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UBE2V2 Ubiquitin-conjugating enzyme E2 variant 2 is a protein that in humans is encoded by the UBE2V2 gene. Ubiquitin-conjugating enzyme E2 variant proteins constitute a distinct subfamily within the E2 protein family. # Structure UBE2V2 has sequence similarity to other ubiquitin-conjugating enzymes but lack the conserved cysteine residue that is critical for the catalytic activity of E2s. The protein encoded by this gene also shares homology with ubiquitin-conjugating enzyme E2 variant 1 and yeast MMS2 gene product. # Function UBE2V2 has also been implicated as an intracellular sensor of reactive electrophilic species, which are present in high levels during periods of pathogenic and/or environmental stress. The C69 residue of UBE2V2 is capable of binding with various RES. It has been shown that binding of RES to UBE2V2 promotes UBE2V2-mediated activation of Ube2N, another E2 protein that complexes with UBE2V2. Activated Ube2N has been shown to play a major role in promoting DNA-damage responses. Thus, UBE2V2 may promote genome integrity by directly sensing RES and effecting DNA damage responses. It may also be involved in the differentiation of monocytes and enterocytes. # Interactions UBE2V2 has been shown to interact with HLTF. Although UBE2V2 itself lacks ubiquitin-conjugating activity, it can interact with different Ubiquitin-conjugating enzymes to facilitate their catalytic activities. For instance, UBE2V2 can complex with UBE2N to form a heterodimer capable of synthesizing Lys-63 linked polyubiquitin chains. UBE2V2 may facilitate UBE2N activity by coordinating UBE2N's positioning to promote ubiquitin chain formation specifically at Lys-63, as the ubiquitin molecule has multiple potential Lysine binding sites. Similarly, it has been shown that UBE2V2 interact with the ubiquitin-conjugating enzyme, Ubc13, to induce Ubc13 to adopt an active conformation that can create Lys-63 polyubituitin chains on various substrates. Addition of Lys-63 polyubiquitin chains to intracellular targets is distinct from the canonical Lys-48 polyubiquitin chains in that Lys-63 chains do not mediate proteasomal degradation of its substrate. Although their function remains poorly characterized, Lys-63 chains have been shown to regulate signaling pathways by either activating or inhibiting its target protein function. For example, TRIM5alpha restriction of retroviral reverse-transcription is dependent on UBE2V2/UBE2N-mediated poly-ubiquitination. UBE2V2 has been shown to regulate TRIM21 antiviral activity in an analogous manner.

This report provides recommendations for use of a newly developed recombinant outer-surface protein A (rOspA) Lyme disease vaccine (LYMErix,™ SmithKline Beecham Pharmaceuticals) for persons aged 15-70 years in the United States. The purpose of these recommendations is to provide health-care providers, public health authorities, and the public with guidance regarding the risk for acquiring Lyme disease and the role of vaccination as an adjunct to preventing Lyme disease. The Advisory Committee on Immunization Practices recommends that decisions regarding vaccine use be made on the basis of assessment of individual risk, taking into account both geographic risk and a person's activities and behaviors relating to tick exposure. *sensu lato: including all subordinate taxa of a taxon that would otherwise be considered separately. † sensu stricto: excluding similar taxa that otherwise would be considered together.# INTRODUCTION Lyme disease is a tickborne zoonosis caused by infection with the spirochete Borrelia burgdorferi. The number of annually reported cases of Lyme disease in the United States has increased approximately 25-fold since national surveillance began in 1982; during 1993-1997, a mean of 12,451 cases annually were reported by states to CDC (1,2, CDC, unpublished data, 1998). In the United States, the disease is primarily localized to states in the northeastern, mid-Atlantic, and upper north-central regions, and to several areas in northwestern California (1 ). Lyme disease is a multisystem, multistage, inflammatory illness. In its early stages, Lyme disease can be treated successfully with oral antibiotics; however, untreated or inadequately treated infection can progress to late-stage complications requiring more intensive therapy. The first line of defense against Lyme disease and other tickborne illnesses is avoidance of tick-infested habitats, use of personal protective measures (e.g., repellents and protective clothing), and checking for and removing attached ticks. Early diagnosis and treatment are effective in preventing late-stage complications. Recently, two Lyme disease vaccines have been developed that use recombinant B. burgdorferi lipidated outer-surface protein A (rOspA) as immunogen -LYMErix,™ SmithKline Beecham Pharmaceuticals, and ImuLyme,™ Pasteur Mérieux Connaught. As of publication of this report, only LYMErix has been licensed by the U.S. Food and Drug Administration for use in the United States; therefore, these recommendations apply only to the use of that vaccine. Additional statements will be provided as other Lyme disease vaccines are licensed. Results of a large-scale, randomized, controlled (Phase III) trial of safety and efficacy of LYMErix in persons aged 15-70 years residing in disease-endemic areas of the northeastern and north-central United States indicate that the vaccine is safe and efficacious when administered on a three-dose schedule of 0, 1, and 12 months (3,4 ). Information regarding vaccine safety and efficacy beyond the transmission season immediately after the third dose is not available. Thus, the duration of protective immunity and need for booster doses beyond the third dose are unknown. # CLINICAL FEATURES OF LYME DISEASE Clinical Description Most often, Lyme disease is evidenced by a characteristic rash (erythema migrans) accompanied by nonspecific symptoms (e.g., fever, malaise, fatigue, headache, myalgia, and arthralgia) (5)(6)(7). The incubation period from infection to onset of erythema migrans is typically 7-14 days but can be as short as 3 days or as long as 30 days. Some infected persons have no recognized illness (i.e., asymptomatic infection determined by serologic testing), or they manifest only nonspecific symptoms (e.g., fever, headache, fatigue, and myalgia). Lyme disease spirochetes disseminate from the site of inoculation by cutaneous, lymphatic, and bloodborne routes. The signs of early disseminated infection usually occur from days to weeks after the appearance of a solitary erythema migrans lesion. In addition to multiple or secondary erythema migrans lesions, early disseminated infection can be manifested as disease of the nervous system, the musculoskeletal system, or the heart (5)(6)(7). Early neurologic manifestations include lymphocytic meningitis; cranial neuropathy, especially facial nerve palsy; and radiculoneuritis. Musculoskeletal manifestations can include migratory joint and muscle pains with or without objective signs of joint swelling. Cardiac manifestations are rare but can include myocarditis and transient atrioventricular block of varying degree. B. burgdorferi infection in the untreated or inadequately treated patient can progress to late-disseminated disease from weeks to months after infection (5)(6)(7). The most common objective manifestation of late-disseminated Lyme disease is intermittent swelling and pain of one or some joints, usually large, weight-bearing joints (e.g., the knee). Some patients experience chronic axonal polyneuropathy, or encephalopathy, the latter usually manifested by cognitive disorders, sleep disturbance, fatigue, and personality changes. Infrequently, Lyme disease morbidity can be severe, chronic, and disabling (8,9 ). An ill-defined post-Lyme disease syndrome occurs in some persons after treatment for Lyme disease (10)(11)(12). Lyme disease is rarely, if ever, fatal. # Diagnosis The diagnosis of Lyme disease is based primarily on clinical findings, and treating patients with early disease solely on the basis of objective signs and a known exposure is often appropriate (13 ). Serologic testing can, however, provide valuable supportive diagnostic information in patients with endemic exposure and objective clinical findings that indicate later-stage disseminated Lyme disease (13 ). When serologic testing is indicated, CDC recommends testing initially with a sensitive first test, either an enzyme-linked immunosorbent assay (ELISA) or an indirect fluorescent antibody test, followed by testing with the more specific Western immunoblot (WB) test to corroborate equivocal or positive results obtained with the first test (14 ). Although antibiotic treatment in early localized disease can blunt or abrogate the antibody response, patients with early disseminated or late-stage disease usually have strong serologic reactivity and demonstrate expanded WB immunoglobulin G (IgG) banding patterns to diagnostic B. burgdorferi antigens (15,16 ). Antibodies often persist for months or years after successfully treated or untreated infection. Thus, seroreactivity alone cannot be used as a marker of active disease. Neither positive serologic test results nor a history of previous Lyme disease ensures that a person has protective immunity. Repeated infection with B. burgdorferi has been reported (17 ). B. burgdorferi can be cultured from 80% or more of biopsy specimens taken from early erythema migrans lesions (18 ). However, the diagnostic usefulness of this procedure is limited because of the need for a special bacteriologic medium (i.e., modified Barbour-Stoenner-Kelly medium) and protracted observation of cultures. Polymerase chain reaction (PCR) has been used to amplify genomic DNA of B. burgdorferi in skin, blood, cerebrospinal fluid, and synovial fluid (19,20 ), but PCR has not been standardized for routine diagnosis of Lyme disease. # Treatment Lyme disease can usually be treated successfully with standard antibiotic regimens (5,6 ). Early and uncomplicated infection, including infection with isolated cranial nerve palsy, usually responds satisfactorily to treatment with orally administered antibiotics (21 ). Parenteral antibiotics are generally recommended for treating meningitis, carditis, later-stage neurologic Lyme disease, and complicated Lyme disease arthritis. Late, complicated Lyme disease might respond slowly or incompletely, and more than one antibiotic treatment course can be required to eliminate active infection (8,9 ). Refractory Lyme disease arthritis is associated with expression of certain Class II major histocompatibility complex (MHC II) molecules (22 ), and can require anti-inflammatory agents and surgical synovectomy for relief of symptoms (8 ). In a limited number of patients, persistent or recurrent symptoms after appropriate antibiotic therapy often can be attributed to causes other than persistent infection (22,23 ). # EPIDEMIOLOGY OF LYME DISEASE Antigenic Variation of B. burgdorferi Sensu Lato* In the United States, a number of genospecies of B. burgdorferi sensu lato have been isolated from animals and ticks, but only OspA expressing B. burgdorferi sensu stricto † has been isolated from humans (24 ). Existing evidence also demonstrates that rOspA vaccines will be protective against most if not all human infections in the United States (25 ). B. burgdorferi sensu stricto also occurs in Europe, but the dominant European and Asian genospecies are B. garinii and B. afzelii, both of which are antigenically distinct from B. burgdorferi sensu stricto (26 ) and vary in their expression of OspA. Vaccines using combinations of immunogenic proteins might be necessary to provide protection against multiple genospecies (27 ). # Routes of Transmission Humans acquire B. burgdorferi infection from infected ticks at the time the tick takes a blood meal (28 ); Lyme disease is not spread by person-to-person contact or by direct contact with infected animals. Transplacental transmission of B. burgdorferi has been reported (29,30 ), but the effects of such transmission on the fetus remain unclear. The results of two epidemiologic studies document that congenital Lyme disease must be rare, if it occurs at all (31,32 ). Transmission in breast milk has not been described. B. burgdorferi can be cultured from the blood in some patients with early acute infection, and it is able to survive for several weeks in stored blood. However, at least one study has found that the risk for transfusion-acquired infection is minimal (33 ). # Tick Vectors of Lyme Disease B. burgdorferi is transmitted to humans by ticks of the Ixodes ricinus complex (34 ). I. scapularis, the black-legged or deer tick, is the vector in the eastern United States; I. pacificus, the western black-legged tick, transmits B. burgdorferi in the western United States (35,36 ). I. scapularis is also a vector for human granulocytic ehrlichiosis and babesiosis (34,37 ). In their nymphal stage, these ticks feed predominantly in the late spring and early summer. The majority of Lyme disease cases result from bites by infected nymphs. In highly enzootic areas of the United States, approximately 15%-30% of questing I. scapularis nymphs and up to 14% of I. pacificus nymphs are infected with B. burgdorferi (38)(39)(40)(41). However, in the southern United States, the prevalence of infection in I. scapularis ticks is generally 0%-3% (36 ). The risk for acquiring Lyme disease in the United States varies with the distribution, density, and prevalence of infection in vector ticks (Appendix). During the past several decades, the distribution of I. scapularis has spread slowly in the northeastern and upper north-central regions of the United States (42 ). Although deer are not competent reservoirs of B. burgdorferi, they are the principal maintenance hosts for adult black-legged ticks, and the presence of deer appears to be a prerequisite for the establishment of I. scapularis in any area (43 ). The explosive repopulation in the eastern United States by white-tailed deer during recent decades has been linked to the spread of I. scapularis ticks and of Lyme disease in this region. The future limits of this spread are not known (42 ). # Distribution of Human Cases of Lyme Disease Lyme disease is endemic in several regions in the United States, Canada, and temperate Eurasia (1,44 ). The disease accounts for more than 95% of all reported cases of vectorborne illness in the United States. Using a national surveillance case definition (45 ), state health officials reported >62,000 cases to CDC during 1993-1997, and the national mean annual rate during this 5-year period was 5.5 cases/100,000 population (1,2, CDC, unpublished data, 1998). Persons of all ages are equally susceptible to infection, although the highest reported rates of Lyme disease occur in children aged <15 years and in adults aged 30-59 years (1 ). Both underreporting and overdiagnosis are common (46)(47)(48). Approximately 90% of cases are reported by approximately 140 counties located along the northeastern and mid-Atlantic seaboard and in the upper north-central region of the United States (Appendix). A rash similar to erythema migrans of Lyme disease, but not caused by B. burgdorferi infection, has been described in patients who have been bitten by ticks in the southern United States (49,50 ). This rash is suspected of being associated with the bite of Amblyomma americanum ticks (51 ). # Populations at Risk for Lyme Disease Most B. burgdorferi infections result from periresidential exposure to infected ticks (38,(52)(53)(54)(55) during property maintenance, recreation, and leisure activities. Thus, persons who live or work in residential areas surrrounded by woods or overgrown brush infested by vector ticks are at risk for acquiring Lyme disease. In addition, persons who participate in recreational activities away from home (e.g., hiking, camping, fishing, and hunting) in tick habitat and persons who engage in outdoor occupations (e.g., landscaping, brush clearing, forestry, and wildlife and parks management) in endemic areas might also be at elevated risk for acquiring Lyme disease (56)(57)(58). # PREVENTION AND CONTROL OF LYME DISEASE # Avoidance of Tick Habitat Whenever possible, persons should avoid entering areas that are likely to be infested with ticks, particularly in spring and summer when nymphal ticks feed. Ticks favor a moist, shaded environment, especially that provided by leaf litter and lowlying vegetation in wooded, brushy, or overgrown grassy habitat. Both deer and rodent hosts must be abundant to maintain the enzootic cycle of B. burgdorferi. Sources of information regarding the distribution of ticks in an area include state and local health departments, park personnel, and agricultural extension services. # Personal Protection Persons who are exposed to tick-infested areas should wear light-colored clothing so that ticks can be spotted more easily and removed before becoming attached. Wearing long-sleeved shirts and tucking pants into socks or boot tops can help keep ticks from reaching the skin. Ticks are usually located close to the ground, so wearing high rubber boots can provide additional protection. Applying insect repellents containing DEET (n,n-diethyl-m-toluamide) to clothes and exposed skin and applying permethrin, which kills ticks on contact, to clothes, should also help reduce the risk of tick attachment. DEET can be used safely on children and adults but should be applied according to the U.S. Environmental Protection Agency guidelines to reduce the possibility of toxicity (59 ). Because transmission of B. burgdorferi from an infected tick is unlikely to occur before 36 hours of tick attachment (28,60 ), daily checks for ticks and their prompt removal will help prevent infection. # Strategies for Reducing Tick Abundance The number of ticks in endemic residential areas can be reduced by removing leaf litter, brush, and woodpiles around houses and at the edges of yards and by clearing trees and brush to admit more sunlight, thus reducing deer, rodent, and tick habitat (61 ). Tick populations have also been effectively suppressed by applying pesticides to residential properties (62,63 ). Community-based interventions to reduce deer populations or to kill ticks on deer and rodents have not been extensively implemented, but might be effective in reducing communitywide risk for Lyme disease (64 ). The effectiveness of deer feeding stations equipped with pesticide applicators to kill ticks on deer and other baited devices to kill ticks on rodents is currently under evaluation. # Prophylaxis After Tick Bite The relative cost-effectiveness of postexposure treatment of tick bites to avoid Lyme disease in endemic areas is dependent on the probability of B. burgdorferi infection after a tick bite (65 ). In most circumstances, treating persons for tick bite alone is not recommended (6,66 ). Persons who are bitten by a deer tick should remove the tick and seek medical attention if any signs and symptoms of early Lyme disease, ehrlichiosis, or babesiosis develop during the ensuing days or weeks. # Early Diagnosis and Treatment Lyme disease is readily treatable in its early stages (5,6 ). The early diagnosis and proper antibiotic treatment of Lyme disease are important strategies for avoiding the morbidity and costs of complicated and late-stage illness. # LYME DISEASE VACCINE Description LYMErix is made from lipidated rOspA of B. burgdorferi sensu stricto. The rOspA protein is expressed in Escherichia coli and purified. Each 0.5-mL dose of LYMErix contains 30 µg of purified rOspA lipidated protein adsorbed onto aluminum hydroxide adjuvant. # Mechanism of Action Several studies in animals have provided evidence that B. burgdorferi in a vector tick undergoes substantial antigenic change between the time of tick attachment on a mammalian host and subsequent transmission of the bacterium to the host. The spirochetes residing in the tick gut at the initiation of tick feeding express primarily OspA. As tick feeding begins, the expression of outer-surface protein C (OspC) is increased and the expression of OspA is decreased, so that spirochetes that reach the mammalian host after passing through the tick salivary glands express primarily OspC (67 ). Thus, the rOspA vaccine might exert its principal protective effect by eliciting antibodies that kill Lyme disease spirochetes within the tick gut (68,69 ). # Route of Administration, Vaccination Schedule, and Dosage LYMErix is administered by intramuscular injection, 0.5 mL (30 µg), into the deltoid muscle. Three doses are required for optimal protection. The first dose is followed by a second dose 1 month later and a third dose administered 12 months after the first dose. Vaccine administration should be timed so that the second dose of the vaccine (year 1) and the third dose (year 2) are administered several weeks before the beginning of the B. burgdorferi transmission season, which usually begins in April. The safety and immunogenicity of alternate dosing schedules are currently being evaluated. # VACCINE PERFORMANCE # Safety # Randomized, Controlled Clinical (Phase III) Trial of LYMErix A total of 10,936 subjects aged 15-70 years living in Lyme disease-endemic areas were recruited at 31 sites and randomized to receive three doses of vaccine or placebo (3 ); 5,469 subjects received at least one 30-µg dose of rOspA vaccine, and 5,467 subjects received at least one injection of placebo. The subjects were then followed for 20 months. Information regarding adverse events that were believed to be related or possibly related to injection were available from 4,999 subjects in each group. Soreness at the injection site was the most frequently reported adverse event, which was reported without solicitation by 24.1% of vaccine recipients and 7.6% of placebo recipients (p < 0.001). Redness and swelling at the injection site were reported by <2% of either group but were reported more frequently among vaccine recipients than among those who received placebo (p < 0.001). Myalgia, influenza-like illness, fever, and chills were more common among vaccine recipients than placebo recipients (p < 0.001), but none of these was reported by more than 3.2% of subjects (3 ). Reports of arthritis were not significantly different between vaccine and placebo recipients, but vaccine recipients were significantly (p < 0.05) more likely to report arthralgia or myalgia within 30 days after each dose (70 ). No statistically significant differences existed between vaccine and placebo groups in the incidence of adverse events more than 30 days after receiving a dose, and no episodes of immediate hypersensitivity among vaccine recipients were noted (3 ). # Safety in Patients with Previously Diagnosed Lyme Disease The safety of three different dosage strengths of rOspA vaccine with adjuvant in 30 adults with previous Lyme disease was evaluated in an uncontrolled safety and immunogenicity trial (71 ). Doses were administered at 0, 1, and 2 months. Follow-up of subjects was conducted 1 month after the third dose. No serious adverse events were recorded during the study period. In the randomized controlled Phase III trial of LYMErix, the incidence of adverse events among vaccinees who were seropositive at baseline was similar to the incidence among those who were seronegative (70 ). The incidence of musculoskeletal symptoms within the first 30 days after vaccination was higher among vaccinees with a self-reported previous history of Lyme disease compared with vaccinees with no such history. This difference was not statistically significant at the p = 0.05 level in the placebo group. No statistically significant difference existed in the incidence of late musculoskeletal adverse events between vaccine and placebo recipients with a selfreported previous history of Lyme disease (70 ). # Risk for Possible Immunopathogenicity of rOspA Vaccine After infection with B. burgdorferi, persons