other. These cases highlight the complexity of using phylogenetic analysis to determine the direction or mode of transmission in individual situations when events occurred at unknown times in the past.

Among the 12 couples that had concordant (or indeterminant) HCV genotypes or serotypes, 50% were HCV RNA negative. This rate of spontaneous clearance is similar to that observed among persons infected at younger (<30 years) ages (by transfusion of whole blood, receipt of contaminated Rh immune globulin, IDU, or accidental needlestick injuries), and prospectively followed for 20 years (24-26). Although a younger age at infection might explain the high proportion of anti-HCV-positive, HCV RNA-negative partners in our study, one might speculate that repeated exposures to small "doses" of HCV resulted in an immunization-like effect or facilitated viral clearance once infection occurred.

We acknowledge that we have not genetically "proven" transmission among the phylogenetically linked partners, but rather have presented strong evidence for such a transmission. The method we used are much more effective for excluding possible transmission than it is for confirming it. The consensus sequence of the virus is heavily dominated by a handful of dominant quasispecies, and it drifts relatively slowly. If the genetic distance is not significantly more similar between the pairs than to the rest of the population, then there is no realistic chance the dominant strains came from the same source. Proving (or providing strong evidence for) infection with HCV from a common source is difficult for several reasons. 1. HCV passes a bottleneck upon infection (it has been estimated that only a dozen to <100 infectious particles initiate an infection, and these may not be randomly sampled from the donor quasispecies). Therefore, it is possible even with deep sequencing that finding identical quasispecies variants shortly after infection may not be possible. 2. HCV rapidly adapts to a new host over the first 1-2 months of infection, leading to a burst of diversity and genetic drift. During the rapid expansion in a new host there is little constraining adaptive immunity, and consequently novel variants are not selected out as rapidly as in an established infection, and immune escape variants that were selected in the donor often revert to a more-fit sequence. 3. HCV's mutation rate is far higher than its fixation rate (i.e., the number apparent from population sequencing like we did). Therefore, at a quasispecies level, the viral sequence is essentially "shimmering" from the combined effects of random mutation and its opponent, negative selection. This mandates a rather careful genetic analysis to prove common-source infection, and this problem rapidly increases with time since infection. Finally, deep sequencing is quite error prone and consequently rather extensive statistical treatment of the data are required to be sure that rare variants actually exist in a sample. This means that even if identical reads are reported in 2 paired samples, one or both of them could easily be a sequencing error. The integration of these issues is that it would be perfectly possible by a careful quasispecies analysis or a deep sequencing analysis to prove an identical source for 2 infections shortly after transmission, but the ability to prove a common source decays relatively quickly with time and if difficult in situations where the transmission occurred many years in the past.

Limitations of this study include its cross-sectional nature. A prospective cohort would be the ideal study design to determine incident HCV infections among uninfected partners but the logistics and cost of undertaking such a longitudinal study are daunting, given the low incidence of infection. Unlike prior studies, we sought to overcome the limitations of the cross-sectional design by obtaining a detailed relationship history of sexual practices utilizing techniques similar to those used to obtain lifetime alcohol use histories. Since the partner's HCV status was unknown in the majority of cases prior to history-taking, there would be minimal effect of differential bias in recall of sexual or other shared practices. Regardless, some participants may have unacknowledged histories of IDU or other sensitive risk factors, a limitation we tried to minimize by screening each participant on multiple occasions. Recall bias is a potential limitation with any cross-sectional study, but we found no difference in completeness of the sexual histories among HCV-positive versus negative couples. Another potential limitation was the sample size and the small number of positive partners for stratified analysis. Finally, the study population may not be representative. While index persons were similar in age and gender distribution to HCV-positive adults identified in the general population (1), the study population was predominantly Non-Hispanic White and the majority had an education level beyond high school.

In summary, we conclude that HCV transmission by sex from chronically infected persons to their heterosexual partners in a long-term monogamous relationship likely occurs but is a rare event. Our results provide a basis for specific counseling messages that clinicians can use with their patients. These messages should be qualified given the limitations of the sample size, but they support the current national recommendations that couples not change their sexual practices if they are in a monogamous heterosexual relationship.

Source

lifeplus

by PinkNews.co.uk Staff Writer
7 January 2013, 5:00pm

A new iPhone App, ‘Life plus’, has been launched by the sexual health charity Terrence Higgins Trust with funding from the Elton John AIDS Foundation to help the 5,000 members of its myHIV service get access to information on their condition wherever they go.

It is free to download, and contains tools for monitoring CD4 and viral load levels, storing health and treatment information, and setting medication and appointment reminders.

Life plus also includes links to a library of information about living well with HIV, including specialised advice on employment, housing, travel, and disclosure.

Lisa Power, Policy Director for Terrence Higgins Trust, said: “Since we launched myHIV, we’ve found a significant proportion of people have been accessing their personal information via an iPhone.

“There are a number of situations in which people with HIV need a quick reminder of dates, times and information about medication, especially when they’re newly diagnosed. They might use the app to log their blood counts during clinic visits, or to keep track of questions to ask their HIV specialist at their next appointment. “Life plus transforms the tools from myHIV into a trusted resource people can carry in their pockets.”

myHIV is funded by the Elton John AIDS Foundation. 5,000 have registered to use the myHIV website since its launch in 2011.

