JP2007525432A - Use of ritonavir for the treatment of unconjugated hyperbilirubinemia - Google Patents

Abstract

  The present invention is directed to a method of inducing UGT1A1 isoform expression to treat a disease or disorder or adverse effect resulting from elevated serum concentrations of UGT1A1 substrate comprising administering to a patient an effective amount of ritonavir. In particular, the present invention is directed to a method of treating UGT1A1-induced unconjugated hyperbilirubinemia comprising administering to a patient an effective amount of ritonavir.

Description

  The present invention is directed to a method of inducing expression of a UGT1A1 isoform to treat a disease, disorder or adverse effect resulting from elevated serum concentrations of a UGT1A1 substrate comprising administering to a patient an effective amount of ritonavir. . In particular, the present invention is directed to a method of treating UGT1A1-induced unconjugated hyperbilirubinemia comprising administering to a patient an effective amount of ritonavir.

  Glucuronidation is a major metabolic pathway that determines the disposal of various endogenous substrates such as estriol and bilirubin, as well as exogenous substrates such as ethinyl estradiol. UDP-glucuronosyltransferase (UGT) is a superfamily enzyme that catalyzes these reactions with UDG-glucuronic acid as a cofactor. The UGT superfamily contains at least 10 isozymes in rats and more than 10 isozymes in humans. Actually, it has been confirmed that 16 types of human UGT isozymes have different substrate specificities, and UGT1A1 is a bilirubin-specific isozyme. Bilirubin is the main product of the heme catabolism and is glucuronidated, i.e. conjugated with glucuronic acid, removed from the circulation by the liver resulting in water-soluble metabolites such as mono and diglucuronide, and finally excreted in feces Is done.

  Indinavir (Merck & Co. Inc., New Jersey, USA) is a commonly used potent protease inhibitor that has shown high efficacy against HIV when used in combination with a reverse transcriptase inhibitor. Zucker, S.M. D. , Qin. X, Rooster, S.M. D et al., “Mechanism of indavir-induced hyperbilirubinemia”, PNAS 2001; 98: 12671-12676. Unfortunately, indinavir treatment is associated with an incidence of 6% to 25% of asymptomatic unconjugated hyperbilirubinemia without histological liver injury. The same book. Patients who developed clinical jaundice due to excessive bilirubin accumulation were discontinued from treatment and further laboratory testing was performed. The same book. It has been discovered that the increase in serum unconjugated bilirubin associated with indinavir treatment is caused by direct inhibition of bilirubin conjugation activity. The same book.

  Similarly, atazanavir (Bristol-Myers Squibb, New Jersey, USA) also causes increased blood levels of unconjugated bilirubin. In one study, 57% of subjects who took atazanavir showed unconjugated hyperbilirubinemia. Agarwala, S., was poster presented at the 42nd Academic Conference on Antibacterials and Chemotherapy in San Diego, California, September 27-30, 2002. Russo, R .; other. "Study of pharmacokinetic (PK) interaction at steady state of atazanavir (ATV) and ritonavir (RTV) in healthy subjects (PST) Interaction Study of Affects of Aritavir (ATV) ) " It was discovered that atazanavir inhibits bilirubin conjugation in the micromolar range both in the human microsomal in vitro system and in human cells transfected with the UGT1A1 isoform. O'Mara, E. C., who had a poster presentation at the 40th Academic Conference on Antibacterials and Chemotherapy in Toronto, Ontario, Canada, September 17-20, 2000 , Mumaneni, V .; other. “Relationship Between Uridine Diphosphate-GlucuronosylTransGDP” in healthy subjects who received BMS-232632 and saquinavir. ) 1A1 Genotype and Total Birubin Elevations in Health Subjects Receiving BMS-232632 and Saquinavir) ”.

  Unconjugated hyperbilirubinemia may also be a drug or combination of drugs such as amphotericin B / cholesteryl sulfate complex, testosterone, interferon beta-1b, bicalutamide, ciprofloxacin, oxaliplatin, floxlidine, gemcitabine hydrochloride, sargramostim, gemtuzumab Adverse effects associated with ozogamicin, vinorelbine tartrate, carboplatin, peginterferon alfa-2B, tacrolimus, aldesleukin, darhopristin / quinupristin, didanosine and capecitabine.