who express certain MHC II molecules are more likely than others to develop chronic, poorly responsive Lyme arthritis associated with high levels of antibody to OspA in serum and synovial fluid (22 ). In chronic Lyme arthritis patients, the levels of antibody to OspA, and especially to the C-terminal epitope of OspA, have been found to correlate directly with the severity and duration of the arthritis (72 ). Researchers have proposed that an autoimmune reaction might develop within the joints of some Lyme arthritis patients as a result of molecular mimicry between the dominant T-cell epitope of OspA and human leukocyte function associated antigen 1 (hLFA-1) (73 ). The Phase III trial did not detect differences in the incidence of neurologic or rheumatologic disorders between vaccine recipients and their placebo controls during the 20 months after the initial dose (3 ). However, because the association between immune reactivity to OspA and treatment-resistant Lyme arthritis is poorly understood, the vaccine should not be administered to persons with a history of treatment-resistant Lyme arthritis. # Efficacy Randomized, Controlled Trial (Phase III) of LYMErix Using an intention-to-treat analysis, the vaccine efficacy in protecting against "definite" Lyme disease after two doses was 49% (95% confidence interval = 15%-69%) and after three doses was 76% (95% CI = 58%-86%) (3 ). (In this study, "definite" Lyme disease was defined as the presence of erythema migrans or objective neurologic, musculoskeletal, or cardiovascular manifestations of Lyme disease, plus laboratory confirmation of infection by cultural isolation, PCR positivity, or WB seroconversion.) Efficacy in protecting against asymptomatic infection (no recognized symptoms, but with WB seroconversions recorded in year 1 or year 2) was 83% (95% CI = 32%-97%) in year 1 and 100% (95% CI = 26%-100%) in year 2. # Immunogenicity A subset of adult subjects enrolled in the Phase III clinical trial of LYMErix was studied for the development of OspA antibodies at months 2, 12, 13, and 20 (3 ). At month 2, one month after the second injection, the geometric mean antibody titer (GMT) of IgG anti-OspA antibodies was 1,227 ELISA units/mL. Ten months later, the GMT had declined to 116 ELISA units/mL. At month 13, one month after the third injection, a marked anamnestic response resulted in a GMT of 6,006 ELISA units/mL. At month 20, the mean response had decreased to 1,991 ELISA units/mL (70 ). An analysis of antibody titers and the risk for developing Lyme disease for a subset of subjects enrolled in the Phase III clinical trial concluded that a titer >1,200 ELISA units/mL correlated with protection (SmithKline Beecham poster at Infectious Disease Society of America Conference, Denver, Colorado, November 1998). # Effect of Vaccination on the Serologic Diagnosis of Lyme Disease Care providers and laboratorians should be advised that vaccine-induced anti-rOspA antibodies routinely cause false-positive ELISA results for Lyme disease (74 ). Experienced laboratory workers, through careful interpretation of the results of WB, can usually discriminate between B. burgdorferi infection and previous rOspA immunization, because anti-OspA antibodies do not develop after natural infection. # COST-EFFECTIVENESS OF LYME DISEASE VACCINATION The cost of Lyme disease has been evaluated from both a societal and a thirdparty-payer perspective (75 ). The cost-effectiveness of vaccinating against Lyme disease has also been analyzed from a societal perspective (76 ). At an assumed cost of vaccination of $100/person/year, a vaccine effectiveness of 0.85, a probability of 0.85 of correctly identifying and treating early Lyme disease, and an assumed incidence of Lyme disease of 1,000/100,000 persons/year, the net cost of vaccination to society was $5,692/case averted and $35,375/complicated neurologic or arthritic case avoided (Figure 1). Using these same baseline assumptions, the societal cost of vaccination exceeds the cost of not vaccinating, unless the incidence of Lyme disease is >1,973/100,000 persons/year. Of the variables examined, the incidence of Lyme disease had the greatest impact on cost-effectiveness of vaccination. The likelihood of early diagnosis and treatment also has a substantial impact on vaccine costeffectiveness because of the reduced incidence of sequelae when Lyme disease is diagnosed and patients are treated early in the disease. Most disease-endemic states and counties report Lyme disease incidence that are substantially below 1,000/100,000 persons/year. For example, in 1997, the highest reported state incidence was 70/100,000 persons in Connecticut, and the highest reported county incidence was 600/100,000 population in Nantucket County, Massachusetts. However, some studies document that approximately 10%-15% of physician-diagnosed cases of Lyme disease are reported to state authorities in highly endemic areas (46,47 ). Epidemiologic studies of populations at high risk in the northeastern United States have estimated annual incidence of >1,000/100,000 persons/ year in several communities (77)(78)(79)(80). # ASSESSING THE RISK FOR LYME DISEASE The decision to administer Lyme disease vaccine should be made on the basis of an assessment of individual risk, which depends on a person's likelihood of being bitten by tick vectors infected with B. burgdorferi. This likelihood is primarily determined by the following: - density of vector ticks in the environment, which varies by place and season; - the prevalence of B. burgdorferi infection in vector ticks; and - the extent of person-tick contact, which is related to the type, frequency, and duration of a person's activities in a tick-infested environment. Assessing risk should include considering the geographic distribution of Lyme disease. The areas of highest Lyme disease risk in the United States are concentrated within some northeastern and north-central states. The risk for Lyme disease differs not only between regions and states and counties within states (Appendix), but even within counties and townships. Detailed information regarding the distribution of Lyme disease risk within specific areas is best obtained from state and local public health authorities. The second step in determining Lyme disease risk is to assess a person's activities. Activities that place persons at high risk are those that involve frequent or prolonged exposure to the habitat of infected ticks at times of the year when the nymphal stages of these ticks are actively seeking hosts, which in most endemic areas is April-July. Typical habitat of Ixodes ticks are wooded, brushy, or overgrown grassy areas that are favorable for deer and the ticks' rodent hosts. Several recreational, property # FIGURE 1. Cost-effectiveness of Lyme disease vaccination OBJECTIVE This MMWR provides recommendations regarding prevention and control of Lyme disease. These recommendations were developed by CDC staff members and the ACIP members. This report is intended to guide clinical practice and policy development related to administration of the Lyme disease vaccine. Upon completion of this educational activity, the reader should be able to describe the epidemiology of Lyme disease in the United States; list effective methods of Lyme disease prevention; describe the characteristics and use of the currently licensed Lyme disease vaccine; and recognize the most common adverse reactions following administration of Lyme disease vaccine. # EXPIRATION -June 4, 2000 The response form must be completed and returned electronically, by fax, or by mail, postmarked no later than 1 year from the publication date of this report, for eligibility to receive continuing education credit. # INSTRUCTIONS 1. Read this MMWR (Vol. 48, RR-7 ), which contains the correct answers to the questions beginning on the next page. 2. Complete all registration information on the response form, including your name, mailing address, phone number, and e-mail address, if available. # Indicate whether you are registering for Continuing Medical Education (CME) credit, Continuing Education Unit (CEU) credit, or Continuing Nursing Education (CNE) credit. 4. Select your answers to the questions, and mark the corresponding letters on the response form. To receive continuing education credit, you must answer all of the questions. Questions with more than one correct answer will instruct you to "indicate all that are true." 5. Sign and date the response form. # MMWR Response Form for Continuing Education Credit June 4, 1999 / Vol. 48 / No. RR-7 # Recommendations for the Use of Lyme Disease Vaccine Recommendations of the Advisory Committee on Immunization Practices (ACIP) Fill in the appropriate block(s) to indicate your answer(s). To receive continuing education credit, you must answer all of the questions. . A B C D E 2. A B C D E 3. A B C D E 4. A B C D E 5. A B C D E 6. A B C D E 7. A B C D E 8. A B C D E 9. A B C D E 10. A B C D E F 11. A B C D E F 12. A B C D E 13. A B C D E 14. A B C 15. A B C D E 16. A B C D E 17. A B C D E 18. A B C D E Detach or photocopy. 1.2 hours of CNE credit maintenance, occupational, or leisure pursuits that are carried out in tick habitat can be risky activities. When in highly endemic areas, persons can reduce their risk for Lyme disease and other tickborne illnesses by avoiding tick-infested habitats. If exposure to a tick-infested habitat cannot be avoided, persons should use repellents, wear protective clothing, and regularly check themselves for ticks. Persons who are unlikely to seek medical care for early manifestations of Lyme disease can be at increased risk for Lyme disease complications. Morbidity from Lyme disease can be substantially reduced by detecting and treating the infection in its early stages, because early and correct treatment usually results in a prompt and uncomplicated cure. # RECOMMENDATIONS FOR USE OF LYME DISEASE VACCINE Lyme disease vaccine does not protect all recipients against infection with B. burgdorferi and offers no protection against other tickborne diseases. Vaccinated persons should continue to practice personal protective measures against ticks and should seek early diagnosis and treatment of suspected tickborne infections. Because Lyme disease is not transmitted person-to-person, use of the vaccine will not reduce risk among unvaccinated persons. Decisions regarding the use of vaccine should be based on individual assessment of the risk for exposure to infected ticks and on careful consideration of the relative risks and benefits of vaccination compared with other protective measures, including early diagnosis and treatment of Lyme disease. The risk for Lyme disease is focally distributed in the United States (Appendix). Detailed information regarding the distribution of Lyme disease risk within specific areas is best obtained from state and local public health authorities. The following recommendations are made regarding use of Lyme disease vaccine: -Lyme disease vaccination may be considered for persons aged 15-70 years who are exposed to tick-infested habitat but whose exposure is neither frequent nor prolonged. The benefit of vaccination beyond that provided by basic personal protection and early diagnosis and treatment of infection is uncertain. -Lyme disease vaccination is not recommended for persons who have minimal or no exposure to tick-infested habitat. - Travelers can obtain some protection from two doses of vaccine but will not achieve optimal protection until the full series of three doses has been administered. All travelers to high-or moderate-risk areas during Lyme disease transmission season should practice personal protection measures as described earlier and seek prompt diagnosis and treatment if signs or symptoms of Lyme disease develop. Lyme disease is endemic in some temperate areas of Europe and Asia; however, considerable heterogeneity of expression exists in the Eurasian strains of B. burgdorferi sensu lato that infect humans, and whether the rOspA vaccine licensed for use in the United States would protect against infection with Eurasian strains is uncertain. - Pregnant Women -Because the safety of rOspA vaccines administered during pregnancy has not been established, vaccination of women who are known to be pregnant is not recommended. No evidence exists that pregnancy increases the risk for Lyme disease or its severity. Acute Lyme disease during pregnancy responds well to antibiotic therapy, and adverse fetal outcomes have not been reported in pregnant women receiving standard courses of treatment. A vaccine pregnancy registry has been established by SmithKline Beecham Pharmaceuticals. In the event that a pregnant women is vaccinated, health-care providers are encouraged to register this vaccination by calling, toll-free, (800) 366-8900, ext. 5231. - Persons with Immunodeficiency -Persons with immunodeficiency were excluded from the Phase III safety and efficacy trial, and no data are available regarding Lyme disease vaccine use in this group. # Persons with Musculoskeletal Disease -Persons with diseases associated with joint swelling (including rheumatoid arthritis) or diffuse musculoskeletal pain were excluded from the Phase III safety and efficacy trial, and only limited data are available regarding Lyme disease vaccine use in such patients. - Persons with a Previous History of Lyme Disease -Vaccination should be considered for persons with a history of previous uncomplicated Lyme disease who are at continued high risk. -Persons who have treatment-resistant Lyme arthritis should not be vaccinated because of the association between this condition and immune reactivity to OspA. -Persons with chronic joint or neurologic illness related to Lyme disease, as well as second-or third-degree atrioventricular block, were excluded from the Phase III safety and efficacy trial, and thus, the safety and efficacy of Lyme disease vaccine in such persons are unknown. - Vaccine Schedule, Including Spacing and Timing of Administration -Three doses of the vaccine should be administered by intramuscular injection. The initial dose should be followed by a second dose 1 month later and a third dose 12 months after the first dose. Vaccine administration should be timed so that the second dose of the vaccine (year 1) and the third dose (year 2) are administered several weeks before the beginning of the B. burgdorferi transmission season, which usually begins in April. - Boosters -Whether protective immunity will last longer than 1 year beyond the month-12 dose is unknown. Data regarding antibody levels during a 20-month period after the first injection of LYMErix indicate that boosters beyond the month-12 booster might be necessary (see Immunogenicity). Additional data are needed before recommendations regarding vaccination with more than three doses of rOspA vaccine can be made. # Simultaneous Administration with Other Vaccines -The safety and efficacy of the simultaneous administration of rOspA vaccine with other vaccines have not been established. If LYMErix must be administered concurrently with other vaccines, each vaccine should be administered in a separate syringe at a separate injection site. # FUTURE CONSIDERATIONS # Recommendations for Surveillance, Research, Education, and Program Evaluation Activities - Determine safety, immunogenicity, and efficacy of Lyme disease vaccine in children. - Determine optimal vaccine dosage schedules and timing of administration. - Determine the need for and spacing of booster doses. - Determine safety and efficacy of the vaccine in persons aged >70 years. - Develop additional serodiagnostic tests that discriminate between infection and vaccine-induced antibody production. - Develop a program of Lyme disease vaccine education for care providers and prospective vaccine clients. - Develop an information sheet to be distributed to prospective vaccine recipients or to persons at the time of vaccine administration. - Conduct surveillance for rare or late-developing adverse effects of vaccination. - Establish postlicensure epidemiologic studies of safety, efficacy, prevention effectiveness, cost-effectiveness, and patterns of use. - Develop a program to monitor vaccine use at the local, state, and national levels and to measure its public health and economic impact. - Develop population-based studies to assess the impact of vaccine use on incidence of Lyme disease in communities. - Continue to develop maps of geographic distribution of Lyme disease with improved accuracy and predictive power. exposure were compiled on a county-unit scale for the United States. Then geographic information systems (GIS) technology was used to combine these data and categorize each of the 3,140 counties into four risk classes. # ENTOMOLOGIC RISK Vector Distribution Vector data were obtained from a national distribution map of Ixodes scapularis and I. pacificus, which was previously published by CDC (2 ). These data delineate three classes of tick distribution based on all published and unpublished county collection records available to CDC before 1998. The three classes are as follows: - established populations (≥6 ticks reported or more than one life stage); - reported occurrence (<6 ticks reported and only one life stage); and - absence of ticks or missing data. Although these data are currently the best source of vector distribution available, many gaps exist because of uneven sampling efforts among the counties. Therefore, a neighborhood analysis GIS procedure was used to modify the original tick distribution to smooth absent data and minimize the impact of reporting gaps. In this process, the original tick coverage map was rasterized to 1 km, and each cell was given a numeric value corresponding to the county tick class (0 = absent; 1 = reported; and 2 = established). A neighborhood analysis was performed using ERDAS IMAGINE- image-processing software. This function employed a moving filter (25 by 25 km), which summed the values of the area surrounding each 1-km pixel and created a new focally smoothed image. An outline of counties was overlaid to define boundaries on the smoothed map, and new values were summed from the total pixel values for each county. The three original vector classes were maintained with the new classification. The revised map employed a threshold reclassification based on mean summary statistics generated from the neighborhood analysis. This procedure resulted in a weighted value for each county that was determined by the classes of surrounding counties, thus smoothing the map to minimize rough edges and isolated holes in the data. The modified vector distribution increased the number of counties containing I. scapularis and I. pacificus from 1,058 counties (34% of total counties) in the original data set to 1,404 (45% of total) in the modified version. This modification resulted in greater continuity among adjacent counties, as well as a less-conservative description of vector distribution. # Infection Prevalence in Vectors The prevalence of infection with B. burgdorferi is low throughout the distribution of I. pacificus (3 ) with the exception of one California county (4 ). Within the entire southern distribution of I. scapularis, prevalence of infection with B. burgdorferi is low compared with the Northeast and upper Midwest (3 ). One possible reason for these *ERDAS IMAGINE map production computer software, a product of ERDAS, Inc., 2801 Buford Highway, Atlanta, GA 30329-2137, (404) 248-9000, . The national map illustrates a clear focal pattern of Lyme disease risk with the greatest risk occurring in the Northeast and upper Midwest regions. Overall, 115 (4%) counties were classified as high risk, followed by 146 (5%) moderate risk, 1,143 (36%) low risk, and 1,736 (55%) as minimal or no-risk counties. Lyme disease risk is measurable as a function of two epidemiologic parametersentomologic risk and human exposure. Entomologic risk for Lyme disease is defined as the density per unit area of host-seeking nymphal ticks infected with Borrelia burgdorferi (1 ). Field studies needed for determination of entomologic risk require trained entomologists, and such studies are limited to a narrow seasonal window within the life-cycle of vector ticks. Limited resources preclude the direct measurement of entomologic risk over large geographic areas; therefore, indirect measures were used to estimate risk to develop this national Lyme disease risk map. First, data on vector distribution, abundance, B. burgdorferi infection prevalence, and human # High risk # Moderate risk # Low risk # Minimal or no risk # Areas of predicted Lyme disease transmission Note: This map demonstrates an approximate distribution of predicted Lyme disease risk in the United States. The true relative risk in any given county compared with other counties might differ from that shown here and might change from year to year. Risk categories are defined in the accompanying text. Information on risk distribution within states and counties is best obtained from state and local public health authorities. differences is the geographic variations in abundance of hosts that are competent reservoirs of infection for immature ticks. The white-footed mouse (Peromyscus leucopus) is the principal host for ticks in the Northeast and upper Midwest and is a competent reservoir for the spirochete. But in the Southeast and West Coast regions, reptiles appear to serve as major hosts for immature ticks, and reptiles are either inefficient or incompetent reservoir hosts for spirochetes. This pattern of tick-host association might result from the greater population density of lizards relative to rodents ( 5), resulting in reduced transmission rates in regions where lizards dominate. An index was created to map the effect of host-species composition on infection prevalence in I. scapularis ticks. # National Lyme disease risk map with four categories of risk A literature survey was conducted to identify a complete list of hosts for I. scapularis (6 ). A total of 38 nondomestic host species was identified, including 32 mammal species and 6 reptile species. Birds were excluded because of their migratory nature and their uncertain role as natural reservoir hosts. Species range maps were obtained from the literature (7,8 ), then digitized by county into ArcView GIS- software for presence or absence of reservoir hosts. The county data were then summed to determine the total host species composition available for I. scapularis. A ratio of total reptiles divided by the total hosts multiplied by 100 was calculated for each county and mapped. The reptile ratio index delineates those areas having a high reptile-to-total-hosts ratio (>10) and forms a linear boundary, below which reptiles are more likely to serve as hosts for ticks. The geographic boundary runs roughly on the 38º north latitude from Virginia to Missouri. This reptile ratio illustrates that although total hosts in the northern states can be equal to those of the southern states, reptiles dilute the force of transmission, thus lowering the prevalence of infection in ticks and creating less of a risk to humans in the South. # HUMAN EXPOSURE TO RISK CDC case reports were used as a measure of human exposure to entomologic risk. County-specific data were compiled for the years 1994-1997. Counties comprising the ninetieth percentile of all human cases reported during this 4-year period were selected to represent counties with high human exposure. These 137 counties reported a minimum total of 23 cases. Heuristic, or procedure-based decision rule, was employed to construct the national Lyme disease risk map. Expert decision rule was applied to construct the risk classification as follows: # Risk Classes - High Risk. Counties where I. scapularis or I. pacificus populations are established and where prevalence of infection is predicted to be high, and which are in the top tenth percentile of counties reporting human cases during the 4-year period, 1994-1997.