The app can be downloaded by clicking here.

Source

Public release date: 7-Jan-2013

Contact: Hilary Glover
hilary.glover@biomedcentral.com
44-020-319-22370
BioMed Central

51444_web

Polymorphisms (SNP) in the gene coding for interleukin-28 (IL28B) influence natural hepatitis C viral (HCV) clearance and response to pegylated interferon-α plus ribavirin (PEG-IFN/RBV).

Credit: María A Jiménez-Sousa, Amanda Fernández-Rodríguez, María Guzmán-Fulgencio, Mónica García-Álvarez and Salvador Resino.

A metanalysis published in BioMed Central's open access journal BMC Medicine has confirmed that polymorphisms (SNP) in the gene coding for interleukin-28 (IL28B) influence natural hepatitis C viral (HCV) clearance and response to pegylated interferon-α plus ribavirin (PEG-IFN/RBV). Information about IL28B genotype could be used to provide personalized medicine and target treatment options effectively.

Over 200 million people worldwide are chronically infected with hepatitis C virus (HCV) and about a quarter of these will go on to develop cirrhosis of the liver. Treatment with (PEG-IFN/RBV) only works in 40-80% of patients, depending in part on HCV strain, and treatment often has severe side effects. It is consequently important to separate people who will not respond to treatment, from those who may, so that treatment is targeted effectively.

Researchers from the Health Institute Carlos III, Spain, incorporated 67 studies that investigated IL28B polymorphisms with the suppression of viral activity to undetectable levels (sustained virologic response - SVR), and ten that looked at IL28B polymorphisms and spontaneous clearance, into a metanalysis. Approximately 23,500 people were included overall.

The results of this analysis showed that IL28B polymorphisms influence how well IFN treatment works and natural clearance of HCV infection. Having a favourable genotype at any one of seven IL28B polymorphisms equated to more than double the probability of achieving SVR. The study also found that two SNP were associated with spontaneous clearance. Detailed analysis showed that the effect of ethnicity and viral type also influenced the strength of individual association. Consequently the association between favourable variants and SVR for HCV types 2 and 3 was three times lower than types 1 and 4.

María Ángeles Jiménez-Sousa, Amanda Fernández-Rodríguez and Salvador Resino who led this study explained, "Treatment with (PEG-IFN/RBV) is costly and can have side effects which prevent patient compliance. Consequently knowing a patient's IL-28B status will help target interferon treatment to those who will benefit most, and play a substantial role in the selection of candidates for standard treatment versus triple therapy with direct-acting antivirals (DAA). Also, because IL28B genotyping needs be performed only once in a patient's life, it is relatively cheap."

###

Media Contact

Dr Hilary Glover
Scientific Press Officer
BioMed Central
Mob: 44-778-698-1967

Notes

1. Meta-analysis: implications of interleukin-28B polymorphisms in spontaneous and treatment-related clearance for patients with hepatitis C
María A Jiménez-Sousa, Amanda Fernández-Rodríguez, María Guzmán-Fulgencio, Mónica García-Álvarez and Salvador Resino
BMC Medicine (in press)

Please name the journal in any story you write. If you are writing for the web, please link

to the article. All articles are available free of charge, according to BioMed Central's open access policy.

Article citation and URL available on request on the day of publication.

2. BMC Medicine is the flagship medical journal of the BMC series, publishing original research, commentaries and reviews that are either of significant interest to all areas of medicine and clinical practice, or provide key translational or clinical advances in a specific field. @BMCMedicine

3. This research is the latest addition to an ongoing article collection by BMC Medicine on Personalized medicine: genes, biomarkers and tailored treatment.

4. Images are to be credited to María A Jiménez-Sousa, Amanda Fernández-Rodríguez, María Guzmán-Fulgencio, Mónica García-Álvarez and Salvador Resino.

5. BioMed Central is an STM (Science, Technology and Medicine) publisher which has pioneered the open access publishing model. All peer-reviewed research articles published by BioMed Central are made immediately and freely accessible online, and are licensed to allow redistribution and reuse. BioMed Central is part of Springer Science+Business Media, a leading global publisher in the STM sector. @BioMedCentral

Source

Scientists say vaccine temporarily brakes HIV

alg-hiv-cure-jpg

"It is the most solid demonstration in the scientific literature that a therapeutic vaccine is possible," researchers said in a statement.

AFP RELAXNEWS
Friday, January 4, 2013, 3:12 PM

A team of Spanish researchers say they have developed a therapeutic vaccine that can temporarily brake growth of the HIV virus in infected patients.

The vaccine, based on immune cells exposed to HIV that had been inactivated with heat, was tested on a group of 36 people carrying the virus and the results were the best yet recorded for such a treatment, the team said.

"What we did was give instructions to the immune system so it could learn to destroy the virus, which it does not do naturally," said Felipe Garcia, one of the scientists in the team at Barcelona University's Hospital Clinic.

The therapeutic vaccine, a shot that treats an existing disease rather than preventing it, was safe and led to a dramatic drop in the amount of HIV virus detected in some patients, said the study, published Wednesday in Science Translation Medicine.