  In order to overcome diseases or disorders or adverse effects due to elevated serum UGT1A1 substrate, such as unconjugated hyperbilirubinemia, it is desirable to induce UGT1A1.

  It would also be desirable to be able to treat HIV infection and AIDS with effective HIV protease inhibitors such as indinavir and atazanavir without interrupting treatment due to elevated serum bilirubin caused by inhibition of UGT1A1.

  In addition, without interruption of treatment due to elevated serum bilirubin, drugs such as amphotericin B / cholesteryl sulfate complex, testosterone, interferon beta-1b, bicalutamide, ciprofloxacin, oxaliplatin, floxlidine, gemcitabine hydrochloride, sargramostim , Gemtuzumab ozogamicin, vinorelbine tartrate, carboplatin, peginterferon alfa-2B, tacrolimus, aldesleukin, darhopristin / quinupristin, didanosine and capecitabine.

  The present invention provides a method of inducing expression of a UGT1A1 isoform to treat a disease or disorder or adverse effect resulting from elevated serum levels of UGT1A1 substrate, comprising administering to a patient an effective amount of ritonavir. Preferably, the present invention provides a method for treating unconjugated hyperbilirubinemia comprising administering an effective amount of ritonavir to a patient in need of treatment for unconjugated hyperbilirubinemia. In one embodiment, the effective range of ritonavir is from about 25 mg to about 1200 mg per day.

  Surprisingly, it was discovered that ritonavir induces UGT1A1 isoform expression. This is particularly useful for the treatment of diseases or disorders or adverse effects resulting from elevated serum concentrations of UGT1A1 substrate. Unconjugated hyperbilirubinemia is caused by a drug or combination of drugs, such as indinavir, atazanavir, amphotericin B / cholesteryl sulfate complex, testosterone, interferon beta-1b, bicalutamide, ciprofloxacin, oxaliplatin, floxlidine, hydrochloride Undesirable disease or disorder or adverse effect caused by administration of gemcitabine, sargramostim, gemtuzumab ozogamicin, vinorelbine tartrate, carboplatin, peginterferon alfa-2B, tacrolimus, aldesleukin, darfopristin / quinupristin, didanosine and capecitabine.

  To overcome undesirable diseases or disorders or adverse effects, ritonavir is indinavir, atazanavir, amphotericin B / cholesteryl sulfate complex, testosterone, interferon beta-1b, bicalutamide, ciprofloxacin, oxaliplatin, floxlidine, gemcitabine hydrochloride , Salgramostim, gemtuzumab ozogamicin, vinorelbine tartrate, carboplatin, peginterferon alfa-2B, tacrolimus, aldesleukin, darhopristin / quinupristin, didanosine and capecitabine to induce UGT1A1 isoform expression, followed by serum bilirubin concentration be able to.

  In a first embodiment, the present invention provides a method of inducing UGT1A1 to treat a disease or disorder or adverse effect resulting from elevated serum levels of UGT1A1 substrate, comprising administering an effective amount of ritonavia to a patient. provide. Preferably, the present invention provides a method for treating unconjugated hyperbilirubinemia comprising administering an effective amount of ritonavir to a patient in need of treatment for unconjugated hyperbilirubinemia.

  In a second embodiment, the present invention comprises administering an effective amount of ritonavir together with an active pharmaceutical ingredient to a patient in need of treatment for a disease or disorder or adverse effect resulting from elevated serum levels of UGT1A1 substrate. The present invention provides a therapeutic method by administration of a pharmaceutical ingredient having a disease or disorder caused by an increase in serum concentration of UGT1A1 substrate or an adverse effect. In one embodiment, the active pharmaceutical ingredient is indinavir, atazanavir, amphotericin B / cholesteryl sulfate complex, testosterone, interferon beta-1b, bicalutamide, ciprofloxacin, oxaliplatin, floxlidine, gemcitabine hydrochloride, sargramostim, gemtuzumab It is selected from the group consisting essentially of ozogamicin, vinorelbine tartrate, carboplatin, peginterferon alfa-2B, tacrolimus, aldesleukin, darhopristin / quinupristin, didanosine and capecitabine. In another embodiment, the active pharmaceutical ingredient is indinavir. In yet other embodiments, the active pharmaceutical ingredient is atazanavir. In one embodiment, the disease or disorder resulting from elevated serum levels of UGT1A1 substrate is unconjugated hyperbilirubinemia.