Borazine Borazine is an inorganic compound composed of the elements boron, nitrogen and hydrogen. In this cyclic compound three hydroborane (BH) units and three amino units (NH) alternate. The compound was synthesised in 1926 by the chemists Alfred Stock and Pohland by a reaction of diborane with ammonia. The structure is isoelectronic and isostructural with benzene and for this reason borazine is called inorganic benzene by a proposal of Nils Wiberg and the compound also goes by the name of borazol from the German name for benzene which is benzol. # Synthesis Borazine is synthesized from diborane and ammonia in a 1:2 ratio at 250 - 300 °C with a conversion of 50%. An alternative more efficient route begins with lithium borohydride and ammonium chloride with improved chemical yield: In a two-step process to borazine, boron trichloride is first converted to trichloroborazine: The B-Cl bonds are subsequently converted to B-H bonds: # Properties Borazine is a colourless liquid with an aromatic smell. In water it decomposes to boric acid, ammonia, and hydrogen. Borazine, with a standard enthalpy change of formation ΔHf of -531 kJ/mol, is thermally very stable. ## Structure Borazine is isostructural with benzene and bond lengths are identical just as in benzene. The distance between boron and nitrogen in the ring is 0.1436 nm, the carbon carbon bond in benzene has a length of 0.1397 nm. The boron nitrogen bond is between that of the boron nitrogen single bond with 0.151 nm and the boron nitrogen double bond which is 0.131 nm. This suggests partial delocalisation of nitrogen lone pair electrons. ## Mesomers The electronegativity of boron (2.04 on the Pauling scale) compared to that of nitrogen (3.04) and also the electron deficiency on the boron atom and the lone pair on nitrogen favor alternative mesomer structures for borazine. Boron is the Lewis acid and nitrogen is the Lewis base. # Reactions Borazine is more reactive than benzene. It reacts with hydrogen chloride in an addition reaction. If borazine were truly aromatic like benzene this reaction would not occur without a Lewis acid catalyst. The addition reaction with bromine takes place without catalyst. Borazines interact with nucleophilic attack at boron and electrophilic attack at nitrogen. Heating borazine at 70 °C expulses hydrogen with formation of a borazinyl polymer or polyborazylene in which the monomer units are coupled in a para fashion by new boron - nitrogen bonds. # Applications Borazine and borazine derivatives are potential precursors to boron nitride ceramics. Boron nitride can be prepared by heating polyborazylene to 1000 °C. Borazines are also starting materials for other potential ceramics such as boron carbonitrides:

Endogeny The word endogenous means "arising from within", the opposite of exogenous. # Biology Endogenous substances are those that originate from within an organism, tissue, or cell . Endogenous retrovirus are caused by ancient infections of germ cells in humans, mammals and other vertebrates. Their proviruses remain in the genome and are passed on to the next generation. Endogenous processes include circadian rhythms. In some biological systems, endogeneity refers to the recipient of DNA (usually in prokaryotes). However, due to homeostasis, discerning between internal and external influences is often difficult. # Psychology An emotion or behaviour is endogenous if it is spontaneously generated from an individual's internal state. # Economics and finance A variable is called endogenous if it is explained within the model in which it appears. For example, in a supply and demand model of an agricultural market, changes in the weather or in consumer tastes would be exogenous variables that might shift the supply and demand curves; the price and quantity of trade would be the endogenous variables explained by the model. nl:Endogeen (aardwetenschappen) fi:Endogeeninen

Korean Guideline for Iron Chelation Therapy in Transfusion-Induced Iron Overload # Introduction Regular red blood cell (RBC) transfusions are the major supportive care for many Korean patients with aplastic anemia (AA), myelodysplastic syndromes (MDS), or other rare anemias. Physicians most commonly use the hemoglobin (Hgb) level to determine whether or not to transfuse [bib_ref] Red blood cell transfusion practice in elective orthopedic surgery: a multicenter cohort..., Vuille-Lessard [/bib_ref]. However, most guidelines recommend that transfusions should be given for symptoms of anemia and that the decision to transfuse should not be based on the Hgb level alone [bib_ref] Clinical practice guideline: red blood cell transfusion in adult trauma and critical..., Napolitano [/bib_ref]. An optimal RBC transfusion regimen involves administering sufficient RBCs to maximize clinical benefits while avoiding unnecessary transfusions that increase costs and expose patients to potential risks, including infection and iron overload. Hence, the Korean Society of Blood Transfusion (KSBT) recommends transfusion to be considered at Hgb concentrations of 7 g/dL or less in chronically anemic patients. KSBT does not rec-ommend routine RBC transfusion when Hgb > 7 g/dL. Furthermore, KSBT only recommends RBC transfusion in those patients with symptoms such as dyspnea, palpitation on exertion, or edema. Chronic transfusion therapy inevitably leads to secondary iron overload, which can cause significant damage to many organs such as the liver, heart, and endocrine system. Recently, deferasirox (DFX), an oral iron chelator, has been introduced and has been shown to improve the treatment of iron overload [bib_ref] Tailoring iron chelation by iron intake and serum ferritin: the prospective EPIC..., Cappellini [/bib_ref]. To summarize the evidence and provide practical guidelines for iron chelation in Korea, the Korean Society of Hematology Aplastic Anemia Working Party (KSHAAWP) describes here general considerations regarding iron overload in transfusion dependent patients and proposes guidelines for the treatment of iron overload based on published clinical evidence and the experience of the expert panel. ## Pathophysiology of iron overload Iron metabolism and the mechanism of organ damage Iron is involved in many critical steps of cellular metabolism such as cellular respiration, heme synthesis, production of oxygen radicals, antioxidation, and DNA synthesis and repair, as well as in cellular proliferation and inflammation. Normal body iron stores are 3-4 g; an excess of iron of 20 g or more can lead to organ damage [bib_ref] Practical management of iron overload, Porter [/bib_ref]. Most additional iron can be stored within the reticuloendothelial system (RES). However, if the iron level exceeds the capacity of the RES to retain it, the excess iron will be released into the plasma. Normally, transferrin binds this released iron but, if the plasma iron concentration continues to rise, transferrin saturation may result. This scenario may occur with sequential transfusions: when the RES cannot retain all of the iron, it then diffuses into the plasma in amounts that exceed the capacity of transferrin to bind it. As a result, non-transferrin-bound iron, which seems to be the major mediator of extrahepatic tissue damage in transfusional iron overload, appears in the plasma [bib_ref] Impact of transfusion dependency and secondary iron overload on the survival of..., Malcovati [/bib_ref]. The harmful effects of excess iron may result from deposition into tissues and organs, but also from oxidative stress. Non-transferrin-bound plasma iron is deposited specifically in tissues with high level of transferrin receptors (eg, liver, heart, anterior pituitary, and pancreas). As the human body has no mechanism for excreting excess iron, and each unit of transfused RBCs contains 200-250 mg of iron, iron overload can readily occur in patients given multiple transfusions, typically after 10 to 20 transfusions (6). In the absence of treatment to reduce iron overload, progressive cardiomyopathy, cirrhosis, endocrinopathy and diabetes can have a serious impact on morbidity and mortality. Iron chelation therapy in patients given multiple transfusions aims to prevent or reverse some of these consequences and has been associated with a reduced morbidity and mortality [bib_ref] Improvement in iron status and liver function in patients with transfusional iron..., Hoffbrand [/bib_ref]. ## Measuring and monitoring body iron Serum ferritin is the most common indirect parameter used to assess body iron stores in the clinical setting. Measurement of serum ferritin is easy and inexpensive but individual values may be affected by infection, inflammation, liver disease, vitamin C deficiency, and hemolysis [bib_ref] Serum ferritin, liver iron stores, and liver histology in children with thalassaemia, Virgiliis [/bib_ref]. Although ferritin is not the most accurate marker of iron overload, ferritin levels do correlate with transfusion burden [bib_ref] Prognostic factors and life expectancy in myelodysplastic syndromes classified according to WHO..., Malcovati [/bib_ref] and liver iron concentration (LIC) [bib_ref] Serum ferritin, liver iron stores, and liver histology in children with thalassaemia, Virgiliis [/bib_ref] , and trends in ferritin levels are useful for following iron load. Liver biopsy is considered the gold standard for direct measurement of total body iron, but the use of this invasive technique is excluded in many patients because of thrombocytopenia and neutropenia, which may predispose to bleeding and infectious complications [bib_ref] Monitoring chelation therapy to achieve optimal outcome in the treatment of thalassaemia, Porter [/bib_ref]. Non-invasive measurement techniques are being developed, including T2* MRI and magnetic susceptometry using a superconducting quantum interference device (SQUID). These techniques have been shown in some studies to provide results that correlate well with liver biopsy-determined iron concentrations [bib_ref] Iron-chelating therapy and the treatment of thalassemia, Olivieri [/bib_ref] [bib_ref] Magnetic-susceptibility measurement of human iron stores, Brittenham [/bib_ref] [bib_ref] Assessment of iron distribution between liver, spleen, pancreas, bone marrow, and myocardium..., Papakonstantinou [/bib_ref] , but they are not widely available worldwide. The data reported in a recent study indicate that serial measurements of serum ferritin are useful for monitoring chelation therapy with DFX so that doses may be adjusted according to the ongoing iron load due to transfusion [bib_ref] Tailoring iron chelation by iron intake and serum ferritin: the prospective EPIC..., Cappellini [/bib_ref]. The DFX prescribing information recommends monitoring serum ferritin monthly to assess the patient's response to therapy and adjusting the DFX dose, if necessary, every 3 to 6 months (16). ## Clinically available iron chelators Currently, three chelating agents are available: deferoxamine, deferiprone, and DFX. ## Deferoxamine (desferal tm ) Deferoxamine (DFO) has been in widespread clinical use since the late 1970s and has provided unequivocal evidence that effective chelation therapy can arrest the progression of, and prevent early death from, iron overload [bib_ref] Intensive iron-chelation therapy with desferrioxamine in iron-loading anaemias, Pippard [/bib_ref]. DFO is a trihydroxamic acid sideraphore secreted by Streptomyces pilosus, a fungus. DFO is a hexadentate chelator with a very high and selective affinity for iron. One molecule of DFO binds one atom of iron. DFO is administered as long parenteral infusions because the plasma half-life is short (min) and it is not active orally. It is usually given as an overnight subcutaneous infusion 5 to 7 nights per week. It is mainly distributed extracellularly and the protein binding in plasma is low (< 10%). After complexing with iron it is excreted rapidly as ferrioxamine, mainly through the kidney and one-third into bile through the feces. Thus, its efficacy is also dependent upon adequate urine output and may be facilitated by dialysis in the case of kidney dysfunction. This requirement is extremely demanding and can result in poor adherence, thereby compromising efficacy and outcomes [bib_ref] Iron chelation therapy, Hershko [/bib_ref]. ## Deferiprone (ferriprox tm ) Deferiprone (L1; CP20; 1,2-dimethyl-3-hydroxypyrid-4-one) is an orally active hydroxypyridineone first used in humans in 1987. Deferiprone is a bidentate chelator. Three molecules of deferiprone are needed to bind one atom of iron. An advantage of this compound is that the iron chelate of deferiprone carries no net charge and, therefore, can penetrate membranes easily, allowing removal of potentially toxic iron from tissues [bib_ref] The design of orally active iron chelators, Hider [/bib_ref]. Other major advantages include oral administration and rapid absorption through the gastrointestinal tract. Deferiprone is mainly metabolized as glucuronide conjugates and is excreted via the renal route. It has a half-life of 2-3 hr. The typical dosage of deferiprone is 75 mg/kg/day in 3 divided doses, up to 100 mg/ http://dx.doi.org/10.3346/jkms.2013. kg daily [bib_ref] Randomized controlled trial of deferiprone or deferoxamine in beta-thalassemia major patients with..., Pennell [/bib_ref] [bib_ref] Safety and effectiveness of 100 mg/kg/day deferiprone in patients with thalassemia major:..., Taher [/bib_ref]. Deferiprone is rapidly absorbed mainly from the stomach and reaches the circulation quickly. However, there may possibly be food-drug interactions or other gastric factors that delay the appearance of the drug in the blood following oral administration. Wide variations in the metabolism and clearance of deferiprone among patients have been observed, mainly depending on the iron overload and availability of chelatable iron [bib_ref] Oral iron chelators, Cappellini [/bib_ref]. This agent is approved in some Asian and European countries for second-line treatment of iron overload, but not in the USA and Canada due to its narrow therapeutic index and safety concerns including a risk of agranulocytosis [bib_ref] Efficacy and safety of deferiprone (Ferriprox), an oral iron-chelating agent, in pediatric..., Won [/bib_ref] [bib_ref] The safety and effectiveness of deferiprone in a large-scale, 3-year study in..., Ceci [/bib_ref]. ## Deferasirox (exjade tm ) DFX (ICL670) is a new oral tridentate iron (Fe 3+ ) chelator developed specifically for the treatment of chronic iron overload. It is an N-substituted bis-hydroxyphenyl-triazole selected from more than 700 compounds screened as part of a national drug development program [bib_ref] Development of tridentate iron chelators: from desferrithiocin to ICL-670, Nick [/bib_ref]. Two molecules of DFX are needed to bind one atom of iron. With a plasma half-life of 8 to 16 hr, oncedaily dosing permits circulating drug to continuously scavenge non-transferrin-bound "labile plasma iron, " which is the chemical species responsible for generating toxic oxygen intermediaries that can cause tissue damage in iron-overloaded subjects [bib_ref] Role of iron in inducing oxidative stress in thalassemia: can it be..., Rachmilewitz [/bib_ref]. Hepatocytes readily take up DFX, which chelates hepatocellular iron. The DFX-iron complex is then excreted in the bile [bib_ref] Pharmacokinetics, metabolism, and disposition of deferasirox in beta-thalassemic patients with transfusion-dependent iron..., Waldmeier [/bib_ref]. Within cells, DFX chelates cytosolic iron, leading to ferritin degradation by the proteasome [bib_ref] Specific iron chelators determine the route of ferritin degradation, De Domenico [/bib_ref]. ## Clinical benefits Iron chelation therapy has been shown to be beneficial in patients with transfusion-dependent anemia, especially β-thalassemia major. One small, randomized trial and many other observational studies have demonstrated that maintenance of low serum ferritin levels using iron chelators was associated with a reduction in end-organ toxicity as well as prolongation of survival in patients with transfusion-dependent anemia [bib_ref] Update on iron chelators in thalassemia, Neufeld [/bib_ref] [bib_ref] Iron-chelating therapy for transfusional iron overload, Brittenham [/bib_ref]. In Korea, thalassemia is very rare and major indication for iron chelation therapy includes AA and MDS [bib_ref] Iron chelation therapy in the myelodysplastic syndromes and aplastic anemia: a review..., Lee [/bib_ref]. Guidelines for the management of AA or MDS suggest that iron chelation therapy should be considered for the patients with iron overload, although the guidelines often differ in specific details [bib_ref] Guidelines for the diagnosis and management of aplastic anaemia, Marsh [/bib_ref] [bib_ref] Iron chelation therapy in MDS: what have we learnt recently?, Schmid [/bib_ref]. Few studies have assessed the efficacy of iron chelation therapy in patients with AA. The prospective 1-yr Evaluation of Patients' Iron Chelation with DFX (EPIC) study enrolled the largest number (n = 116) of AA patients and showed the effectiveness of DFX in reducing body iron (4). However, no trial has established the long-term effectiveness of iron chelation therapy in preventing organ toxicity or improving survival in patients with AA. Clinical effects of iron chelation therapy in iron overloaded patients with MDS have been studied in several aspects: survival prolongation, improvement of organ function, improved outcomes after allogeneic hematopoietic stem cell transplantation (HSCT), and cytopenia improvement in lower risk MDS. However, these potential benefits of iron chelation therapy in MDS patients remain controversial [bib_ref] Controversies surrounding iron chelation therapy for MDS, Leitch [/bib_ref]. For example, iron chelation therapy may not be beneficial in patients with MDS whose expected survival is less than one year. In individual patients receiving long-term transfusion, the benefits of iron chelation therapy may vary according to the morbidity associated with the