After 12 weeks of the trial, the HIV viral load dropped by more than 90 percent among 12 of the 22 patients who received the vaccine. Only one among the 11 patients who received a control injection without the vaccine experienced a similar result.

After 24 weeks, the effectiveness had begun to decline, however, with seven of the 20 remaining patients receiving the vaccine enjoying a similar 90-percent slump in viral load. No-one in the control group of 10 patients experienced such a decline in the virus.

The vaccine lost its effectiveness after a year, when the patients had to return to their regular combination therapy of anti-retroviral drugs.

Researchers said the results were similar to those achieved with a single anti-retroviral drug, used to block the growth of HIV.

"It is the most solid demonstration in the scientific literature that a therapeutic vaccine is possible," they said in a statement.

The vaccine allowed patients temporarily to live without taking multiple medicines on a daily basis, which created hardship for patients, could have toxic side-effects over the long term and had a high financial price, the team said.

"This investigation opens the path to additional studies with the final goal of achieving a functional cure -- the control of HIV replication for long periods or an entire life without anti-retroviral treatment," the researchers said in a statement.

"Although we still have not got a functional cure, the results published today open the possibility of achieving an optimal therapeutic vaccine, or a combination of strategies that includes a therapeutic vaccine, and could help to reach that goal," they said.

The team said it took seven years to get to this point, and the researchers would now work on improving the vaccine and combining it with other therapeutic vaccines over the next three or four years.

According to latest UN figures, the number of people infected by HIV worldwide rose to 34 million in 2011 from 33.5 million in 2010.

Source

Boceprevir HCV Liver Transplant

Download the PDF here

Download the PDF here

HepDart Dec 2011: Pharmacokinetic Interaction Between the HCV Protease Inhibitor Boceprevir and the Calcineurin Inhibitors Cyclosporine and Tacrolimus (12/07/11)

HepDart Dec 2011: Coadministration of the HCV Protease Inhibitor Boceprevir Has No Clinically Meaningful Effect on the Pharmacokinetics of the Selective Serotonin Reuptake Inhibitor Escitalopram in Healthy Volunteers (12/07/11)

same research, just published

pdf of editorial published in Sept 2012, attached for download

Pharmacokinetic interaction between the hepatitis C virus protease inhibitor boceprevir and cyclosporine and tacrolimus in healthy volunteers

Hepatology Nov 2012

Abstract

The hepatitis C virus protease inhibitor boceprevir is a strong inhibitor of cytochrome P450 3A4 and 3A5 (CYP3A4/5). Cyclosporine and tacrolimus are calcineurin inhibitor immunosuppressants used to prevent organ rejection after liver transplantation; both are substrates of CYP3A4. This two-part pharmacokinetic interaction study evaluated boceprevir with cyclosporine (part 1) and tacrolimus (part 2). In part 1, 10 subjects received single-dose cyclosporine (100 mg) on day 1, single-dose boceprevir (800 mg) on day 3, and concomitant cyclosporine/boceprevir on day 4. After washout, subjects received boceprevir (800 mg three times a day) for 7 days plus single-dose cyclosporine (100 mg) on day 6. In part 2A, 12 subjects received single-dose tacrolimus (0.5 mg). After washout, they received boceprevir (800 mg three times a day) for 11 days plus single-dose tacrolimus (0.5 mg) on day 6. In part 2B, 10 subjects received single-dose boceprevir (800 mg) and 24 hours later received boceprevir (800 mg) plus tacrolimus (0.5 mg). Coadministration of boceprevir with cyclosporine/tacrolimus was well tolerated. Concomitant boceprevir increased the area under the concentration-time curve from time 0 to infinity after single dosing (AUCinf) and maximum observed plasma (or blood) concentration (Cmax) of cyclosporine with geometric mean ratios (GMRs) (90% confidence interval [CI]) of 2.7 (2.4-3.1) and 2.0 (1.7-2.4), respectively. Concomitant boceprevir increased the AUCinf and Cmax of tacrolimus with GMRs (90% CI) of 17 (14-21) and 9.9 (8.0-12), respectively. Neither cyclosporine nor tacrolimus coadministration had a meaningful effect on boceprevir pharmacokinetics. Conclusion: Dose adjustments of cyclosporine should be anticipated when administered with boceprevir, guided by close monitoring of cyclosporine blood concentrations and frequent assessments of renal function and cyclosporine-related side effects. Administration of boceprevir plus tacrolimus requires significant dose reduction and prolongation of the dosing interval for tacrolimus, with close monitoring of tacrolimus blood concentrations and frequent assessments of renal function and tacrolimus-related side effects.