  In a third embodiment, the present invention provides a method of increasing glucuronidation of UGT1A1 substrate comprising administering an effective amount of ritonavir. In one embodiment, the UGT1A1 substrate is bilirubin.

  In a fourth embodiment, the present invention provides a method for increasing UGT1A1 substrate excretion comprising administering an effective amount of ritonavir. In one embodiment, the UGT1A1 substrate is bilirubin.

  In a fifth embodiment, the present invention provides UGT1A1 for treating a disease or disorder or adverse effect resulting from elevated serum levels of UGT1A1 substrate comprising administering to a patient a composition comprising an effective amount of ritonavir. Provide a way to guide. Preferably, the present invention provides a method of treating unconjugated hyperbilirubinemia comprising administering to a patient in need of treatment of unconjugated hyperbilirubinemia a composition comprising an effective amount of ritonavir. The composition can further comprise an effective amount of lopinavir in addition to ritonavir. Examples of such compositions are disclosed in US Pat. No. 6,458,818.

  The terms “concurrent administration” and “administering in parallel” refer to ritonavir and other active pharmaceutical ingredients in subjects in separate dosage forms, simultaneously, at different times, or together as a single dosage form. It means to administer.

  The term “patient” means a mammal, more specifically a human.

  The term “effective amount” is an amount of ritonavir sufficient to induce UGT1A1 isoform expression or increase glucuronidation of the UGT1A1 substrate or increase UGT1A1 substrate excretion. In a preferred embodiment, an effective amount of ritonavir is an amount ranging from about 25 mg to about 1200 mg per day.

  The amount of ritonavir that can be combined with the carrier material to form a single dosage form will vary depending on the patient being treated and the particular mode of administration.

  However, the specific dose concentration for any particular patient will depend on the patient's age, weight, general health, sex and diet, as well as administration time, route of administration, excretion rate, combination of drugs, serum bilirubin elevation. It depends on a variety of factors, including the severity of the disease or disorder caused or adverse effects.

  Ritonavir is structurally represented as the following formula (I).

  Ritonavir is described in U.S. Pat. Nos. 5,142,056, 5,151,438, 5,354,866, 5,362,912, 5,491,253, 5,508,409, 5,526,677, 5,539,206, 5,541,206, which are incorporated herein by reference in their entirety. No. 5541328, No. 5541334, No. 5543549, No. 5543551, No. 5543552, No. 5545750, No. 5552558, No. 5554582, No. 5565418, No. 5556604, No. 5567823, No. 5556777, No. 5580984 No. 5,583,232, No. 5,583233, No. 5,591,860, No. 5,597,926, No. 5,597,927, No. 5,579,928, No. 5,608,072, No. 5,616,714, No. 561672. No. 5661677, No. 5625072, No. 5625092, No. 5635523, No. 5648497, No. 5654466, No. 5659044, No. 5659045, No. 5667675, No. 5567882, No. 55679797, No. 5696270, 5786500, 5837873, 5846987, 5886036, 5892052, 6017928, 6150530 and 6531610 can be used to prepare.

  Ritonavir is also known to be an HIV protease inhibitor and an inhibitor of cytochrome P450 monooxidase, in particular CYP3A4 disclosed in US Pat. No. 6,037,157.

  Ritonavir is a dosage unit formulation containing the conventional pharmaceutically acceptable non-toxic carriers, adjuvants and vehicles as described, administered orally, parenterally, sublingually, by inhalation spray, rectally or topically. Is possible. Topical administration can also include the use of transdermal administration such as transdermal patches or ion implanters. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.

  Injectable preparations, for example, injectable aqueous or oleaginous sterile suspensions can be prepared by known techniques using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, 1.3-propanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending solvent. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in injectable preparations.

  Suppositories for rectal administration of ritonavir are suitable non-irritating pharmaceutical additives such as cocoa butter and polyethylene glycol that are solid at normal temperature but liquid at rectal temperature and therefore melt in the colon and release the drug And the drug can be prepared by mixing.