therapy, the estimated prognosis of MDS, and the latency period between the onset of transfusion and the development of clinical manifestations of iron overload [bib_ref] Iron-chelating therapy for transfusional iron overload, Brittenham [/bib_ref]. Recent retrospective data suggest that efficient iron chelation therapy may prolong survival in patients with MDS [bib_ref] Does iron chelation therapy improve survival in regularly transfused lower risk MDS..., Rose [/bib_ref] [bib_ref] Improving clinical outcome in patients with myelodysplastic syndrome and iron overload using..., Leitch [/bib_ref]. The Groupe Francophone des Myelodysplasies reported a retrospective study in which 97 patients with low or intermediate-1 IPSS risk presenting for RBC transfusion over a one-month period were analyzed, and 53 patients (55%) received iron chelation therapy for at least 6 months. Median survival was 124 months in the group of patients receiving standard or adequate chelation (n = 41, deferoxamine by infusion at least 3 days per week, or deferiprone, deferasirox, or a combination of agents) compared to 85 months in those receiving low or weak chelation (n = 12; deferoxamine by intermittent bolus) and 53 months in non-chelated patients (n = 44) (P < 0.01) [bib_ref] Does iron chelation therapy improve survival in regularly transfused lower risk MDS..., Rose [/bib_ref]. In a retrospective study from Vancouver, the four year survival was 80% for chelated patients with low or intermediate-1 IPSS risk MDS and it was 44% for non-chelated patients (P < 0.03) [bib_ref] Improving clinical outcome in patients with myelodysplastic syndrome and iron overload using..., Leitch [/bib_ref]. So far, there have been no randomized trials examining whether morbidity or mortality would be improved with iron chelation therapy in patients with MDS. One randomized trial is currently recruiting patients to prospectively assess the efficacy and safety of iron chelation therapy with DFX compared to placebo in MDS patients with transfusional iron overload. Although iron chelation therapy is well established to reverse hepatic or cardiac dysfunction and reduce the risk of diabetes mellitus in beta-thalassemia major, there have been only limited data for the effects of iron chelation therapy on organ function in MDS. Retrospective nationwide survey of Japanese patients with transfusion-dependent MDS and AA showed that effective chelation with deferoxamine resulted in improved serum ferritin, liver enzymes, and fasting blood sugar [bib_ref] Iron-chelating therapy and the treatment of thalassemia, Olivieri [/bib_ref] [bib_ref] Long-term outcome of continuous 24-hour deferoxamine infusion via indwelling intravenous catheters in..., Davis [/bib_ref] [bib_ref] Retrospective nationwide survey of Japanese patients with transfusion-dependent MDS and aplastic anemia..., Takatoku [/bib_ref]. The EPIC study enrolled 341 patients with MDS and overall median serum ferritin decreased significantly at 1 yr (P = 0.002). Alanine aminotransferase levels also decreased significantly and the change correlated significantly with reduction in serum ferritin (P < 0.001) [bib_ref] Deferasirox in ironoverloaded patients with transfusion-dependent myelodysplastic syndromes: results from the large..., Gattermann [/bib_ref]. In higher risk MDS, unlikely the case with lower risk MDS who would live long enough to experience adverse effects of http://dx.doi.org/10.3346/jkms.2013.28.11.1563 iron overload-induced tissue damage, the major benefit of iron chelation therapy is not likely to come from reduction in end organ damage due to tissue iron overload, but from potential beneficial effects on other outcomes such as reduction of infections, prevention or delay of leukemic evolution, and improved outcomes after allogeneic HSCT [bib_ref] Objectives of iron chelation therapy in myelodysplastic syndromes: more than meets the..., Pullarkat [/bib_ref]. It has been evidenced that iron overload increases infection risk and iron chelators, especially DFX with in vitro fungicidal effects and long half-life, may lower infection risk in higher risk MDS with neutropenia [bib_ref] Objectives of iron chelation therapy in myelodysplastic syndromes: more than meets the..., Pullarkat [/bib_ref] [bib_ref] Natural resistance, iron and infection: a challenge for clinical medicine, Bullen [/bib_ref]. Excessive labile plasma iron (LPI) in iron overload can lead to increased generation of reactive oxygen species (ROS), which can induce genomic instability in hematopoietic stem cells [bib_ref] Regulation of reactive oxygen species and genomic stability in hematopoietic stem cells, Naka [/bib_ref]. Thus, use of iron chelating agents in higher risk MDS may prevent or delay progression to AML [bib_ref] Objectives of iron chelation therapy in myelodysplastic syndromes: more than meets the..., Pullarkat [/bib_ref]. In a subgroup analysis of 18 patients with low or intermediate-1 risk MDS who were treated with subcutaneous deferoxamine, median leukemia-free survival was not reached at 226 months compared with a matched control group who had a median leukemia-free survival of 40 months [bib_ref] Controversies surrounding iron chelation therapy for MDS, Leitch [/bib_ref]. In the allogeneic HSCT setting, iron overload is known to increase the risk of transplant-related mortality (TRM) and other complications including fungal infections, hepatic dysfunction, and hepatic veno-occlusive disease after HSCT [bib_ref] Iron overload adversely affects outcome of allogeneic hematopoietic cell transplantation, Pullarkat [/bib_ref] [bib_ref] Iron overload might increase transplant-related mortality in haematopoietic stem cell transplantation, Altès [/bib_ref] [bib_ref] Iron overload in bone marrow transplant recipients, Strasser [/bib_ref] [bib_ref] Iron overload manifesting as apparent exacerbation of hepatic graftversus-host disease after allogeneic..., Kamble [/bib_ref]. Until now, the adverse prognostic impacts of iron overload on the transplantation outcomes have been evaluated in the patients with various hematologic disorders including thalassemia, MDS, and acute leukemia [bib_ref] Iron overload adversely affects outcome of allogeneic hematopoietic cell transplantation, Pullarkat [/bib_ref] [bib_ref] Antin JH. Prognostic impact of elevated pretransplantation serum ferritin in patients undergoing..., Armand [/bib_ref] [bib_ref] Red blood cell transfusion dependence and outcome after allogeneic peripheral blood stem..., Platzbecker [/bib_ref] [bib_ref] Effect of iron overload and iron-chelating therapy on allogeneic hematopoietic SCT in..., Lee [/bib_ref]. Therefore, for candidates to allogeneic HSCT, iron chelation therapy seems to be important to reduce TRM and achieve better transplantation outcome. According to a recent report, iron chelation therapy should be considered in patients with iron overload because it can prevent organ damage by reducing the iron overload and also may improve hematopoiesis [bib_ref] Improvement in hematopoiesis after iron chelation therapy with deferasirox in patients with..., Lee [/bib_ref]. In this study, 4 cases of AA raised the possibility of a potential additive benefit on hematopoiesis following iron chelation therapy with DFX. Possible effect on hematopoiesis is DFX can suppress LPI and ROS and may inhibit apoptosis. Other possible effects of DFX on hematopoiesis include altering intracellular levels of the nuclear transcription factor NF-kB or increasing erythropoietin levels [bib_ref] Controversies surrounding iron chelation therapy for MDS, Leitch [/bib_ref] [bib_ref] Objectives of iron chelation therapy in myelodysplastic syndromes: more than meets the..., Pullarkat [/bib_ref]. ## Current status of iron overload and practice in korea According to a multicenter, cross-sectional survey of 1,128 adult Korean patients with AA or MDS, a substantial proportion (n = 331, 29%) suffered from iron overload (serum ferritin > 1,000 ng/mL), and all of the patients analyzed who were treated with DFO iron chelation therapy experienced organ damage. Interestingly, in these patients, iron chelation treatment was not actively administered until complications related to iron overload had appeared. Among the 331 patients who were diagnosed with iron overload, 97 also had organ dysfunction and they were heavily transfused, with a high iron burden (high serum ferritin level) and a long duration of disease. This study also showed that there was a correlation between serum ferritin and the number of transfusions, duration of transfusion therapy, and duration of transfusion dependence [bib_ref] Iron chelation therapy in the myelodysplastic syndromes and aplastic anemia: a review..., Lee [/bib_ref]. In a retrospective analysis of 101 Korean children receiving HSCT, patients were divided into three groups according to their ferritin levels at the time of transplantation as follows: patients with serum ferritin > 1,000 ng/mL at the time of HSCT, serum ferritin < 1,000 ng/mL before HSCT without iron chelation therapy, and serum ferritin that decreased to < 1,000 ng/ mL after iron chelation therapy before HSCT. In this study, patients with serum ferritin < 1,000 ng/mL before HSCT had higher survival rates and lower treatment-related mortality compared to patients with serum ferritin > 1,000 ng/mL. The authors concluded that iron chelation therapy before HSCT can improve the outcome (50). ## Korean guideline for iron chelation therapy Iron chelation therapy is critical and there is much evidence of its clinical benefits in patients with transfusion-induced iron overload. To optimize iron chelation therapy, the KSHAAWP has constructed a Korean guideline for the treatment of transfusion-induced iron overload. Components of the Korean iron chelation guideline are given below. ## Determining patients who need iron chelation therapy Transfusion-dependent patients (MDS, AA, pure red cell aplasia, myelofibrosis, etc.) need iron chelation therapy. Transfusion-dependent patients are defined as those receiving > 8 RBC units with a serum ferritin level > 1,000 ng/mL in at least two successive tests. Patients should have a life expectancy of more than 1 yr. ## Initiating iron chelation Oral DFX and deferoxamine are available in Korea. However, due to short half life and poor compliance, deferoxamine is not recommended as a standard treatment for iron overload. Oral DFX is a recommended treatment in Korean patients with iron overload. The recommended initial daily dose of DFX is 20 mg/ kg body weight, taken on an empty stomach at least 30 min before food. Tablets are available in dosages of 125, 250, or 500 mg and should be dispersed in water or orange or apple juice. ## Monitoring and maintenance therapy After initiating iron chelation therapy, serum ferritin levels and organ functions including cardiac, hepatic and renal functions http://dx.doi.org/10.3346/jkms.2013. should be checked monthly during the first 3 months and then at least once every 3 months. It is recommended that serum ferritin levels be maintained below 1,000 ng/mL and that, when ferritin levels are < 500 ng/mL at two successive tests, iron chelation should be discontinued. If ferritin levels continue to increase after initiating therapy, increase the DFX dose up to 30 mg/kg/day. After discontinuing iron chelation, if serum ferritin levels increase above 1,000 ng/mL, restart DFX at the same dose. This guideline is summarized in . ## Adverse events ## Gastrointestinal disturbances Safety data for DFX have been reported in many studies for patients with various anemias [bib_ref] Relative response of patients with myelodysplastic syndromes and other transfusion-dependent anaemias to..., Porter [/bib_ref] [bib_ref] Patient-reported outcomes of deferasirox (Exjade, ICL670) versus deferoxamine in sickle cell disease..., Vichinsky [/bib_ref] [bib_ref] A phase 3 study of deferasirox (ICL670), a once-daily oral iron chelator,..., Cappellini [/bib_ref] [bib_ref] Phase II clinical evaluation of deferasirox, a once-daily oral chelating agent, in..., Galanello [/bib_ref]. In the EPIC study, DFX was well tolerated with a clinically manageable side effect profile [bib_ref] Tailoring iron chelation by iron intake and serum ferritin: the prospective EPIC..., Cappellini [/bib_ref]. According to previous studies, the most common drug-related adverse events (AE) were gastrointestinal (GI) disturbances such as abdominal pain (6%), nausea (22%), vomiting (8%), and diarrhea (15%). These events were seen to be generally mildto-moderate in severity and dose dependent, tended to occur early in the course of treatment and usually lasted less than 1 week, and in general, resolved spontaneously without the need for dose adjustment or discontinuation of treatment [bib_ref] Tailoring iron chelation by iron intake and serum ferritin: the prospective EPIC..., Cappellini [/bib_ref]. ## Expert panel recommendations for gi disturbances Management of diarrhea requires an exact diagnosis to exclude other etiologies. The expert panel recommends hydration and the use of loperamide for a maximum of 2 days, with evening pre-prandial DFX dosing. The panel does not recommend administration before bedtime because of the risk of esophageal irritation and bleeding. The panel does not recommend DFX dose reduction in cases of mild diarrhea. In cases of persisting moderate-to-severe diarrhea, dose reduction to 10 mg/kg/day should be considered for moderate diarrhea but cease DFX administration for severe diarrhea. When moderate diarrhea has resolved, the DFX dose should be increased in 5 mg/kg/day steps to the target dose. In severe cases, the panel recommends re-initiating the DFX dose at 10 mg/kg/day and adjusting the dose in increments of 5 mg/kg each week to the target dose. These recommendations are summarized in . In cases of abdominal pain the panel recommends evening pre-prandial dosing and consumption of soft food for several hours after taking DFX. The panel does not recommend the use of narcotic analgesic drugs or non-steroidal anti-inflammatory drugs because of side effects. If patients have persistent mildto-moderate pain, dose reductions should be considered before discontinuation. Reduce the dose in steps of 5 mg/kg/day . Korean guideline for iron overload Determining which patients need iron chelation therapy (all criteria must be satisfied) · Transfusion-dependent patients: MDS, AA, primary red cell aplasia, myelofibrosis, etc. · RBC transfusion ≥ 8 units · Ferritin > 1,000 ng/mL · Life expectancy ≥ 1 yr Initiating Iron Chelation · Starting dose: deferasirox (DFX) 20 mg/kg/day · If not effective, DFX dose can be increased to 30 mg/kg/day · Take on an empty stomach at least 30 min before food · Tablets are available in dosages of 125, 250, or 500 mg · Disperse in water or orange or apple juice Monitoring and maintenance therapy · Check ferritin levels and organ functions including renal, hepatic function every month for the first 3 months · After 3 months, check ferritin levels and organ functions at 3 month intervals · Maintain the ferritin level at < 1,000 ng/mL · If the ferritin level is < 500 ng/mL, stop iron chelation · After discontinuing of iron chelation, if the ferritin level is > 1,000 ng/mL, restart DFX . Management of adverse events -Diarrhea (reported incidence: 15%). · Hydration · Loperamide · Pre-prandial evening DFX dosing · Reduce DFX dose to 10 mg/kg/day in moderate diarrhea · Discontinue DFX dose in severe diarrhea ## Diarrhea resolves · Continue hydration · Re-escalate DFX dose to target dose in increments of 5 mg/kg each week in moderate diarrhea · Re-initiate DFX dose at 10 mg/kg/day and dose adjust in increments of 5 mg/kg each week to target dose in severe diarrhea Diarrhea persists or is worse after treatment · Investigate other reasons for diarrhea · Discontinue DFX therapy if diarrhea is unmanageable on re-initiation http://dx.doi.org/10.3346/jkms.2013. and increment the dose in steps of 5 mg/kg/day after the pain has resolved. If persistent severe abdominal pain occurs, temporarily discontinue DFX treatment and re-initiate and escalate in 5 mg/kg/day steps after the abdominal pain has resolved. These recommendations are summarized in [fig_ref] Figure 2: Management of adverse events -Abdominal pain [/fig_ref]. For the management of nausea and vomiting the expert panel recommends hydration, and preprandial administration of DFX in the evening should be considered. In cases of severe vomiting reduce the DFX dose in steps of 5 mg/kg/day. When the symptoms have resolved, re-initiate DFX and increase the dose in increments up to the target dose. These recommendations are summarized in [fig_ref] Figure 3: Management of adverse events -Nausea/vomiting [/fig_ref]. ## Renal adverse events Some mild increases in serum creatinine levels have been observed and these appear to be dose dependent. In patients with β-thalassemia, 3% overall had serum creatinine values that increased more than 33% above baseline but were still within the normal range [bib_ref] Effectiveness and safety of ICL670 in iron-loaded patients with thalassaemia: a randomised,..., Nisbet-Brown [/bib_ref]. In patients with AA, 25% experienced an increase in serum creatinine to more than 33% above baseline and higher than the upper limit of normal (ULN) on two consecutive visits, but there were no progressive increases. The only factor identified as having a significant impact on serum creatinine was concomitant use of the immunosuppressive agent cyclosporine, a drug with a well-recognized potential to Pre-prandial evening DFX dosing · Take soft food for several hours after DFX · Do not recommend DFX before bedtime · Do not use narcotic pain medications or non-steroidal anti-inflammatory drugs · Consider dose reduction in step of 5 mg/kg/day before discontinuation in mild-to-moderate cases · Temporarily discontinue DFX in severe cases Abdominal pain resolves · Continue DFX · Re-escalate DFX dose to target dose in increments of 5 mg/kg each week in moderate abdominal pain · Re-initiate DFX dose at 10 mg/kg/day and dose adjust