Boceprevir (800 mg three times a day), in combination with pegylated interferon-α (PEG-IFNα) and ribavirin, was approved in the United States and Europe for the treatment of genotype 1 chronic hepatitis C infection in adult patients with compensated liver disease. As a structurally novel ketoamide serine protease inhibitor of the hepatitis C virus (HCV) nonstructural 3 (NS3/4A) active site, boceprevir has been shown to significantly increase rates of sustained virologic response (SVR) when added to PEG-IFNα plus ribavirin as compared with treatment with PEG-IFNα plus ribavirin alone.1, 2 In treatment-naive patients, SVR rates increased from 38% among patients treated with PEG-IFNα plus ribavirin to 63%-66% in those receiving boceprevir plus PEG-IFNα and ribavirin.2 Similarly, in treatment-experienced patients, SVR rates were 21% with PEG-IFNα plus ribavirin and 59%-66% in those receiving boceprevir plus PEG-IFNα and ribavirin.1 Boceprevir (800 mg three times a day) in combination with PEG-IFNα and ribavirin, was approved for the treatment of genotype 1 chronic hepatitis C infection in adult patients with compensated liver disease in the United States and Europe in 2011. Metabolism of boceprevir occurs by aldo-ketoreductase to form inactive keto-reduced metabolites and by cytochrome P450 3A4 and 3A5 (CYP3A4/5).3 Boceprevir is also a substrate for the efflux pump P-glycoprotein (P-gp) and is an inhibitor of OATP1B1.4

Hepatitis C-related liver cirrhosis is a frequent cause of liver transplantation, and because recurrent viremia is common among patients who are viremic at the time of transplantation, treatment of HCV infection is frequently required after transplantation.5 Cyclosporine and tacrolimus are calcineurin inhibitors widely used to prevent solid organ transplant rejection. Both agents are substrates for CYP3A6, 7 and P-gp.8 Cyclosporine is also an inhibitor of several other transporter proteins, including OATP1B1 and OATP1B3.9 Both agents have a narrow therapeutic index, with therapeutic monitoring being required to avoid either underexposure, which can result in organ rejection, or excess exposure, which may cause nephrotoxicity, neurotoxicity, hypertension, or gastrointestinal toxicity. Boceprevir is a strong inhibitor of CYP3A4/5 and would be anticipated to increase exposure to cyclosporine and tacrolimus upon coadministration, as was previously observed for another recently approved HCV NS3/4A protease inhibitor (telaprevir, Incivek, Vertex Pharmaceuticals, Inc.).10 In this study, the pharmacokinetic (PK) interactions between boceprevir and tacrolimus/cyclosporine were separately evaluated.

AE, adverse event; AUCinf, area under the concentration-time curve from time 0 to infinity after single dosing; AUClast, area under the concentration-time curve from time 0 to time of last measurable sample; BMI, body mass index; CI, confidence interval; Cmax, maximum observed plasma (or blood) concentration; CL/F, apparent total body clearance; CYP3A4/5, cytochrome P450 3A4 and 3A5; GMR, geometric mean ratio; HCV, hepatitis C virus; HIV, human immunodeficiency virus; HPLC, high-performance liquid chromatography; LLOQ, lower limit of quantification; NS3/4A, HCV nonstructural 3/4A protein; PEG-IFNα, pegylated interferon-α; P-gp, P-glycoprotein; PK, pharmacokinetics; SVR, sustained virologic response; t1/2, terminal phase half-life; Tmax, time to maximum observed plasma (or blood) concentration.

Subjects and Methods

This was a single-center, two-part, open-label study. The study was conducted in accordance with the principles of Good Clinical Practice and was approved by the appropriate institutional review boards and regulatory agencies. All subjects provided written informed consent prior to participation in study-related procedures.

Subjects

Healthy adult male and female subjects aged 18-55 years with an inclusive body mass index (BMI) of 18-32 kg/m2 were enrolled. All subjects were required to be free of any clinically significant disease and have clinical laboratory tests (including complete blood counts, blood chemistries, urinalysis, electrocardiogram, and vital signs) within normal limits or clinically acceptable to the investigator. Premenopausal women and men were required to use a medically accepted method of contraception. All subjects were required to provide written informed consent and to adhere to dose and visit schedules. No information on CYP3A4/5 polymorphisms in the study subjects was available prior to dosing.

Subjects who were pregnant, breastfeeding, or who (in the opinion of the investigator) were unable to participate optimally in the study were excluded. Additional exclusion criteria were: a surgical or medical condition that might significantly alter the absorption, distribution, metabolism, or excretion of any drug; a recent history of any infectious disease; and infection with hepatitis B, hepatitis C, or human immunodeficiency virus (HIV). Subjects with a history of alcohol or drug abuse in the past 2 years, who smoked >10 cigarettes or had equivalent tobacco use per day, or who had elevated liver function tests also were excluded.

Study Design

This study consisted of two parts, each with a fixed-sequence design. Part 1 was designed to assess the effect of cyclosporine on boceprevir PK and the effect of boceprevir on cyclosporine PK (Fig. 1A). In part 2, the effect of boceprevir on tacrolimus PK and the effect of tacrolimus on boceprevir PK were assessed (Fig. 1B). In both parts of the study, boceprevir was administered orally as 4 x 200-mg capsules swallowed (not crushed or chewed) with a glass of water. A meal or light snack preceded boceprevir. During hospitalization (part 1, days -1 to 5 and days 10 to 13; part 2a, days -1 to 5 and days 11 to 19; part 2b, days -1 to 3), the subjects were on standard meals, including a standard breakfast comprising 828 kcal (20.2% fat, 14.5% protein, 65.2% carbohydrates). Neoral (soft gelatin capsule, 100 mg) was used for cyclosporine treatment and Prograf (capsule, 0.5 mg) was used for tacrolimus treatment. In cases of boceprevir and cyclosporine or tacrolimus coadministration, drugs were taken concomitantly with 240 mL of water.

Part 1: Cyclosporine.