  Solid dosage forms for oral administration can include capsules, tablets, pills, powders, and granules. In such solid dosage forms, ritonavir can be mixed with at least one inert diluent such as sucrose lactose or starch. Such dosage forms can also conventionally contain other substances besides inert diluents, for example, a lubricant such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms can also contain buffering agents. Tablets and pills can additionally be prepared with enteric coatings.

  Liquid dosage forms for oral administration can include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs containing inert diluents commonly used in the art, such as water. Such compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.

  Ritonavir can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous solvent. Liposomes can be formed and physiologically acceptable non-toxic metabolizable lipids can be used. In addition to the compound of the present invention, the liposome-type composition of the present invention can contain a stabilizer, a preservative, a pharmaceutical additive and the like. Preferred lipids are both natural and synthetic phospholipids and phosphatidylcholines (lecithins).

  Methods for producing liposomes are known in the art. See, for example, Prescott, Methods in Cell Biology, Volume XIV, Academic Press, New York, N. Y. (1976), page 33.

  Exemplary ritonavir preparations are U.S. Pat. Nos. 5,142,056, 5,151,438, 5,354,866, 5,362,912, 5,484,801, 5,491,253, 5,508,409, 5,526,677, which are incorporated herein by reference in their entirety. No. 5,539,122, No. 5541206, No. 5541328, No. 5541334, No. 5543549, No. 5543551, No. 5543552, No. 5545750, No. 5554558, No. 5559158, No. 5559158, No. 5556518, No. 5,565,604, 5,567,823, 5,569,777, 5,580,984, 5,583,232, 5,583233, 5,591,860, 5,597,926, 5,597,927, 5,597,928 5608072, No. 5610193, No. 5616714, No. 5616720, No. 5616776, No. 5625072, No. 5625092, No. 5635523, No. 5648497, No. 5654466, No. 5659044, No. 5659045, No. 5670675, 5675882, 55679797, 5696270, 5725878, 5786500, 5837873, 5846987, 5876749, 5886036, 5892052, 5948436, 6017928 6150530, 6232333, 6458818, 6521651 and 6531610, U.S. Patent Application Nos. US-2003-0032619-A1 and PC Application No. WO93 / 23361, WO94 / 14436, WO95 / 07696, WO95 / 09614, WO95 / 20384, WO98 / 22106, WO00 / 74677, are disclosed in WO01 / 52 821 and WO02 / 096 395. It should be noted that although the foregoing references disclose various ritonavir preparations as examples, the present invention is not so limited.

  The following examples are provided to further illustrate UGT1A1 induction by ritonavir.

  Gunn rats (n = 8) deficient in UDP-glucuronosyltransferase (UGT) activity were administered atazanavir / ritonavir together with a total daily dose of 500/100 mg / kg / day by gavage. . The second group of rats received ritonavir at a total daily dose of 100 mg / kg / day. The third group of rats received only vehicle. Atazanavir was prepared with a vehicle composed of EtOH 5%: propyl glycol (PG) 95%: 1 molar equivalent of p-toluenesulfonic acid. Ritonavir was prepared with a vehicle composed of EtOH 5%: propyl glycol (PG) 95%: p-toluenesulfonic acid 2 molar equivalents. The formulation was prepared so that all rats were given a constant amount of 2 ml / kg per treatment. Rats were dosed twice on day 0 at approximately 12 hour intervals, twice a day, and three times in the morning of day 1.

  Four hours after the first and third doses, blood samples (approximately 0.5 ml) were collected from the jugular vein of each rat into a flat bottom microcentrifuge tube (without anticoagulant) and used for analysis of total bilirubin. . The mean (± SD) serum bilirubin over time for the three groups of rats is shown in FIG. Serum bilirubin increased significantly after the first dose and decreased after the third dose.

  A similar approach was used to assess the increase in bilirubin caused by indinavir / ritonavir and the results are shown in FIG. Serum bilirubin increased markedly after the first dose, and then decreased after the third dose.