in increments of 5 mg/kg each week to target dose in severe abdominal pain Abdominal pain persists or is worse after treatment · Investigate other reasons for abdominal pain · Reduce DFX dose in steps of 5 mg/kg/day before discontinuation in persistent mild-to-moderate abdominal pain · Discontinue DFX therapy if abdominal pain is unmanageable on re-initiation impair renal function. Careful monitoring of renal function is necessary in patients with AA who are receiving concomitant cyclosporine and DFX (4). ## Expert panel recommendations for renal adverse events The management of serum creatinine increases should be individualized. It is recommended that serum creatinine levels be assessed in duplicate before initiating therapy and monitored monthly thereafter. Patients who have additional renal risk factors, such as preexisting renal or co-morbid conditions, are elderly, or are receiving medicine that depresses renal function should be monitored weekly during the first month after initiation or modification of the DFX dose and monitored monthly thereafter. In patients who exhibit serum creatinine elevations to more than 33% above baseline but still within the normal range at two consecutive visits, the daily dose should be reduced by 10 mg/kg. If there is a progressive increase in serum creatinine beyond ULN, DFX should be discontinued [bib_ref] Clinical application of deferasirox: practical patient management, Vichinsky [/bib_ref]. Once the creatinine level has returned to within the normal range, and if the clinical benefit is expected to exceed the potential risks, DFX should be re-initiated at 10 mg/kg/day and increased in 5 mg/ kg/day steps to the target dose. These recommendations are summarized in [fig_ref] Figure 4: Management of renal adverse events [/fig_ref]. ## Hepatic and skin adverse events In the 1-yr trials, up to 2% of patients developed elevations in serum transaminases. These elevations were not related to dose [bib_ref] Tailoring iron chelation by iron intake and serum ferritin: the prospective EPIC..., Cappellini [/bib_ref]. In previous studies, up to 11% of patients developed dosedependent skin eruptions and most symptoms were mild to moderate [bib_ref] Tailoring iron chelation by iron intake and serum ferritin: the prospective EPIC..., Cappellini [/bib_ref]. ## Expert panel recommendations for hepatic and skin adverse events As a precaution, liver function should be monitored monthly. Furthermore, following any severe or persistent elevations in serum transaminase levels, dose modification should be considered and a hepatologist should be consulted. Therapy may be restarted in patients once serum transaminase levels have returned to within normal limits and levels should be monitored monthly. In cases of mild-to-moderate skin adverse events, the expert · If Cr > 33% above baseline but still within normal range at two consecutive visits, DFX should be reduced by 10 mg/kg · Discontinue DFX dose if progressive Cr elevation Creatinine level returns to normal range · Re-escalate DFX dose to target dose in increments of 5 mg/kg each week in case of reduced dosing · Re-initiate DFX dose at 10 mg/kg/day and dose adjust in increments of 5 mg/kg each week to target dose in case of discontinuation Creatinine level progressively increases · Investigate other reasons for renal impairment · Discontinue DFX therapy · Do not reduce dose in mild-to-moderate cases · Temporarily discontinue DFX in severe cases · Short-term use of steroid Skin eruptions resolve · Continue DFX in mild-to-moderate cases · Re-initiate DFX dose at 10 mg/kg/day and dose adjust in increments of 5 mg/kg each week to target dose in case of discontinuation Skin eruptions persist or are worse after treatment · Investigate other reasons for skin eruptions · Discontinue DFX therapy http://dx.doi.org/10.3346/jkms.2013.28.11.1563 panel does not recommend dose reduction because most skin eruptions resolved spontaneously without treatment. But if the skin eruptions persist or are worse after 1 week the panel advises a temporary discontinuation of DFX treatment until the skin eruptions have disappeared. Short-term use of a steroid should be considered. DFX should then be reinitiated and the dose should be escalated in 5 mg/kg/day steps. These recommendations are summarized in [fig_ref] Figure 5: Management of skin adverse events [/fig_ref]. ## Other general considerations Although the increases in adverse events described above have been reported as dose dependent, recent data showed that the safety profile of DFX in patients who received more than 30 mg/ kg/day was consistent with that of patients who received less than 30 mg/kg/day. Interestingly, this study also demonstrated that no adverse events were observed following escalation to over 30 mg/kg/day that were not present at lower doses. There was a higher incidence of drug-related AEs and higher treatment discontinuation rates in patients with MDS than in patients with other chronic anemias [bib_ref] Tailoring iron chelation by iron intake and serum ferritin: the prospective EPIC..., Cappellini [/bib_ref]. This may be related to the risk of disease progression, preexisting co-morbidities, use of concomitant medication, and the advanced ages of patients with MDS. Most AEs resolved spontaneously and no patients with renal or hepatic failure or drug-related cytopenia have been reported in any of the studies to date. The only contraindication to DFX is prior hypersensitivity to the drug. DFX has not been investigated in pregnant or breast feeding women, or in pediatric patients younger than 2 yr. Iron chelation should be ceased as soon as pregnancy is confirmed. Caution should be considered in patients older than 65 yr due to a greater frequency of preexisting suppressed hepatic, renal, or cardiac function. # Conclusion Iron overload is a major concern in patients with chronic anemia for whom regular transfusions are necessary. Evidence from many clinical trials has demonstrated clear reduction of the iron burden and improvement in organ function after administration of DFX to patients with transfusion-induced iron overload. This guideline could help many Korean physicians to anticipate potential effects, alert patients to the likelihood of key events, and readily provide effective management according to the recommendations outlined. [fig] Figure 2: Management of adverse events -Abdominal pain (reported incidence: 6%). [/fig] [fig] Figure 3: Management of adverse events -Nausea/vomiting (reported incidence: 22%, 8%). [/fig] [fig] Figure 4: Management of renal adverse events (reported incidence: 25%). [/fig] [fig] Figure 5: Management of skin adverse events (reported incidence: 11%). [/fig]

Daclatasvir # Disclaimer WikiDoc MAKES NO GUARANTEE OF VALIDITY. WikiDoc is not a professional health care provider, nor is it a suitable replacement for a licensed healthcare provider. WikiDoc is intended to be an educational tool, not a tool for any form of healthcare delivery. The educational content on WikiDoc drug pages is based upon the FDA package insert, National Library of Medicine content and practice guidelines / consensus statements. WikiDoc does not promote the administration of any medication or device that is not consistent with its labeling. Please read our full disclaimer here. # Overview Daclatasvir is a hepatitis C virus (HCV) NS5A inhibitor that is FDA approved for the treatment of patients with chronic HCV genotype 1 or 3 infection with sofosbuvir and with or without ribavirin. Common adverse reactions include headache, anemia, nausea, and fatigue (≥10%). # Adult Indications and Dosage ## FDA-Labeled Indications and Dosage (Adult) Daclatasvir is indicated for use with sofosbuvir, with or without ribavirin, for the treatment of patients with chronic hepatitis C virus (HCV) genotype 1 or genotype 3 infection. - Limitations of Use: Sustained virologic response (SVR12) rates are reduced in HCV genotype 3-infected patients with cirrhosis receiving Daclatasvir in combination with sofosbuvir for 12 weeks. - Testing Prior to Initiation of Therapy NS5A Resistance Testing in HCV Genotype 1a-Infected Patients with Cirrhosis: Consider screening for the presence of NS5A polymorphisms at amino acid positions M28, Q30, L31, and Y93 in patients with cirrhosis who are infected with HCV genotype 1a prior to the initiation of treatment with Daclatasvir and sofosbuvir with or without ribavirin. - Recommended Dosage - The recommended dosage of Daclatasvir is 60 mg, taken orally, once daily, with or without food. - Table 1 provides the recommended Daclatasvir-containing treatment regimens and duration based on HCV genotype and patient population. The optimal duration of Daclatasvir and sofosbuvir with or without ribavirin has not been established for HCV genotype 3 patients with cirrhosis or for HCV genotype 1 patients with Child-Pugh C cirrhosis. - For patients with HCV/HIV-1 coinfection, follow the dosage recommendations in Table 1. - For specific dosage recommendations for sofosbuvir, refer to the prescribing information. - For HCV genotype 1 or 3 patients with Child-Pugh B or C cirrhosis or post-transplantation patients, the starting dose of ribavirin is 600 mg once daily, increasing up to 1000 mg daily as tolerated. The starting dose and on-treatment dose of ribavirin can be decreased based on hemoglobin and creatinine clearance. - For HCV genotype 3 patients with compensated cirrhosis (Child-Pugh A), the recommended dosing of ribavirin is based on weight (1000 mg for patients weighing less than 75 kg and 1200 mg for those weighing at least 75 kg administered orally in two divided doses with food). - Table 1:Recommended Treatment Regimen and Duration for Daclatasvir in Patients with Genotype 1 or 3 HCV DAKLINZA: Daclatasvir's Brand name - Dosage Modification Due to Drug Interactions Refer to the drug interactions and contraindications sections for other drugs before coadministration with Daclatasvir. - Table 2:Recommended Daclatasvir Dosage Modification with CYP3A Inhibitors and Inducers DAKLINZA: Daclatasvir's Brand name Dosage reduction of Daclatasvir for adverse reactions is not recommended. - Discontinuation of Therapy If sofosbuvir is permanently discontinued in a patient receiving Daclatasvir with sofosbuvir, then Daclatasvir should also be discontinued. ## Off-Label Use and Dosage (Adult) ### Guideline-Supported Use There is limited information regarding Off-Label Guideline-Supported Use of Daclatasvir in adult patients. ### Non–Guideline-Supported Use There is limited information regarding Off-Label Non–Guideline-Supported Use of Daclatasvir in adult patients. # Pediatric Indications and Dosage ## FDA-Labeled Indications and Dosage (Pediatric) Safety and effectiveness of Daclatasvir in pediatric patients younger than 18 years of age have not been established. ## Off-Label Use and Dosage (Pediatric) ### Guideline-Supported Use There is limited information regarding Off-Label Guideline-Supported Use of Daclatasvir in pediatric patients. ### Non–Guideline-Supported Use There is limited information regarding Off-Label Non–Guideline-Supported Use of Daclatasvir in pediatric patients. # Contraindications - When Daclatasvir is used in combination with other agents, the contraindications applicable to those agents are applicable to the combination regimen. Refer to the respective prescribing information for a list of contraindications. - Daclatasvir is contraindicated in combination with drugs that strongly induce CYP3A and, thus, may lead to lower exposure and loss of efficacy of Daclatasvir. Contraindicated drugs include, but are not limited to those listed in Table 3. - Table 3:Drugs that are Contraindicated with Daclatasvir Daclatasvir: Daclatasvir's Brand name # Warnings The concomitant use of Daclatasvir and other drugs may result in known or potentially significant drug interactions, some of which may lead to: - loss of therapeutic effect of Daclatasvir and possible development of resistance, - dosage adjustments of concomitant medications or Daclatasvir, - possible clinically significant adverse reactions from greater exposures of concomitant drugs or Daclatasvir. See Table 3 for drugs contraindicated with Daclatasvir due to loss of efficacy and possible development of resistance. See Table 7 for steps to prevent or manage other possible and known significant drug interactions. Consider the potential for drug interactions before and during Daclatasvir therapy, review concomitant medications during Daclatasvir therapy, and monitor for the adverse reactions associated with the concomitant drugs. Postmarketing cases of symptomatic bradycardia and cases requiring pacemaker intervention have been reported when amiodarone is coadministered with sofosbuvir in combination with another HCV direct-acting antiviral, including Daclatasvir. A fatal cardiac arrest was reported in a patient receiving a sofosbuvir-containing regimen (ledipasvir/sofosbuvir). Bradycardia has generally occurred within hours to days, but cases have been observed up to 2 weeks after initiating HCV treatment. Patients also taking beta blockers or those with underlying cardiac comorbidities and/or advanced liver disease may be at increased risk for symptomatic bradycardia with coadministration of amiodarone. Bradycardia generally resolved after discontinuation of HCV treatment. The mechanism for this bradycardia effect is unknown. Coadministration of amiodarone with Daclatasvir in combination with sofosbuvir is not recommended. For patients taking amiodarone who have no alternative treatment options and who will be coadministered Daclatasvir and sofosbuvir: - Counsel patients about the risk of serious symptomatic bradycardia. - Cardiac monitoring in an inpatient setting for the first 48 hours of coadministration is recommended, after which outpatient or self-monitoring of the heart rate should occur on a daily basis through at least the first 2 weeks of treatment. Patients who are taking sofosbuvir in combination with Daclatasvir who need to start amiodarone therapy due to no other alternative treatment options should undergo similar cardiac monitoring as outlined above. Due to amiodarone’s long elimination half-life, patients discontinuing amiodarone just prior to starting sofosbuvir in combination with Daclatasvir should also undergo similar cardiac monitoring as outlined above. Patients who develop signs or symptoms of bradycardia should seek medical evaluation immediately. Symptoms may include near-fainting or fainting, dizziness or lightheadedness, malaise, weakness, excessive tiredness, shortness of breath, chest pain, confusion, or memory problems. If Daclatasvir and sofosbuvir are administered with ribavirin, the warnings and precautions for ribavirin, in particular the pregnancy avoidance warning, apply to this combination regimen. Refer to the ribavirin prescribing information for a full list of the warnings and precautions for ribavirin. # Adverse Reactions ## Clinical Trials Experience If Daclatasvir and sofosbuvir are administered with ribavirin, refer to the prescribing information for ribavirin regarding ribavirin-associated adverse reactions. The following serious adverse reaction is described below and elsewhere in the labeling: - Serious Symptomatic Bradycardia When Coadministered with Sofosbuvir and Amiodarone. Because clinical trials are conducted under widely varying conditions, adverse reaction rates observed in the clinical trials of a drug cannot be directly compared to rates in the clinical trials of another drug and may not reflect the rates observed in practice. Approximately 2400 subjects with chronic HCV infection have been treated with the recommended dose of Daclatasvir in combination with other anti-HCV drugs in clinical trials. Six hundred seventy-nine subjects have received a Daclatasvir and sofosbuvir-based regimen. Safety experience from three clinical trials of Daclatasvir and sofosbuvir with or without ribavirin is presented. - Daclatasvir and Sofosbuvir In the ALLY-3 trial, 152 treatment-naive and treatment-experienced subjects with HCV genotype 3 infection were treated with Daclatasvir 60 mg once daily in combination with sofosbuvir for 12 weeks. The most common adverse reactions (frequency of 10% or greater) were headache and fatigue. All adverse reactions were mild to moderate in severity. No subjects discontinued therapy for adverse events. In the ALLY-2 trial, 153 treatment-naive and treatment-experienced subjects with HCV/HIV-1 coinfection were treated with Daclatasvir 60 mg once daily (dose-adjusted for concomitant antiretroviral use) in combination with sofosbuvir for 12 weeks. The most common adverse reaction (frequency of 10% or greater) was fatigue. The majority of adverse reactions were mild to moderate in severity. No subjects discontinued therapy for adverse events. Adverse reactions considered at least possibly related to treatment and occurring at a frequency of 5% or greater in ALLY-3 or ALLY-2 are presented in Table 4. - Table 4:Adverse Reactions (All Severity) Reported at ≥5% Frequency, Daclatasvir + Sofosbuvir, Studies ALLY-3 and ALLY-2 - Daclatasvir, Sofosbuvir, and Ribavirin In the ALLY-1 trial, 113 subjects with chronic HCV infection, including 60 subjects with Child-Pugh A, B, or C cirrhosis and 53 subjects with recurrence of HCV after liver transplantation, were treated with Daclatasvir 60 mg once daily in combination with sofosbuvir and ribavirin for 12 weeks. The most common adverse reactions (frequency of 10% or greater) among the 113 subjects were headache, anemia, fatigue, and nausea. The majority of adverse reactions were mild to moderate in severity. Of the 15 (13%) subjects who discontinued study drug for adverse events, 13 (12%) subjects discontinued ribavirin only and 2 (2%) subjects discontinued all study drugs. During treatment, 4 subjects in the cirrhotic cohort underwent liver transplantation. Adverse reactions considered at least possibly related to treatment and occurring at a frequency of 5% or greater in either treatment cohort in ALLY-1 are presented in Table 