On day 1, after a standard breakfast, all subjects received a single dose of oral cyclosporine (100 mg). PK samples for cyclosporine determination were obtained predose on day 1 and then at selected time points until 48 hours postdose on day 3. After the 48-hour sample on day 3, all subjects received a single oral dose of boceprevir (800 mg) with PK samples obtained predose and then at selected intervals until 24 hours postdose (on day 4). After the final boceprevir PK sample had been obtained on the morning of day 4, all subjects received single doses of boceprevir (800 mg) and cyclosporine (100 mg) and PK samples for boceprevir were again obtained at intervals up to 24 hours postdose.

From the morning of day 6 through the evening of day 12, all subjects received boceprevir 800 mg three times a day. Plasma samples for trough boceprevir levels were obtained before morning dose on days 10, 11, 12, and 13. In addition, on day 11, all subjects received a single 100-mg oral dose of cyclosporine together with their scheduled dose of boceprevir. PK samples for cyclosporine concentrations (at steady state boceprevir) were then collected before cyclosporine dosing on day 11 until 48 hours postdose on the morning of day 13. All subjects then returned for final clinic safety assessments on day 20.

Part 2: Tacrolimus.

Because of the anticipated long half-life of tacrolimus, 2 separate enrollment cohorts were employed to study the PK interactions between tacrolimus and boceprevir. Cohort A was designed to evaluate the effect of boceprevir on tacrolimus, and cohort B was designed to evaluate the effect of tacrolimus on boceprevir.

In cohort A, following a standard breakfast on day 1, all subjects received a single dose of oral tacrolimus (0.5 mg). PK samples were obtained predose and then at selected intervals until the morning of day 7 (equivalent to a postdose period of 144 hours). From the morning of day 8 through the evening of day 16, subjects then received boceprevir 800 mg three times a day. Plasma samples for trough levels of boceprevir were obtained before the morning dose on days 12, 13, 14, 15, 16, and 17. In addition, on day 13, subjects received a single oral dose of tacrolimus (0.5 mg) and PK samples for evaluation of tacrolimus levels (at steady state boceprevir) were collected from day 13 predose until the morning of day 19 (equivalent to 144 hours postdose). All subjects returned to the clinic for a final safety assessment on day 24.

In cohort B, on the morning of day 1 all subjects received a standard breakfast and were then administered a single oral dose of boceprevir (800 mg). PK samples for boceprevir determination were obtained predose and at selected intervals until 24 hours postdose. After the final PK sample was obtained on day 2, subjects received another single dose of boceprevir (800 mg) together with a single dose of tacrolimus (0.5 mg). PK samples for boceprevir (in the presence of tacrolimus) were collected predose and then at selected intervals until the morning of day 3 (equivalent to 24 hours postdose). On day 3, after the last PK sample had been obtained, safety assessments were performed, and subjects were then discharged. All subjects returned to the clinic for final safety assessments on day 10.

Bioanalysis

Concentrations of cyclosporine and tacrolimus in collected human blood samples were determined using high-performance liquid chromatography (HPLC) and HPLC-tandem mass spectrometry, respectively, at PharmaNet Canada (Quebec, Quebec, Canada). The lower limit of quantification (LLOQ) for the cyclosporine assay was 2 ng/mL; the linear calibration range was 2-1,002 ng/mL. The LLOQ for the tacrolimus assay was 50.52 pg/mL; the linear calibration range was 50.52 to 50,520 pg/mL. Concentrations of boceprevir and its metabolites in collected human plasma samples were determined using HPLC-tandem mass spectrometry at PPD (Middleton, WI). Concentrations of boceprevir were determined as the sum of concentrations of two enantiomers of boceprevir: SCH 534128 and SCH 534129. Concentrations of SCH 629144, an inactive metabolite of boceprevir, were obtained as the sum of concentrations of four analytes: SCH 783004, SCH 783005, SCH 783006, and SCH 783007. The overall LLOQ for boceprevir was 4.80 ng/mL, and the overall LLOQ for SCH 629144 was 2.50 ng/mL.

Endpoints

Standard PK variables were assessed, including area under the concentration-time curve from time 0 to the time of the last measurable sample (AUClast); area under the concentration-time curve from time 0 to infinity after single dosing (AUCinf); maximum observed plasma (or blood) concentration (Cmax); time to maximum observed plasma (or blood) concentration (Tmax); terminal phase half-life (t1/2); and apparent total body clearance (CL/F). Safety variables including vital signs, electrocardiograms, adverse events (AEs), hematology, and blood chemistries also were monitored regularly.

Statistical Analysis

Assessment of safety and tolerability included all subjects who received at least one dose of boceprevir, and PK analyses were based on the per-protocol population, which included all protocol-compliant subjects. PK parameters were summarized by treatment using descriptive statistics and graphics. The log-transformed AUC and Cmax values were analyzed using mixed effect modeling extracting the effect due to treatment as fixed effect, and subject as random effect.