  Ritonavir was orally administered to male Sprague-Dawley rats between 225 and 275 grams body weight, approximately 6-12 weeks of age, at the indicated concentrations. Ritonavir was prepared in 5 molar equivalents of enothal 5%: propylene glycol 95%: p-toluenesulfonic acid. Rats were dosed daily for 1 or 3 days and sacrificed the next day. Approximately 100 mg of each liver was placed in TRIzol® reagent and immediately homogenized using a Turrax tissue grinder.

  RNA preparation and analysis was performed at Affymetrix Inc. It carried out according to the method of. The RNA integrity of the collected samples was measured using an Agilent 2100 Bioanalyzer. The cDNA was prepared from 15 μg of RNA using the Superscript Choice system (Cat # 18090-019) from Gibco BRL Life Technologies. Gibco's method was followed exactly, except that the primer used for the reverse transcriptase reaction was a modified T7 primer with 24 thymidines at the 5 termini. The sequence is

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Met.

  After this, labeled cRNA was synthesized from the cDNA using the EnzoRNA transcript labeling kit (catalog number 900182) according to the manufacturer's instructions. Next, about 20 mg of cRNA was fragmented in a solution of 40 mM Trisacetic acid, pH 8.1, KOAc 100 mM and MgOAc 30 mM at 94 ° for 35 minutes. Thereafter, the cDNA was hybridized to an Affymetrix Rat U34 chip containing about 9000 gene sequences. cRNA was hybridized overnight at 45 ° C.

  Microarray data was analyzed using the Rosetta Resolver v2.0 expression data analysis system. Expression changes between the two types of arrays were quantified as “fold change” where the expression ratio was normalized error-weighted PM / MM difference intensity.

  The Affymetrix U34 chip contains five different probe sequences corresponding to UDP-glucuronosyltransferase 1A (UGT1A1). Induction of these probe groups in response to ritonavir treatment is shown in FIG. Rats treated with ritonavir at dose concentrations of 100 mg / kg and higher consistently showed increased UGT1A1 concentrations in all probe groups after 1 and 3 days of treatment. This result indicates that ritonavir is an inducer of UGT1A1 expression.

  Ritonavir was prepared as 7.5 and 25 mg / mL solutions in 5% ethanol, 95% propyl glycol and 2 molar equivalents of p-toluenesulfonic acid. A control group of rats received a vehicle composed of 5% ethanol, 95% propyl glycol and 37.6 mg / mL p-toluenesulfonic acid. The phenobarbital sodium administration solution was prepared in a normal saline solution at a concentration of 25 mg base / ml.

  Sixteen male and female Sprague-Dawley rats (VAF Crl: CD (SD) BR) weighing approximately 250 g were purchased from Charles River Laboratories, Inc. (Portage, MI). The rats were randomly divided into the four treatment groups shown in Table 2.

Animals were given free access to Certified Rodent Chow ^* # 5002 pellets (Purina Mills, Inc., St. Louis, MO.) And water. The rats were individually housed in a stainless steel cage and identified by a unique identification number. Body weight was recorded on day 0, day 7 and the last day of the experiment.

Each rat of treatment groups T ₀ , T ₁ and T ₂ was orally administered ritonavir once daily at a constant dose of 2 mL / kg for 14 consecutive days by gavage. Each of the treatment group, T ₃ rats, received phenobarbital intraperitoneally for 14 consecutive days at a constant dose of 2 mL / kg once daily.

Rats in treatment groups T ₀ , T ₁ and T ₂ were sacrificed approximately 48 hours after the last dose. Rats treated groups T ₃ were sacrificed approximately 24 hours after the last dose. The liver was taken out, wiped with water, and each weight was measured. The liver was then homogenized separately using a tissue homogenizer in ice-cold 10 mM potassium phosphate buffer (pH 7.4) containing 1.15% calcium chloride. Microsomes were prepared by various centrifugations and stored at −70 ° C. in 0.1 M potassium phosphate buffer (pH 7.4) containing 20% (v / v) glycerol and 1.0 mM EDTA. The protein content of microsomes was measured using bicinchoninic acid (BCA) measurement kit (Pierce Chemical) with bovine serum albumin as a standard. Total cytochrome P450 content was measured by iron carbon monoxide complex formation. The microsomes were characterized by the following cytochrome P450-mediated reactions, 7-pentoxyresorufin O-dealkylase, chlorozoxazone 6-hydroxylase, benzphetamine N-demethylase and erythromycin N-demethylase.