5. - Table 5:Adverse Reactions (All Severity) Reported at ≥5% Frequency in Either Treatment Cohort, Daclatasvir + Sofosbuvir + Ribavirin, Study ALLY-1 Daclatasvir: Daclatasvir's Brand name - Laboratory Abnormalities Selected Grade 3 and 4 treatment-emergent laboratory abnormalities observed in clinical trials of Daclatasvir in combination with sofosbuvir with or without ribavirin are presented in Table 6. - Table 6:Selected Grade 3 and 4 Laboratory Abnormalities in Clinical Trials of Daclatasvir + Sofosbuvir ± Ribavirin, Studies ALLY-3, ALLY-2, and ALLY-1 Daclatasvir: Daclatasvir's Brand name ## Postmarketing Experience The following adverse reactions have been identified during postapproval use of Daclatasvir. Because these reactions are reported voluntarily from a population of uncertain size, it is not always possible to reliably estimate their frequency or establish a causal relationship to drug exposure. Cardiac Disorders: Serious symptomatic bradycardia has been reported in patients taking amiodarone who initiate treatment with sofosbuvir in combination with another HCV direct-acting antiviral, including Daclatasvir. # Drug Interactions Daclatasvir is a substrate of CYP3A. Therefore, moderate or strong inducers of CYP3A may decrease the plasma levels and therapeutic effect of daclatasvir. Strong inhibitors of CYP3A (eg, clarithromycin, itraconazole, ketoconazole, ritonavir) may increase the plasma levels of daclatasvir. Daclatasvir is an inhibitor of P-glycoprotein transporter (P-gp), organic anion transporting polypeptide (OATP) 1B1 and 1B3, and breast cancer resistance protein (BCRP). Administration of Daclatasvir may increase systemic exposure to medicinal products that are substrates of P-gp, OATP 1B1 or 1B3, or BCRP, which could increase or prolong their therapeutic effect or adverse reactions (see TABLE 7). Refer to the prescribing information for other agents in the regimen for drug interaction information. The most conservative recommendation should be followed. Table 7 provides clinical recommendations for established or potentially significant drug interactions between Daclatasvir and other drugs. Clinically relevant increase in concentration is indicated as “↑” and clinically relevant decrease as “↓” for drug interaction data. - Table 7:Established and Other Potentially Significant Drug Interactions Daclatasvir: Daclatasvir's Brand name Based on the results of drug interaction trials, no clinically relevant changes in exposure were observed for cyclosporine, darunavir (with ritonavir), dolutegravir, escitalopram, ethinyl estradiol/norgestimate, lopinavir (with ritonavir), methadone, midazolam, tacrolimus, or tenofovir with concomitant use of daclatasvir. No clinically relevant changes in daclatasvir exposure were observed with cyclosporine, darunavir (with ritonavir), dolutegravir, escitalopram, famotidine, lopinavir (with ritonavir), omeprazole, sofosbuvir, tacrolimus, or tenofovir. No dosage adjustment for daclatasvir is necessary with darunavir/cobicistat or moderate CYP3A inhibitors, including atazanavir (unboosted), fosamprenavir, ciprofloxacin, diltiazem, erythromycin, fluconazole, or verapamil. # Use in Specific Populations ### Pregnancy Pregnancy Category (FDA): - Risk Summary No adequate human data are available to determine whether or not Daclatasvir poses a risk to pregnancy outcomes. In animal reproduction studies in rats and rabbits, no evidence of fetal harm was observed with oral administration of daclatasvir during organogenesis at doses that produced exposures up to 6 and 22 times, respectively, the recommended human dose (RHD) of 60 mg of Daclatasvir. However, embryofetal toxicity was observed in rats and rabbits at maternally toxic doses that produced exposures of 33 and 98 times the human exposure, respectively, at the RHD of 60 mg of Daclatasvir. In rat pre- and postnatal developmental studies, no developmental toxicity was observed at maternal systemic exposure (AUC) to daclatasvir approximately 3.6 times higher than the RHD of Daclatasvir. In the U.S. general population, the estimated background risk of major birth defects and miscarriage in clinically recognized pregnancies is 2% to 4% and 15% to 20%, respectively. If Daclatasvir and sofosbuvir are administered with ribavirin, the combination regimen is contraindicated in pregnant women and in men whose female partners are pregnant. Refer to the ribavirin prescribing information for more information on use in pregnancy. - Data - Animal Data Daclatasvir was administered orally to pregnant rats at doses of 0, 50, 200, or 1000 mg/kg/day on gestation days 6 to 15. Maternal toxicity (mortality, adverse clinical signs, body-weight losses, and reduced food consumption) was noted at doses of 200 and 1000 mg/kg/day. In the offspring, malformations of the fetal brain, skull, eyes, ears, nose, lip, palate, or limbs were observed at doses of 200 and 1000 mg/kg. The dose of 1000 mg/kg was associated with profound embryolethality and lower fetal body weight. No malformations were noted at 50 mg/kg/day. Systemic exposure (AUC) at 50 mg/kg/day in pregnant females was 6 times higher than exposures at the RHD. In rabbits, daclatasvir was initially administered at doses of 0, 40, 200, or 750 mg/kg/day during the gestation days 7 to 19. Daclatasvir dosing was modified due to vehicle toxicity during the study to doses of 20, 99, and 370 mg/kg/day, respectively. Maternal toxicity was noted at doses of 200/99 and 750/370 mg/kg/day with adverse clinical signs and severe reductions in body weight and food consumption. Mortality and euthanasia occurred in multiple dams at 750/370 mg/kg/day. At 200/99 mg/kg/day, fetal effects included increased embryofetal lethality, reduced fetal body weights, and increased incidences of fetal malformations of the ribs as well as head and skull. No malformations were noted in rabbits at 40/20 mg/kg/day. Systemic exposures (AUC) at 40/20 mg/kg/day were 22 times higher than exposures at the RHD. In a pre- and postnatal developmental study, daclatasvir was administered orally at 0, 25, 50, or 100 mg/kg/day from gestation day 6 to lactation day 20. At 100 mg/kg/day, maternal toxicity included mortality and dystocia; developmental toxicity included slight reductions in offspring viability in the perinatal and neonatal periods and reductions in birth weight that persisted into adulthood. There was neither maternal nor developmental toxicity at doses up to 50 mg/kg/day. Systemic exposures (AUC) at this dose were 3.6 times higher than the RHD. Pregnancy Category (AUS): There is no Australian Drug Evaluation Committee (ADEC) guidance on usage of Daclatasvir in women who are pregnant. ### Labor and Delivery There is no FDA guidance on use of Daclatasvir during labor and delivery. ### Nursing Mothers - Risk Summary It is not known whether Daclatasvir is present in human milk, affects human milk production, or has effects on the breastfed infant. Daclatasvir was present in the milk of lactating rats. The developmental and health benefits of breastfeeding should be considered along with the mother’s clinical need for Daclatasvir and any potential adverse effects on the breastfed child from Daclatasvir or from the underlying maternal condition. If Daclatasvir is administered with ribavirin, the nursing mothers information for ribavirin also applies to this combination regimen. Refer to ribavirin prescribing information for additional information. - Data Milk concentrations of daclatasvir were evaluated on lactation day 10 as part of the rat pre- and postnatal development study. Daclatasvir was present in rat milk with concentrations 1.7 to 2 times maternal plasma levels. ### Pediatric Use Safety and effectiveness of Daclatasvir in pediatric patients younger than 18 years of age have not been established. ### Geriatic Use Of 1184 subjects treated with the recommended dose of Daclatasvir in ten clinical trials, 7% of subjects were 65 years of age or older. Safety was similar across older and younger subjects and there were no safety findings unique to subjects 65 years and older. SVR12 rates were comparable among older and younger subjects. No dosage adjustment of Daclatasvir is required for elderly patients. ### Gender There is no FDA guidance on the use of Daclatasvir with respect to specific gender populations. ### Race There is no FDA guidance on the use of Daclatasvir with respect to specific racial populations. ### Renal Impairment No dosage adjustment of Daclatasvir is required for patients with any degree of renal impairment. Refer also to the sofosbuvir and ribavirin prescribing information for information regarding use in patients with renal impairment. ### Hepatic Impairment Based on a hepatic impairment study in non–HCV-infected subjects, no dosage adjustment of Daclatasvir is required for patients with mild (Child-Pugh A), moderate (Child-Pugh B), or severe (Child-Pugh C) hepatic impairment. ### Females of Reproductive Potential and Males If Daclatasvir and sofosbuvir are administered with ribavirin, the information for ribavirin with regard to pregnancy testing, contraception, and infertility also applies to this combination regimen. Refer to ribavirin prescribing information for additional information. ### Immunocompromised Patients There is no FDA guidance one the use of Daclatasvir in patients who are immunocompromised. # Administration and Monitoring ### Administration There is limited information regarding Daclatasvir Administration in the drug label. ### Monitoring There is limited information regarding Daclatasvir Monitoring in the drug label. # IV Compatibility There is limited information regarding the compatibility of Daclatasvir and IV administrations. # Overdosage There is no known antidote for overdose of Daclatasvir. Treatment of overdose with Daclatasvir should consist of general supportive measures, including monitoring of vital signs and observation of the patient’s clinical status. Because daclatasvir is highly protein bound (>99%), dialysis is unlikely to significantly reduce plasma concentrations of the drug. # Pharmacology ## Mechanism of Action Daclatasvir is a direct-acting antiviral agent (DAA) against the hepatitis C virus. ## Structure Daclatasvir is an inhibitor of HCV nonstructural protein 5A (NS5A). The chemical name for drug substance daclatasvir dihydrochloride is carbamic acid, N,N′--4,4′-diylbis]]bis-, C,C′-dimethyl ester, hydrochloride (1:2). Its molecular formula is C40H50N8O62HCl, and its molecular weight is 738.88 (free base). Daclatasvir dihydrochloride has the following structural formula: Daclatasvir dihydrochloride drug substance is white to yellow. Daclatasvir is freely soluble in water (>700 mg/mL). This drug contain 60 mg daclatasvir (equivalent to 66 mg daclatasvir dihydrochloride) and the inactive ingredients anhydrous lactose (116 mg), microcrystalline cellulose, croscarmellose sodium, silicon dioxide, magnesium stearate, and Opadry green. Daclatasvir 30 mg tablets (equivalent to 33 mg daclatasvir dihydrochloride) contain the inactive ingredients anhydrous lactose (58 mg), microcrystalline cellulose, croscarmellose sodium, silicon dioxide, magnesium stearate, and Opadry green. Daclatasvir 90 mg tablets (equivalent to 99 mg daclatasvir dihydrochloride) contain the inactive ingredients anhydrous lactose (173 mg), microcrystalline cellulose, croscarmellose sodium, silicon dioxide, magnesium stearate, and Opadry green. Opadry green contains hypromellose, titanium dioxide, polyethylene glycol 400, FD&C blue #2/indigo carmine aluminum lake, and yellow iron oxide. ## Pharmacodynamics Cardiac Electrophysiology At a dose 3 times the maximum recommended dose, daclatasvir did not prolong the QT interval to any clinically relevant extent. ## Pharmacokinetics The pharmacokinetic properties of daclatasvir were evaluated in healthy adult subjects and in subjects with chronic HCV. Administration of daclatasvir tablets in HCV-infected subjects resulted in approximately dose-proportional increases in Cmax, AUC, and Cmin up to 60 mg once daily. Steady state is anticipated after approximately 4 days of once-daily daclatasvir administration. Exposure of daclatasvir was similar between healthy and HCV-infected subjects. Population pharmacokinetic estimates for daclatasvir 60 mg once daily in chronic HCV-infected subjects are shown in Table 8. - Table 8:Population Pharmacokinetic Estimates for Daclatasvir in Chronic HCV-Infected Subjects Receiving Daclatasvir 60 mg Once Daily and Sofosbuvir 400 mg Once Daily In HCV-infected subjects following multiple oral doses of daclatasvir tablet ranging from 1 mg to 100 mg once daily, peak plasma concentrations occurred within 2 hours post dose. In vitro studies with human Caco-2 cells indicated that daclatasvir is a substrate of P-gp. The absolute bioavailability of the tablet formulation is 67%. - Effect of Food on Oral Absorption In healthy subjects, administration of a daclatasvir 60 mg tablet after a high-fat, high-caloric meal (approximately 951 total kcal, 492 kcal from fat, 312 kcal from carbohydrates, 144 kcal from protein) decreased daclatasvir Cmax and AUC(0-inf) by 28% and 23%, respectively, compared with fasted conditions. A food effect was not observed with administration of a daclatasvir 60 mg tablet after a low-fat, low-caloric meal (approximately 277 total kcal, 41 kcal from fat, 190 kcal from carbohydrates, 44 kcal from protein) compared with fasted conditions. With multiple dosing, protein binding of daclatasvir in HCV-infected subjects was approximately 99% and independent of dose at the dose range studied (1-100 mg). In subjects who received daclatasvir 60 mg tablet orally followed by 100 μg -daclatasvir intravenous dose, estimated volume of distribution at steady state was 47 L. Daclatasvir is a substrate of CYP3A, with CYP3A4 being the primary CYP isoform responsible for metabolism. Following single-dose oral administration of 25 mg 14C-daclatasvir in healthy subjects, the majority of radioactivity in plasma was predominately attributed to parent drug (97% or greater). Following single-dose oral administration of 25 mg 14C-daclatasvir in healthy subjects, 88% of total radioactivity was recovered in feces (53% of the dose as unchanged daclatasvir) and 6.6% of the dose was excreted in the urine (primarily as unchanged daclatasvir). Following multiple-dose administration of daclatasvir in HCV-infected subjects, with doses ranging from 1 mg to 100 mg once daily, the terminal elimination half-life of daclatasvir ranged from approximately 12 to 15 hours. In subjects who received daclatasvir 60 mg tablet orally followed by 100 μg -daclatasvir intravenous dose, the total clearance was 4.2 L/h. - Renal Impairment The pharmacokinetics of daclatasvir following a single 60 mg oral dose was studied in non–HCV-infected subjects with renal impairment. Using a regression analysis, the predicted AUC(0-inf) of daclatasvir was estimated to be 26%, 60%, and 80% higher in subjects with creatinine clearance (CLcr) values of 60, 30, and 15 mL/min, respectively, relative to subjects with normal renal function (CLcr of 90 mL/min, defined using the Cockcroft-Gault CLcr formula), and daclatasvir unbound AUC(0-inf) was predicted to be 18%, 39%, and 51% higher for subjects with CLcr values of 60, 30, and 15 mL/min, respectively, relative to subjects with normal renal function. Using observed data, subjects with end-stage renal disease requiring hemodialysis had a 27% increase in daclatasvir AUC(0-inf) and a 20% increase in unbound AUC(0-inf) compared to subjects with normal renal function as defined using the Cockcroft-Gault CLcr formula. Daclatasvir is highly protein bound to plasma proteins and is unlikely to be removed by dialysis. - Hepatic Impairment The pharmacokinetics of daclatasvir following a single 30 mg oral dose was studied in non–HCV-infected subjects with mild (Child-Pugh A), moderate (Child-Pugh B), and severe (Child-Pugh C) hepatic impairment compared to a corresponding matched control group. The Cmax and AUC(0-inf) of total daclatasvir (free and protein-bound drug) were lower by 46% and 43%, respectively, in Child-Pugh A subjects; by 45% and 38%, respectively, in Child-Pugh B subjects; and by 55% and 36%, respectively, in Child-Pugh C subjects. The Cmax and AUC(0‑inf) of unbound daclatasvir were lower by 43% and 40%, respectively, in Child-Pugh A subjects; by 14% and 2%, respectively, in Child-Pugh B subjects; and by 33% and 5%, respectively, in Child-Pugh C subjects. - Pediatric Patients The pharmacokinetics of daclatasvir in pediatric patients has not been evaluated. - Geriatric Patients Population pharmacokinetic analysis in HCV-infected subjects showed that within the age range (18-79 years) analyzed, age did not have a clinically relevant effect on the pharmacokinetics of daclatasvir. - Gender Population pharmacokinetic analyses in HCV-infected subjects estimated that female subjects have a 30% higher daclatasvir AUC compared to male subjects. This difference in daclatasvir AUC is not considered clinically relevant. - Race Population pharmacokinetic analyses in HCV-infected subjects indicated that race had no clinically relevant effect on daclatasvir exposure. - Cytochrome P450 (CYP) Enzymes Daclatasvir is a substrate of CYP3A. In vitro, daclatasvir did not inhibit (IC50 greater than 40 microM) CYP enzymes 1A2, 2B6, 2C8, 2C9, 2C19, or 2D6. Daclatasvir did not have a clinically relevant effect on the exposure of midazolam, a sensitive CYP3A substrate. - Transporters Daclatasvir is a substrate of P-gp. However, cyclosporine, which inhibits multiple transporters including P-gp, did not have a clinically relevant effect on the pharmacokinetics of daclatasvir. Daclatasvir, in vitro, did not inhibit OCT2 and did not have a clinically relevant effect on the pharmacokinetics of tenofovir, an OAT substrate. Daclatasvir demonstrated inhibitory effects on digoxin (a P-gp substrate) and rosuvastatin (an OATP 1B1, OATP 1B3, and BCRP substrate) in drug-drug interaction trials. Drug interaction studies were conducted with daclatasvir and other drugs likely to be coadministered