Geometric mean ratios (GMRs) and associated 90% confidence intervals (CIs) were calculated using the following predefined limits to define clinically meaningful drug-drug interactions. In view of the narrow therapeutic window and high degree of intersubject variability of cyclosporine, confidence bounds for the 90% CI for AUC or Cmax of 0.80-1.25 were chosen to assess the effect of boceprevir on cyclosporine levels. Tacrolimus monitoring using trough concentrations is generally easier and more reliable than cyclosporine monitoring using the modified AUC format, which is prone to greater individual point variability. The effect of boceprevir on tacrolimus was considered not clinically meaningful if the 90% CI for AUC and Cmax of tacrolimus with boceprevir versus tacrolimus alone would be between 0.7 and 1.43. Analysis of the available clinical data for 800 mg three times a day boceprevir in healthy volunteers and patients indicated that confidence bounds for the 90% CI for AUC or Cmax of (0.50-2.00) would be appropriate to control resistance generation and/or treatment failure as well as prevent clinically significant safety concerns (data on file).

Results

Interaction of Boceprevir and Cyclosporine

Ten subjects were enrolled and completed the cyclosporine study. There were seven females and three males, all of Hispanic or Latino ethnicity. The overall mean age was 36 years (SD 7.1 years), and the mean BMI was 26.8 kg/m2 (SD 2.8 kg/m2).

Effect of Boceprevir on PK Parameters of Cyclosporine.

Coadministration of boceprevir with cyclosporine resulted in increased cyclosporine exposure, with the mean AUCinf increasing from 1,800 ng/hour/mL to 4,870 ng/hour/mL and mean Cmax levels increasing from 388 ng/mL to 737 ng/mL (Fig. 2, Table 1). The GMRs for AUCinf and Cmax parameters for the comparison of cyclosporine plus boceprevir versus cyclosporine alone were 2.7 and 2.0, with 90% CIs for the GMRs falling outside the predefined range for defining clinically meaningful drug-drug interactions of 0.80-1.25 (Table 2). Consistent with the increase in exposure, there was an approximately 2-fold reduction in apparent cyclosporine clearance in the presence of boceprevir (mean CL/F of 21.0 L/hour versus 58.8 L/hour when administered alone; Table 1). The mean cyclosporine half-life increased by approximately 25%, from 11.3 hours to 15.7 hours, in the presence of boceprevir versus cyclosporine alone.

Effect of Cyclosporine on PK Parameters of Boceprevir.

Boceprevir AUCinf and Cmax increased 16% and 8%, respectively (Table 2). The 90% CIs were within the predefined limits of 0.5 and 2.00, so that the observed increase in boceprevir concentrations is not considered clinically meaningful (Table 2). An approximate 2-fold increase in mean Cmax and AUCinf of the inactive metabolite SCH 629144 was observed following coadministration of boceprevir and cyclosporine (data not shown).
Safety.
No subjects discontinued treatment because of an AE, and there were no serious AEs or deaths. Furthermore, no clinically meaningful changes in blood chemistry, hematology, blood pressure, pulse rate, oral body temperature, or electrocardiogram parameters were observed. A total of 21 AEs were reported by eight subjects in the cyclosporine study, all of which were of mild intensity, with 17 considered possibly drug-related. Dysgeusia was the most frequently reported possibly drug-related AE and occurred after cyclosporine-only treatment (n = 1), cyclosporine-boceprevir coadministration (n = 2), and boceprevir-only treatment (n = 7). Flatulence and headache were both reported twice, headache after boceprevir-only treatment and cyclosporine-boceprevir coadministration, flatulence after cyclosporine-only treatment and cyclosporine-boceprevir coadministration. Abdominal distension, abdominal discomfort, and flushing were each reported once.

Interaction of Boceprevir and Tacrolimus

Effect of Boceprevir on PK Parameters of Tacrolimus (Cohort A).

Twelve subjects were enrolled and completed cohort A of the tacrolimus study (female, n = 5; male, n = 7; all Hispanic or Latino) with a mean age of 32.9 years (SD 10.8 years) and a mean BMI of 27.0 kg/m2 (SD 3.03 kg/m2).

Tacrolimus exposure was markedly increased in the presence of boceprevir (Fig. 3, Table 1); the mean AUCinf increased from 21.8 ng/hour/mL to 345 ng/hour/mL upon concomitant administration of tacrolimus and boceprevir, while the mean Cmax levels increased from 0.8 ng/mL to 7.8 ng/mL (Fig. 3, Table 1). The AUCinf and Cmax GMRs for the comparison of tacrolimus plus boceprevir versus tacrolimus alone indicated a 17- and 9.9-fold rise, respectively, with 90% CIs falling outside the predefined range for defining clinically meaningful drug-drug interactions of 0.7 to 1.43 (Table 2). The mean apparent clearance of tacrolimus was approximately 18 times lower after coadministration of tacrolimus and boceprevir (Table 1). There was also an approximate doubling of the mean t1/2 of tacrolimus in the presence of boceprevir.

Effect of Tacrolimus on PK of Boceprevir (Cohort B).

Ten subjects were enrolled and completed cohort B (Hispanic/Latino, n = 9; African American, n = 1). The mean age was 45.4 years (SD 7.9 years) and the mean BMI was 27.27 kg/m2 (SD 3.60 kg/m2). Eight female subjects and two male subjects were included.
The AUCs and Cmax values of boceprevir were essentially unchanged in the presence of tacrolimus compared with boceprevir administration alone (Table 1). The CL/F and the t1/2 of boceprevir were also similar following concomitant administration of boceprevir and tacrolimus. GMRs were close to unity for Cmax, AUClast, and AUCinf, and 90% CIs were all within the predefined range (0.50-2.00), indicating no clinically meaningful effect of tacrolimus on boceprevir PK. The PK parameters of the major metabolite SCH 629144 were essentially the same following coadministration of boceprevir and tacrolimus compared with boceprevir alone administration (data not shown).