  Liver 17α-ethynylestradiol and 1-naphthol glucuronidation activity is shown in Table 3 below.

  Ritonavir was found to significantly increase glucuronidation of 17α-ethynylestradiol, a UGT1A1 substrate, in both male and female rats. The increase in glucuronidation of 17α-ethynylestradiol in both male and female rats treated with ritonavir 50 mg / kg / day was doubled.

  A study was conducted to examine the effect of single dose ethinylestradiol on the pharmacokinetics of ritonavir administered orally every 12 hours. This was an open-label, one-center study with healthy female volunteers who had not taken oral contraceptives for at least 6 months prior to the start of the study. Subjects received a first dose of oral contraceptive containing 50 μg ethinyl estradiol on day 1. Each subject was administered ritonavir every 12 hours for 16 days from day 15 to day 30. On day 15, 300 mg was administered every 12 hours, on day 16, 400 mg was administered every 12 hours, and thereafter 500 mg was administered every 12 hours. The second oral contraceptive was administered on day 29 during ritonavir administration. Blood samples were collected 48 hours after each oral contraceptive dose for ethinyl estradiol concentration. Blood samples were collected for ritonavir concentrations at baseline, ie before dosing on day 1 (0 h), at day 29 in steady state, and at 0 h and 4 h after morning dosing.

  Twenty-seven subjects were enrolled in this study. Four subjects failed to complete the study and did not receive oral contraceptives during ritonavir administration. Therefore, the data for these four subjects were excluded from the general statistics. The mean ± SD of the 23 subjects who completed the study was 34 ± 10 years (range 10 to 45 years) and the mean ± SD of body weight was 67.3 ± 10.9 kg (50.8 kg to 90.90). 3 kg) and the mean height ± SD was 167 ± 7 cm (ranging from 155 cm to 180 cm).

  Ethinyl estradiol was purchased as a Demulen 1 / 50-21 tablet (GD Seale & Co.) containing 50 μg ethinyl estradiol and 1 mg etinodiol diacetate at 8:00 am on day 1 and day 29 at 8:00 am It was administered with about 240 mL of water within 15 minutes of the snack. Ritonavir (Abott Laboratories) was purchased as a liquid formulation (80 mg / mL solution), 300 mg (3.75 mL) every 12 hours on day 15 and 400 mg (5.0 mL) every 12 hours on day 16 From Day 17 to Day 30, 500 mg (6.25 mL) was administered every 12 hours at around 8:00 am and around 8:00 pm from Day 15 to Day 30.

  Subjects were 64 hours from day -1 (the day before the first dose), during the first 48 hours of blood collection (monitored on day 3), and from day 28 to 64 hours, second blood collection 48 hours. (Monitored on day 31). In addition, subjects attended the clinical laboratory twice a day for meals, medications and other test activities as outpatients for a total of 14 consecutive days starting on day 15. Violent exercise was not allowed during containment. During containment, subjects refrained from all food and beverages except the planned meals and snacks provided in this study. Water was consumed ad libitum. During containment, all meals were unified based on ingredients. Breakfast, lunch and dinner are served at around 7:30 am, around 1:00 pm and around 7:30 pm, and snacks are served at around 10:00 pm, and 9 pm for day 1 and 28 : It was sent to 45. All took 8 hours of fasting before taking blood for laboratory analysis. The start of a series of breakfasts and dinners during each dosing day was maintained on time so that the dosing interval was the same among all subjects.

  For administration on day 1 and day 29, the following time points, before administration (0 h) and after administration 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 18, 24, 30, 36 And blood samples (7 mL) were collected for serum ethinyl estradiol concentration at 48 hours. In addition, blood samples (7 mL) were collected for plasma ritonavir concentrations before dosing on day 1 (0 h) and at steady state on day 29, 0 hours after morning administration (before morning administration) and 4 hours later. did. All plasma and serum samples were sealed with dry ice and transported to the analysis section.