or drugs used as probes to evaluate potential drug-drug interactions. The effects of daclatasvir on the Cmax, AUC, and Cmin of the coadministered drug are summarized in Table 9, and the effects of the coadministered drug on the Cmax, AUC, and Cmin of daclatasvir are summarized in Table 10. For information regarding clinical recommendations. Drug interaction studies were conducted in healthy adults unless otherwise noted. - Table 9:Effect of Daclatasvir on the Pharmacokinetics of Concomitant Drugs DAKLINZA: Daclatasvir's Brand name - Table 10:Effect of Coadministered Drugs on Daclatasvir Pharmacokinetics DAKLINZA: Daclatasvir's Brand name No clinically relevant interaction is anticipated for daclatasvir or the following concomitant medications: peginterferon alfa, ribavirin, or antacids. No clinically relevant interaction is anticipated for daclatasvir with concomitant use of rilpivirine. - Mechanism of Action Daclatasvir is an inhibitor of NS5A, a nonstructural protein encoded by HCV. Daclatasvir binds to the N-terminus of NS5A and inhibits both viral RNA replication and virion assembly. Characterization of daclatasvir-resistant viruses, biochemical studies, and computer modeling data indicate that daclatasvir interacts with the N-terminus within Domain 1 of the protein, which may cause structural distortions that interfere with NS5A functions. - Antiviral Activity Daclatasvir had median EC50 values of 0.008 nM (range, 0.002-0.03 nM; n=35), 0.002 nM (range, 0.0007-0.006 nM; n=30), and 0.2 nM (range, 0.006-3.2 nM; n=17) against hybrid replicons containing genotypes 1a, 1b, and 3a subject-derived NS5A sequences, respectively, without detectable daclatasvir resistance-associated polymorphisms at NS5A amino acid positions 28, 30, 31, or 93. Daclatasvir activity was reduced against genotypes 1a, 1b, and 3a subject-derived replicons with resistance-associated polymorphisms at positions 28, 30, 31, or 93, with median EC50 values of 76 nM (range, 4.6-2409 nM; n=5), 0.05 nM (range, 0.002-10 nM; n=12), and 13.5 nM (range, 1.3-50 nM; n=4), respectively. Similarly, the EC50 values of daclatasvir against 3 genotype 3b and 1 genotype 3i subject-derived NS5A sequences with polymorphisms (relative to a genotype 3a reference) at positions 30+31 (genotype 3b) or 30+62 (genotype 3i) were ≥3620 nM. Daclatasvir was not antagonistic with interferon alfa, HCV NS3/4A protease inhibitors, HCV NS5B nucleoside analog inhibitors, and HCV NS5B non-nucleoside inhibitors in cell culture combination antiviral activity studies using the cell-based HCV replicon system. - Resistance - In Cell Culture HCV genotype 1a, 1b, and 3a replicon variants with reduced susceptibility to daclatasvir were selected in cell culture, and the genotype and phenotype of daclatasvir-resistant NS5A amino acid variants were characterized. Phenotypic analysis of genotype 1a replicons expressing single NS5A M28T, Q30E, Q30H, Q30R, L31V, Y93C, Y93H, and Y93N substitutions exhibited 500-, 18500-, 1083-, 900-, 2500-, 1367-, 8500-, and 34833-fold reduced susceptibility to daclatasvir, respectively. For genotype 1b, L31V and Y93H single substitutions and L31M/Y93H and L31V/Y93H combinations exhibited 33-, 30-, 16000-, and 33667-fold reduced susceptibility to daclatasvir, respectively. A P32-deletion (P32X) in genotype 1b reduced daclatasvir susceptibility by >1,000,000-fold. For genotype 3a, single A30K, L31F, L31I, and Y93H substitutions exhibited 117-, 320-, 240-, and 3733-fold reduced susceptibility to daclatasvir, respectively. - In Clinical Studies Among subjects with HCV genotype 1 or genotype 3 infection and treated in the ALLY-1, -2, and -3 trials with Daclatasvir and sofosbuvir with or without ribavirin for 12 weeks, 31 subjects (11 with genotype 1a, 1 with genotype 1b, and 19 with genotype 3) qualified for resistance analysis due to virologic failure. Post-baseline NS5A and NS5B population-based nucleotide sequence analysis results were available for 31 and 28 subjects, respectively. Virus from all 31 subjects at the time of virologic failure harbored one or more of the following NS5A resistance-associated substitutions (including pre-existing amino acid polymorphisms or treatment-emergent substitutions): M28T, Q30H/K/R, L31M/V, H54R, H58D/P, or Y93C/N for genotype 1a subjects, P32-deletion (P32X) for the genotype 1b subject, and A30K/S, L31I, S62A/L/P/R/T, or Y93H for genotype 3 subjects. Among HCV genotype 1a virologic failure subjects, the most common NS5A amino acid substitutions occurred at position Q30 (Q30H/K/R; 73% , all treatment-emergent). Among HCV genotype 3 virologic failure subjects, the most common NS5A amino acid polymorphism or treatment-emergent substitution was Y93H (89% , treatment-emergent in 11 of 17 subjects). For NS5B, 6 of 28 subjects at the time of virologic failure had virus with NS5B substitutions possibly associated with sofosbuvir resistance or exposure: A112T, L159F, E237G, or Q355H (genotype 1a subjects), or S282T+Q355H (genotype 3 subject). - Persistence of Resistance-Associated Substitutions Limited data for Daclatasvir and sofosbuvir regimens on the persistence of daclatasvir resistance-associated substitutions are available. In a separate long-term follow-up study of predominately HCV genotype 1-infected subjects treated with daclatasvir-containing regimens in phase 2/3 clinical trials, viral populations with treatment-emergent NS5A resistance-associated substitutions persisted at detectable levels for more than 1 year in most subjects. - Effect of Baseline HCV Amino Acid Polymorphisms on Treatment Response Genotype 1a NS5A polymorphisms: In HCV genotype 1a-infected subjects with cirrhosis, the presence of an NS5A amino acid polymorphism at position M28, Q30, L31, or Y93 (defined as any change from reference identified by population-based nucleotide sequencing) was associated with reduced efficacy of Daclatasvir and sofosbuvir with or without ribavirin for 12 weeks in the ALLY-1 and ALLY-2 trials (see Table 11). Due to the limited sample size, insufficient data are available to determine the impact of specific NS5A polymorphisms at these positions on SVR12 rates in subjects with cirrhosis. Six of 54 subjects (11%) with cirrhosis had one of the following specific NS5A polymorphisms at baseline: M28V/T (n=2), Q30R (n=1), L31M (n=2), or Y93N (n=1); 2 subjects with M28V or Q30R achieved SVR12 while 4 subjects with M28T, L31M, or Y93N did not achieve SVR. Eleven of 112 subjects (10%) without cirrhosis had one or more of the following specific NS5A polymorphisms at baseline: M28T/V (n=3), Q30H/L/R (n=5), L31M (n=1), and Y93C/H/S (n=4); all noncirrhotic subjects with these baseline NS5A polymorphisms achieved SVR12. Based on an analysis of 1026 HCV genotype 1a NS5A amino acid sequences from pooled clinical trials, the prevalence of polymorphisms at these positions was 11% overall, and 11% in the U.S. Genotype 1b NS5A polymorphisms: In a pooled analysis of 43 subjects infected with HCV genotype 1b with available baseline nucleotide sequence data in ALLY-1 and -2, virus from 21% (n=9) of subjects receiving Daclatasvir and sofosbuvir with or without ribavirin had one of the following baseline NS5A amino acid polymorphisms: R30K/M/Q (n=4), L31M (n=2), or Y93H (n=3). All 9 subjects with NS5A polymorphisms achieved SVR12, including 5 who were noncirrhotic and 4 who were in the post-transplant period. Genotype 3 NS5A polymorphisms: In the ALLY-3 trial in which HCV genotype 3-infected subjects received Daclatasvir and sofosbuvir for 12 weeks, the presence of an NS5A Y93H polymorphism was associated with a reduced SVR12 rate (see Table 11). In a pooled analysis of 175 subjects infected with HCV genotype 3 with available baseline nucleotide sequence data in the ALLY-1, -2, and -3 trials, virus from 7% (13/175) of subjects had the NS5A Y93H polymorphism, and all 13 of these subjects were in the ALLY-3 trial. Phylogenetic analysis of NS5A sequences indicated that all genotype 3 subjects with available data in the ALLY-1, -2, and -3 trials (n=175) were infected with HCV subtype 3a. - Table 11:Impact of NS5A Amino Acid Polymorphisms on SVR12 Rates in Subjects with HCV Genotype 1a or Genotype 3 Infection in Phase 3 Trials of Daclatasvir + Sofosbuvir ± Ribavirin DAKLINZA: Daclatasvir's Brand name - Cross-Resistance Based on resistance patterns observed in cell culture replicon studies and HCV-infected subjects, cross-resistance between daclatasvir and other NS5A inhibitors is expected. Cross-resistance between daclatasvir and other classes of direct-acting antivirals is not expected. The impact of prior daclatasvir treatment experience on the efficacy of other NS5A inhibitors has not been studied. Conversely, the efficacy of Daclatasvir in combination with sofosbuvir has not been studied in subjects who have previously failed treatment with regimens that include an NS5A inhibitor. ## Nonclinical Toxicology - Carcinogenesis and Mutagenesis A 2-year carcinogenicity study in Sprague Dawley rats and a 6-month study in transgenic (Tg rasH2) mice were conducted with daclatasvir. In the 2-year study in rats, no drug-related increase in tumor incidence was observed at doses up to 50 mg/kg/day (both sexes). Daclatasvir exposures at these doses were approximately 6-fold (males and females) the human systemic exposure at the therapeutic daily dose of Daclatasvir. In transgenic mice no drug-related increase in tumor incidence was observed at doses of 300 mg/kg/day (both sexes). Daclatasvir was not genotoxic in a battery of in vitro or in vivo assays, including bacterial mutagenicity (Ames) assays, mammalian mutation assays in Chinese hamster ovary cells, or in an in vivo oral micronucleus study in rats. If Daclatasvir and sofosbuvir are administered in a regimen containing ribavirin, the information for ribavirin on carcinogenesis and mutagenesis also applies to this combination regimen (see prescribing information for ribavirin). - Impairment of Fertility Daclatasvir had no effects on fertility in female rats at any dose tested. Daclatasvir exposures at these doses in females were approximately 24-fold the human systemic exposure at the therapeutic daily dose of Daclatasvir. In male rats, effects on reproductive endpoints at 200 mg/kg/day included reduced prostate/seminal vesicle weights, minimally increased dysmorphic sperm, as well as increased mean pre-implantation loss in litters sired by treated males. Daclatasvir exposures at the 200 mg/kg/day dose in males were approximately 26-fold the human systemic exposure at the therapeutic daily dose of Daclatasvir. Exposures at 50 mg/kg/day in males produced no notable effects and was 4.7-fold the exposure in humans at the recommended daily dose of Daclatasvir. If Daclatasvir and sofosbuvir are administered with ribavirin, the information for ribavirin on impairment of fertility also applies to this combination regimen. # Clinical Studies The efficacy of Daclatasvir in combination with sofosbuvir and with or without ribavirin was evaluated in three phase 3 clinical trials, as summarized in Table 12. HCV RNA levels were measured during these clinical trials using the COBAS® TaqMan® HCV test (version 2.0), for use with the High Pure System. The assay had a lower limit of quantification (LLOQ) of 25 IU per mL. Sustained virologic response was the primary endpoint and was defined as HCV RNA below the LLOQ at post-treatment week 12 (SVR12). - Table 12:Genotype 1 and 3 Patient Populations from Daclatasvir Trials DAKLINZA: Daclatasvir's Brand name ALLY-3 was an open-label trial that included 152 subjects with chronic HCV genotype 3 infection and compensated liver disease who were treatment naive (n=101) or treatment experienced (n=51). Most treatment-experienced subjects had failed prior treatment with peginterferon/ribavirin, but 7 subjects had been treated previously with a sofosbuvir regimen and 2 subjects with a regimen containing an investigational agent. Previous exposure to NS5A inhibitors was prohibited. Subjects received Daclatasvir 60 mg plus sofosbuvir 400 mg once daily for 12 weeks and were monitored for 24 weeks post treatment. The 152 treated subjects in ALLY-3 had a median age of 55 years (range, 24-73); 59% of the subjects were male; 90% were white, 5% were Asian, and 4% were black. Most subjects (76%) had baseline HCV RNA levels greater than or equal to 800,000 IU per mL; 21% of the subjects had compensated cirrhosis, and 40% had the IL28B rs12979860 CC genotype. SVR12 and outcomes in subjects without SVR12 in ALLY-3 are shown by patient population in Table 13. SVR12 rates were comparable regardless of HCV treatment history, age, gender, IL28B allele status, or baseline HCV RNA level. For SVR outcomes related to baseline NS5A amino acid polymorphisms. - Table 13:ALLY-3: SVR12 in Treatment-Naive and Treatment-Experienced Subjects with or without Cirrhosis with Genotype 3 HCV Treated with Daclatasvir in Combination with Sofosbuvir for 12 Weeks ALLY-2 was an open-label trial that included 153 subjects with chronic hepatitis C and HIV coinfection who received Daclatasvir and sofosbuvir for 12 weeks. Subjects with HCV genotype 1, 2, 3, 4, 5, or 6 infection were eligible to enroll. Subjects were HCV treatment-naive (n=101) or HCV treatment-experienced (n=52). Prior exposure to NS5A inhibitors was prohibited. The dose of Daclatasvir was 60 mg once daily (dose-adjusted for concomitant antiretroviral use) and the dose of sofosbuvir was 400 mg once daily. The 153 treated subjects had a median age of 53 years (range, 24-71); 88% of subjects were male; 63% were white, 33% were black, and 1% were Asian. Sixty-eight percent of subjects had HCV genotype 1a, 15% had HCV genotype 1b, 8% had genotype 2, 7% had genotype 3, and 2% had genotype 4. Most subjects (80%) had baseline HCV RNA levels greater than or equal to 800,000 IU per mL; 16% of the subjects had compensated cirrhosis, and 73% had IL28B rs12979860 non-CC genotype. Concomitant HIV therapy included PI-based regimens (darunavir + ritonavir, atazanavir + ritonavir, or lopinavir/ritonavir) for 46% of subjects, NNRTI-based regimens (efavirenz, nevirapine, or rilpivirine) for 26%, integrase-based regimens (raltegravir or dolutegravir) for 26%, and nucleoside-only regimens (abacavir + emtricitabine + zidovudine) for 1%. Two patients were not receiving treatment for HIV. SVR and outcomes in subjects with HCV genotype 1 without SVR12 in ALLY-2 are shown by patient population in Table 14. Available data on subjects with HCV genotype 2, 4, 5, or 6 infection are insufficient to provide recommendations for these genotypes; therefore, these results are not presented in Table 14. SVR12 rates were comparable regardless of antiretroviral therapy, HCV treatment history, age, race, gender, IL28B allele status, HCV genotype 1 subtype, or baseline HCV RNA level. For SVR outcomes related to baseline NS5A amino acid polymorphisms. No subjects switched their antiretroviral therapy regimen due to loss of plasma HIV-1 RNA suppression. There was no change in absolute CD4+ T-cell counts at the end of 12 weeks of treatment. - Table 14:ALLY-2: SVR12 in Subjects with Genotype 1 and 3 HCV/HIV Coinfection Treated with Daclatasvir in Combination with Sofosbuvir for 12 Weeks ALLY-1 was an open-label trial of Daclatasvir, sofosbuvir, and ribavirin that included 113 subjects with chronic HCV infection and Child-Pugh A, B, or C cirrhosis (n=60) or HCV recurrence after liver transplantation (n=53). Subjects with HCV genotype 1, 2, 3, 4, 5, or 6 infection were eligible to enroll. Subjects could be HCV treatment-naive or treatment-experienced, although prior exposure to NS5A inhibitors was prohibited. Subjects received Daclatasvir 60 mg once daily, sofosbuvir 400 mg once daily, and ribavirin for 12 weeks and were monitored for 24 weeks post treatment. Subjects received an initial ribavirin dose of 600 mg or less daily with food; the initial and on-treatment dosing of ribavirin was modified based on hemoglobin and creatinine clearance measurements. If tolerated, the ribavirin dose was titrated up to 1000 mg per day. A high proportion of reductions in ribavirin dosing occurred in the trial. By week 6, approximately half of the subjects received 400 mg per day or less of ribavirin. In total, 16 subjects (15%) completed less than 12 weeks and 11 subjects (10%) completed less than 6 weeks of ribavirin therapy, respectively. For the cohort of patients with cirrhosis (Child-Pugh A, B, or C), the median time to discontinuation of ribavirin was 43 days (range, 8-82, n=9). For the post-transplant cohort, the median time to discontinuation of ribavirin was 20 days (range, 3-57, n=7). The 113 treated subjects in ALLY-1 had a median age of 59 years (range, 19-82); 67% of the subjects were male; 96% were white, 4% were black, and 1% Asian. Most subjects (59%) were treatment-experienced, and most (71%) had baseline HCV RNA levels greater than or equal to 800,000 IU per mL. Fifty-eight percent of subjects had HCV genotype 1a, 19% had HCV genotype 1b, 4% had genotype 2, 15% had genotype 3, 4% had genotype 4, and 1% had genotype 6, 77% had IL28B rs12979860 non-CC genotype. Among the 60 subjects in the cirrhosis cohort, 20% were Child-Pugh A, 53% were Child-Pugh B, and 27% were Child-Pugh C, and 35% had a Baseline Model for End-Stage Liver Disease (MELD) score of 15 or greater. Most (55%) of the 53 subjects in the post-transplant cohort had F3 or F4 fibrosis (based on FibroSURE® results). SVR12 and outcomes in subjects without SVR12 in ALLY-1 are shown for subjects with HCV genotype 1 by patient population in Table 15. Available data on subjects with HCV genotype 2, 4, 5, or 6 infection are insufficient to provide recommendations; therefore, these results are not presented in Table 15. SVR12 rates were comparable regardless of age, gender, IL28B allele status, or baseline HCV RNA level. For SVR12 outcomes related to baseline NS5A amino acid polymorphisms. No HCV genotype 1 or genotype 3 subjects with Child-Pugh C cirrhosis had baseline resistance-associated NS5A amino acid polymorphisms. SVR12 rates were comparable between genotype 3 (5/6 with Child-Pugh B or C cirrhosis and 10/11 post-liver transplant) and genotype 1 subjects with or without decompensated cirrhosis. - Table 15:ALLY-1: SVR12 in Genotype 1 Subjects with Child-Pugh A, B, or C Cirrhosis or with HCV Genotype 1 Recurrence after Liver Transplantation Treated with Daclatasvir in Combination with Sofosbuvir and Ribavirin for 12 Weeks # How Supplied Daclatasvir is packaged in bottles as described in the table. ## Storage Store Daclatasvir tablets at 25°C (77°F), with excursions permitted between 15°C and 30°C (59°F and 86°F). # Images ## Drug Images ## Package and Label Display Panel # Patient Counseling Information Advise the patient to read the FDA-approved patient labeling (Patient Information). - Drug Interactions Inform patients of the potential for drug interactions with Daclatasvir, and that some drugs should not be taken with Daclatasvir. - Symptomatic Bradycardia When Used in Combination with Sofosbuvir and Amiodarone Advise patients to seek medical evaluation immediately for symptoms of bradycardia, such as near-fainting or fainting, dizziness or lightheadedness, malaise, weakness, excessive tiredness, shortness of breath, chest pain, confusion or memory problems. - Daclatasvir Combination Therapy with Sofosbuvir Inform patients that Daclatasvir should not be used alone. Daclatasvir should be used in combination with sofosbuvir with or without ribavirin for the treatment of HCV genotype 1 or HCV genotype 3 infection. - Missed Doses Advise patients to take Daclatasvir every day at the regularly scheduled time with or without food. Inform patients that it is important not to miss or skip doses and to take Daclatasvir for the duration that is recommended by the physician. For instructions for missed doses of other agents in the regimen, refer to the respective prescribing information. - Pregnancy Advise patients to avoid pregnancy during combination treatment with Daclatasvir and sofosbuvir with ribavirin for 6 months after completion of treatment. Inform patients to notify their healthcare provider immediately in the event of a pregnancy. # Precautions with Alcohol Alcohol-Daclatasvir interaction has not been established. Talk to your doctor regarding the effects of taking alcohol with this medication. # Brand Names DAKLINZA™ # Look-Alike Drug Names There is limited information regarding Daclatasvir Look-Alike Drug Names in the drug label. # Drug Shortage Status # Price