Safety.

No subjects discontinued treatment because of an AE, and there were no serious AEs or deaths. Furthermore, no clinically meaningful changes in blood chemistry, hematology, blood pressure, pulse rate, oral body temperature, or electrocardiogram parameters were observed. In cohort A, 21 AEs were reported by seven subjects receiving tacrolimus either alone or in combination with boceprevir. All AEs were of mild intensity, and 18 were considered to be possibly drug-related. Dysgeusia was the most frequently reported drug-related AE (n = 7; only reported in subjects receiving boceprevir with or without tacrolimus, not in subjects only receiving tacrolimus), followed by headache (n = 2; occurring once each after tacrolimus-only and boceprevir-only treatment), gastroesophageal reflux (n = 2; occurring once each after tacrolimus-only and boceprevir-only treatment), abdominal discomfort (n = 2; after boceprevir-only treatment), and chills (n = 2; once after boceprevir-only treatment and once after tacrolimus-boceprevir coadministration). All other possibly drug-related AEs (ie, asthenia, fatigue, and palpitations) were reported once. In addition, five AEs were reported by five subjects in cohort B, all of which were of mild intensity. Two AEs were considered possibly drug-related (dysgeusia, n = 1; headache, n = 1; both after boceprevir-only treatment).

Discussion

There is a significant unmet clinical need for the treatment of recurrent hepatitis C after liver transplantation. SVR rates for patients receiving PEG-IFNα and ribavirin after liver transplantation are low, with less than one-third of patients achieving SVR.11 Furthermore, treatment-related toxicity represents a significant barrier to completion of therapy.12 Thus, the liver transplantation population represents a subgroup of patients with chronic hepatitis C who could potentially derive significant clinical benefit from the use of direct-acting antiviral agents. Calcineurin inhibitors, such as cyclosporine and tacrolimus, are routinely administered in these patients as immunosuppressants to prevent allograft rejection. Given the narrow therapeutic index within which these agents are effective, and the subsequent need for therapeutic monitoring, a clear and detailed understanding of their propensity for drug-drug interactions is required before their concomitant use with new pharmacologic agents.

Cyclosporine and tacrolimus are both substrates of CYP3A4/5. Because boceprevir is a strong inhibitor of CYP3A4, coadministration with boceprevir would be anticipated to increase exposure to these calcineurin inhibitors. The doses of cyclosporine (100 mg) and tacrolimus (0.5 mg) used in this study were optimized for investigation of the potential for drug-drug interactions between the individual drugs and boceprevir without jeopardizing subject safety. Consequently, doses were much lower (tacrolimus) than or at the lower end (cyclosporine) of standard therapeutic dosing in order to maintain a safety margin if significant elevations in immunosuppressant concentrations were observed upon boceprevir coadministration. In addition, cyclosporine and tacrolimus were each given as single doses to mitigate potential safety concerns (eg, those associated with accumulation). Boceprevir was dosed to steady state in order to ensure that the maximum inhibitory potential of the drug was assessed. Also, to avoid an extremely long study duration, the interaction of tacrolimus as substrate and as perpetrator was studied in different cohorts of subjects.

Concomitant boceprevir administration increased the AUCinf and Cmax of cyclosporine by 2.7- and 2.0-fold, respectively. Boceprevir coadministration had a substantial effect on the PK of tacrolimus, with coadministered geometric mean AUCinf and Cmax parameter values approximately 17-fold and 10-fold higher than when tacrolimus was administered alone. Drug interactions also have been identified between cyclosporine and tacrolimus and telaprevir, another recently approved HCV NS3/4A protease inhibitor.10 Coadministration of telaprevir led to a 4.6- and 1.3-fold increase in the dose-normalized AUCinf and Cmax of cyclosporine and a 70- and 9.3-fold increase in the dose-normalized AUCinf and Cmax of tacrolimus, respectively.

Neither tacrolimus nor cyclosporine had any notable effect on the PK of boceprevir. Boceprevir is metabolized by two pathways: aldo ketoreductase, which leads to (among others) a reduced, inactive metabolite (SCH 629144), and CYP3A4/5.3 Although the Cmax and AUC of boceprevir were essentially unchanged in the presence of cyclosporine compared with boceprevir administration alone, a two-fold increase in the Cmax and AUC of the metabolite SCH 629144 was observed after coadministration of boceprevir and cyclosporine. Because this metabolite is not active against HCV, this increase has no consequences with respect to clinical efficacy; however, it is not known whether the increase in metabolite exposure could potentially increase side effects. Because cyclosporine is an inhibitor of several proteins in both the drug-metabolizing enzyme and the uptake/efflux transporter systems, data in the present study do not provide insight into whether the increase in SCH 629144 levels is due to its effect on the enzyme/transporter interplay, resulting in an increase in the formation of SCH 629144, a decrease in the elimination of the metabolite, or a combination of both. The contribution of cyclosporine-based P-gp inhibition on drug interactions could not be assessed in this study, given that the low cyclosporine dose used did not produce plasma concentrations at the levels predicted to incur clinically meaningful P-gp inhibition (1,000-5,000 ng/mL).13 Furthermore, the potential for tacrolimus to inhibit the metabolism of boceprevir may not have been fully assessed in this study because of the low tacrolimus dose used to allow for a large enough safety margin to accommodate the increased concentrations that were expected upon boceprevir coadministration.