Radiology Image Insertion # Overview It is important to have accurate insight when you are interpreting an imaging series such as CT scans, MRIs, or angiography. From a student's point of view, it may be needlessly complicated to look for a lesion in many frames. For this reason, creating an animated image with highlighted lesions is preferable to uploading many images, since it makes the image more accessible for all users. Here we describe how to make an animated GIF file with highlighted lesions. # How to insert an imaging series for CT scan or MRI If you want to present a series of images (e.g., CT scans, MRIs) instead of uploading many discrete images, you can highlight the pathologic part of the image by encircling it in yellow and then combine the images to create a GIF file by following the steps listed below: - The raw image - 1st highlighted image - 2nd highlighted image - 3rd highlighted image - 4th highlighted image - 5th highlighted image - 6th highlighted image - 7th highlighted image - 8th highlighted image - 9th highlighted image - 10th highlighted image - 11th highlighted image Here are 2 examples for imaging series:

Neurofibromin 1 Neurofibromin 1 (NF1) is a gene in humans that is located on chromosome 17. NF1 codes for neurofibromin, a GTPase-activating protein that negatively regulates RAS/MAPK pathway activity by accelerating the hydrolysis of Ras-bound GTP. There are currently five known neurofibromin isoforms that are expressed in different tissues and perform different functions. NF1 has a high mutation rate and mutations in NF1 can alter cellular growth control, and neural development, resulting in neurofibromatosis type 1 (NF1, also known as von Recklinghausen syndrome). Symptoms of NF1 include cutaneous neurofibromas, café au lait pigment spots, plexiform neurofibromas, skeletal defects and optic nerve gliomas. # Gene NF1 encodes the protein neurofibromin, a GTPase-activating protein, which primarily regulates the protein Ras. NF1 is located on the long arm of chromosome 17, position q11.2 and was identified in 1990 through positional cloning. NF1 spans over 350-kb of genomic DNA and contains 62 exons. 58 of these exons are constitutive and 4 exhibit alternative splicing ( 9a, 10a-2, 23a, and 28a). The genomic sequence starts 4,951-bp upstream of the transcription start site and 5,334-bp upstream of the translation initiation codon, with the length of the 5’ UTR being 484-bp long. There are three genes that are present within intron 27b of NF1. These genes are EVI2B, EVI2A and OMG, which are encoded on the opposite strand and are transcribed in the opposite direction of NF1. EVI2A and EVI2B are human homologs of the Evi-2A and Evi-2B genes in mice that encode proteins related to leukemia in mice. OMG is a membrane glycoprotein that is expressed in the human central nervous system during myelination of nerve cells. ## Promoter Early studies of the NF1 promoter found that there is great homology between the human and mouse NF1 promoters. The major transcription start site has been confirmed, as well as two minor transcription start sites in both the human and mouse gene. The major transcription start is 484-bp upstream of the translation initiation site. The open reading frame is 8,520-bp long and begins at the translation initiation site. NF1 exon 1 is 544-bp long, contains the 5’ UTR and encodes the first 20 amino acids of neurofibromin. The NF1 promoter lies within a CpG island that is 472-bp long, consisting of 43 CpG dinucleotides, and extends into the start of exon 1. This CpG Island begins 731-bp upstream of the promoter and no core promoter element, such as a TATA or CCATT box, has been found within it. Although no core promoter element has been found, consensus binding sequences have been identified in the 5’ UTR for several transcription factors such as Sp1 and AP2. A methylation map of five regions of the promoter in both mouse and human was published in 1999. This map showed that three of the regions (at approximately – 1000, – 3000, and – 4000) were frequently methylated, but the cytosines near the transcription start site were unmethylated.  Methylation has been shown to functionally impact Sp1 sites as well as a CREB binding site. It has been shown that the CREB site must be intact for normal promoter activity to occur and methylation at the Sp1 sites may affect promoter activity. Proximal NF1 promoter/5’ UTR methylation has been analyzed in tissues from NF1 patients, with the idea that reduced transcription as a result of methylation could be a “second hit” mechanism equivalent to a somatic mutation. There are some sites that have been detected to be methylated at a higher frequency in tumor tissues than normal tissues. These sites are mostly within the proximal promoter; however, some are in the 5’ UTR as well and there is a lot of interindividual variability in the cytosine methylation in these regions. ## 3' UTR A study in 1993 compared the mouse NF1 cDNA to the human transcript and found that both the untranslated regions and coding regions were highly conserved. It was verified that there are two NF1 polyadenylated transcripts that differ in size because of the length of the 3’ UTR, which is consistent with what has been found in the mouse gene. A study conducted in 2000 examined whether the involvement of the 3’ UTR in post-transcriptional gene regulation had an effect on the variation of NF1 transcript quantity both spatially and temporally. Five regions of the 3’ UTR that appear to bind proteins were found, one of which is HuR, a tumor antigen. HuR binds to AU-rich elements which are scattered throughout the 3' UTR and are thought to be negative regulators of transcript stability. This supports the idea that post-transcriptional mechanisms may influence the levels of NF1 transcript. ## Mutations NF1 has one of the highest mutation rates amongst known human genes, however mutation detection is difficult because of its large size, the presence of pseudogenes, and the variety of possible mutations. The NF1 locus has a high incidence of de novo mutations, meaning that the mutations are not inherited maternally or paternally. Although the mutation rate is high, there are no mutation “hot spot” regions. Mutations tend to be distributed within the gene, although exons 3, 5, and 27 are common sites for mutations. The Human Gene Mutation Database contains 1,347 NF1 mutations, but none are in the “regulatory” category. There have not been any mutations conclusively identified within the promoter or untranslated regions. This may be because such mutations are rare, or they do not result in a recognizable phenotype. There have been mutations identified that affect splicing, in fact 286 of the known mutations are identified as splicing mutations. About 78% of splicing mutations directly affect splice sites, which can cause aberrant splicing to occur. Aberrant splicing may also occur due to mutations within a splicing regulatory element. Intronic mutations that fall outside of splice sites also fall under splicing mutations, and approximately 5% of splicing mutations are of this nature. Point mutations that effect splicing are commonly seen and these are often substitutions in the regulatory sequence. Exonic mutations can lead to deletion of an entire exon, or a fragment of an exon if the mutation creates a new splice site. Intronic mutations can result in the insertion of a cryptic exon, or result in exon skipping if the mutation is in the conserved 3’ or 5’ end. # Protein NF1 encodes neurofibromin (Nf1), which is a 320-kDa protein that contains 2,818 amino acids. Neurofibromin is a GTPase-activating protein (GAP) that negatively regulates Ras pathway activity by accelerating hydrolysis of Ras-bound guanosine triphosphate (GTP). Neurofibromin localizes in the cytoplasm; however, some studies have found neurofibromin or fragments of it in the nucleus. Neurofibromin does contain a nuclear localization signal that is encoded by exon 43, but whether or not neurofibromin plays a role in the nucleus is currently unknown. Neurofibromin is ubiquitously expressed, but expression levels vary depending on the tissue type and developmental stage of the organism. Expression is at its highest level in adult neurons, Schwann cells, astrocytes, leukocytes, and oligodendrocytes. The catalytic RasGAP activity of neurofibromin is located in a central portion of the protein, that is called the GAP-related domain (GRD). The GRD is closely homologous to RasGAP and represents about 10% (229 amino acids) of the neurofibromin sequence. The GRD is made up of a central portion called the minimal central catalytic domain (GAPc) as well as an extra domain (GAPex) that is formed through the coiling of about 50 residues from the N- and C- terminus. The Ras-binding region is found in the surface of GAPc and consists of a shallow pocket that is lined by conserved amino acid residues. In addition to the GRD, neurofibromin also contains a Sec14 homology-like region as well as a pleckstrin homology-like (PH) domain. Sec14 domains are defined by a lipid binding pocket that resembles a cage and is covered by a helical lid portion that is believed to regulate ligand access. The PH-like region displays a protrusion that connects two beta-strands from the PH core that extend to interact with the helical lid found in the Sec14 domain. The function of the interaction between these two regions is presently unclear, but the structure implies a regulatory interaction that influences the helical-lid conformation in order to control ligand access to the lipid binding pocket. ## Function Through its NF1-GRD domain, neurofibromin increases the rate of GTP hydrolysis of Ras, and acts as a tumor suppressor by reducing Ras activity. When the Ras-Nf1 complex assembles, active Ras binds in a groove that is present in the neurofibromin catalytic domain. This binding occurs through Ras switch regions I and II, and an arginine finger present in neurofibromin. The interaction between Ras and neurofibromin causes GAP-stimulated hydrolysis of GTP to GDP. This process depends on the stabilization of residues in the Ras switch I and switch II regions, which drives Ras into the confirmation required for enzymatic function. This interaction between Ras and neurofibromin also requires the transition state of GDP hydrolysis to be stabilized, which is performed through the insertion of the positively charged arginine finger into the Ras active site. This neutralizes the negative charges that are present on GTP during phosphoryl transfer. By hydrolyzing GTP to GDP, neurofibromin inactivates Ras and therefore negatively regulates the Ras pathway, which controls the expression of genes involved in apoptosis, the cell cycle, cell differentiation or migration. Neurofibromin is also known to interact with CASK through syndecan, a protein which is involved in the KIF17/ABPA1/CASK/LIN7A complex, which is involved in trafficking GRIN2B to the synapse. This suggests that neurofibromin has a role in the transportation of the NMDA receptor subunits to the synapse and its membrane. Neurofibromin is also believed to be involved in the synaptic ATP-PKA-cAMP pathway, through modulation of adenylyl cyclase. It is also known to bind the caveolin 1, a protein which regulates p21ras, PKC and growth response factors. ## Isoforms There are currently five known isoforms of neurofibromin (II, 3, 4, 9a, and 10a-2) and these isoforms are generated through the inclusion of alternative splicing exons (9a, 10a-2, 23a, and 48a) that do not alter the reading frame. These five isoforms are expressed in distinct tissues and are each detected by specific antibodies. Neurofibromin type II, also named GRD2 (domain II-related GAP), results from the insertion of exon 23a, which causes the addition of 21 amino acids in the 5’ region of the protein. Neurofibromin type II is expressed in Schwann cells and has reduced GAP activity. Neurofibromin type 3 (also called isoform 3’ ALT) contains exon 48a which results in the insertion of 18 amino acids into the 3’ terminal. Neurofibromin type 4 contains exons 23a and 48a, which results in the insertion of 21 amino acids in the 5’ region, and 18 amino acids in the 3’ terminal. Neurofibromin 9a (also referred to as 9br), includes exon 9a which results in the insertion of 10 amino acids in the 5’ region. This isoform shows little neuronal expression and may play a role in memory and learning mechanisms. An isoform with insertion of exon 10a-2 has been studied introduces a transmembrane domain. The inclusion of exon 10a-2 causes the insertion of 15 amino acids in the 5’ region. This isoform is expressed in most human tissues, therefore it likely performs a housekeeping function in intracellular membranes. It has been suggested that the quantitative differences in expression between the different isoforms may be related to the phenotypic variability of neurofibromatosis type 1 patients. # RNA editing In the NF1 mRNA, there is a site within the first half of the GRD where mRNA editing occurs. Deamination occurs at this site, resulting in the conversion of cytidine into uridine at nucleotide 3916. This deamination changes an arginine codon (CGA) to an in-frame translation stop codon (UGA). If the edited transcript is translated, it produces a protein that cannot function as a tumor suppressor because the N-terminal of the GRD is truncated. The editing site in NF1 mRNA was shown to have high homology to the ApoB editing site, where double stranded mRNA undergoes editing by the ApoB holoenzyme. NF1 mRNA editing was believed to involve the ApoB holoenzyme due to the high homology between the two editing sites, however studies have shown that this is not the case. The editing site in NF1 is longer than the sequence required for ApoB mediated mRNA editing, and the region contains two guanidines which are not present in the ApoB editing site. # Clinical significance Mutations in NF1 are primarily associated with neurofibromatosis type 1 (NF1, also known as von Recklinghausen syndrome). NF1 is the most common single gene disorder in humans, occurring in about 1 in 2500-3000 births worldwide. NF1 is an autosomal dominant disorder, but approximately half of NF1 cases arise from de novo mutations. NF1 has high phenotypic variability, with members of the same family with the same mutation displaying different symptoms and symptom intensities. Café-au-lait spots are the most common sign of NF1, but other symptoms include lisch nodules, cutaneous neurofibromas, plexiform neurofibromas, skeletal defects, and optic nerve gliomas. In addition to neurofibromatosis type I, mutations in NF1 can also lead to juvenile myelomonocytic leukemia, Watson syndrome, and breast cancer. # Model organisms Model organisms have been used in the study of NF1 function. A conditional knockout mouse line, called Nf1tm1a(KOMP)Wtsi was generated as part of the International Knockout Mouse Consortium program, a high-throughput mutagenesis project to generate and distribute animal models of disease to interested scientists. Male and female animals underwent a standardized phenotypic screen to determine the effects of deletion. Twenty six tests were carried out on mutant mice and four significant abnormalities were observed. Over half the homozygous mutant embryos identified during gestation were dead, and in a separate study none survived until weaning. The remaining tests were carried out on heterozygous mutant adult mice: females displayed abnormal hair cycling while males had an decreased B cell number and an increased monocyte cell number.

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