Coadministration of boceprevir with cyclosporine or tacrolimus was safe and well tolerated in this group of healthy volunteers. Overall, tolerability was consistent with the known safety profile of boceprevir in healthy subjects14-16 and patients with chronic hepatitis C.1, 2, 16 All AEs were mild, there were no treatment discontinuations due to AEs, and dygeusia was the most frequently reported drug-related AE. There was one event of mild palpitations after multiple-dose boceprevir plus single-dose tacrolimus treatment. Palpitations have previously been identified as uncommon for tacrolimus and as common for boceprevir (when taken together with PEG-IFNα and ribavirin).6, 16

The PK of coadministered boceprevir and the calcineurin inhibitors have not been studied in liver transplant patients, which is a limitation for interpretation of these data. The data in the present study were derived from healthy subjects, and the magnitude of the potential interaction between cyclosporine or tacrolimus and boceprevir in liver transplant patients is not known. Blood concentrations of the calcineurin inhibitors in liver transplant patients with recurrence of HCV are subject to a wider range of influences than those in healthy subjects, which in turn could result in greater interpatient variability. HCV infection itself appears to reduce the dose of cyclosporine or tacrolimus required to achieve a given blood level, probably because of down-regulation of hepatic CYP3A4, impaired function of hepatic P-gp, or both.17 The effect is reversed when the HCV-associated inflammatory response is eliminated by antiviral therapy.18 In addition, liver function can change with time after transplantation.19

Based on the results from the present study, dose reductions of cyclosporine should be anticipated when administered with boceprevir and should be guided by close monitoring of cyclosporine blood levels and frequent assessments of renal function and cyclosporine-related side effects. For tacrolimus, significant dose reduction and prolongation of the dosing interval will be required, along with close monitoring of tacrolimus concentrations and frequent assessments of renal function and tacrolimus-related side effects. Plasma concentrations of other commonly used immunosuppressants such as sirolimus and everolimus may also be increased during coadministration with boceprevir. Thus, close monitoring of immunosuppressant blood levels is recommended here as well. This situation is comparable to that of HIV-coinfected patients after liver transplantation who require treatment with ritonavir-boosted HIV protease inhibitors concomitantly with cyclosporine or tacrolimus. HIV protease inhibitors (eg, lopinavir, darunavir, atazanavir, and ritonavir) are all potent CYP3A4 inhibitors, and several reports describe dose reductions of up to 99% of the calcineurin inhibitors when coadministered with HIV protease inhibitors, with dosing schedules of less than once weekly to maintain adequate cyclosporine and tacrolimus concentrations, or both.20-22 Similarly, a preliminary report of the use of telaprevir in a small number of recipients after liver transplantation suggests that tacrolimus dose reduction and prolongation of the dosing interval have been generally well tolerated.23

Another consideration for the concomitant use of tacrolimus or cyclosporine with boceprevir in liver transplant patients relates to the need to readjust the dose levels of cyclosporine or tacrolimus when boceprevir treatment is completed or discontinued. A previous study using midazolam as a sensitive CYP3A4 probe suggests that CYP3A4 activity returns to baseline levels 48 hours after discontinuation of boceprevir (data on file, Merck & Co., Inc.). Although it is anticipated that standard doses of either immunosuppressant could be resumed soon after boceprevir is discontinued, careful and potentially increased frequency of blood concentration monitoring of immunosuppressants will be required.

In the treatment of chronic HCV, boceprevir is used in combination with PEG-IFNα and ribavirin. These therapies are not expected to influence cyclosporine or tacrolimus levels. Neither inhibition nor induction of cytochrome P450 enzymes or exhibition of cytochrome P450 enzyme-mediated metabolism has been observed in in vitro studies of ribavirin.24 PEG-IFNα has shown increases in activity of CYP2D6 and CYP2C8/9, but not CYP3A4/5.25 None of the PK parameters of boceprevir, PEG-IFNα, or ribavirin have been affected by coadministration.16

In conclusion, coadministration with boceprevir results in clinically meaningful increases in exposure to cyclosporine and tacrolimus in healthy subjects. The magnitude of the potential interaction between cyclosporine or tacrolimus and boceprevir in organ transplantation patients is not yet known but could potentially be higher and more variable than those seen in healthy subjects due to intersubject PK variability and variability associated with disease during the course of antiviral therapy. Therefore, dose adjustments of cyclosporine should be anticipated when administered with boceprevir and should be guided by close monitoring of cyclosporine blood concentrations and frequent assessments of renal function and cyclosporine-related side effects. Concomitant administration of boceprevir with tacrolimus requires significant dose reduction and prolongation of the dosing interval for tacrolimus, with close monitoring of tacrolimus blood concentrations and frequent assessments of renal function and tacrolimus-related side effects.

Source