  Serum samples were analyzed for ethynyl estradiol concentration with Pharmaco LSR, Richmond, VA. Plasma samples are stored frozen and can be obtained from Oneida Research Services (ORS). Inc. Ritonavir concentration was analyzed at Whitesboro, NY.

  Analysis for measuring ethinyl estradiol in serum samples was performed at Pharmaco LSR, Richmond, VA. The ethinyl estradiol serum concentration was measured using gas chromatography / mass spectrometry (GC / MS) method. 17α-ethynylestradiol and its corresponding isotopically labeled internal standard were isolated from human serum by buffering the serum to pH 8.0 and then extracting with an ethyl ether / methylene chloride mixture. The organic layer was replaced with an aqueous base, washed with hexane, then acidified and extracted with methylene chloride. The extract was dried over sodium carbonate and derivatized by evaporation. The derivatized extract was stored at −20 ° C. until analysis. Analysis was performed by capillary GC / MS using negative chemical ionization with selected ion monitoring. In this method, 17α-ethynyl estradio in a concentration range of 2.00 to 256 pg / mL was evaluated at a serum lower limit of 2.00 pg / mL for a serum volume of 1.0 mL.

  Serum samples from this study were measured with a standard curve ranging from 17α-ethynylestradiol 2.00 to 256 pg / mL with an average correlation coefficient of 0.9994. Serum samples were supplemented with low, medium and high concentrations of ethinylestradiol and measured along with unknowns as quality control standards and inter-assay variability metrics. The low concentration quality control was 6.00 pg / mL, the medium concentration was 24.0 pg / mL, and the high concentration was 144 pg / mL. The average calculated concentration of ethinyl estradiol at the end of the analysis was 5.31 pg / mL for the low concentration quality control, 21.4 pg / mL for the medium concentration, and 131 pg / mL for the high concentration. The corresponding coefficients of variation were 11.4, 8.1 and 7.1%.

  Analysis of ritonavir plasma samples was performed at ORS, Whitesboro, NY. Ritonavir plasma concentrations were measured using an evaluated reverse phase high performance liquid chromatography (HPLC) method equipped with a UV detector. For the extraction method, an internal standard is added to a fixed amount of 500 μL of a sample, this fixed amount is extracted with ethyl acetate: hexane, 9: 1 (v / v) under neutral conditions, and the organic layer is dried. Distilling until reconstitution, and reconstituting the sample with a mobile phase, and then washing the reconstituted extract twice with hexane. Next, an aliquot of the reconstituted extract was analyzed at 205 nm by reverse phase high performance liquid chromatography equipped with a UV detector. The lower limit of quantification was 0.010 μg / mL. The mobile phase consisted of 0.1% trifluoroacetic acid (TFA) dissolved in tetramethylammonium perchlorate: acetonitrile: methanol (55: 40: 5; v: v: v).

  The method was evaluated with ORS and shown to produce a linear calibration curve in plasma. The average correlation coefficient of the calibration curve with standards in the range of 0.100 to 15.00 μg / mL was 0.998. The daily variation coefficient (CV) was less than 4.85% at any concentration. The daily plasma CV was less than 5.66% at any concentration.

  The summary evaluation method was performed to show that the lower limit of quantification in non-HIV positive plasma was 0.010 μg / mL. The intraday CV was less than 19.29% except for the calibration standard with the lowest concentration where the CV was 39.70%, and the correlation coefficient of the calibration curve was 0.9960. Previous intra-assay variability obtained after 6 replicate analyzes of plasma containing ritonavir 0.010 μg / mL indicated an assay accuracy of 4.25% at the lower limit of quantification.

  The plasma samples in this study were measured with a calibration curve ranging from 0.010 to 15.00 μg / mL with an average correlation coefficient of 0.9993. As a quality control standard and a measure of inter-assay variability, plasma samples were supplemented with low (0.150 μg / mL), medium (7.500 μg / mL) and high (12.00 μg / mL) ritonavir, unknown Measured with The average calculated concentration value at the end of the analysis is 0.158 μg / mL for the low concentration quality control, 6.551 μg / mL for the medium concentration, 10.64 μg / mL for the high concentration, and the corresponding coefficient of variation is 3. 99%, 12.9% and 2.88%.

  The ethinyl estradiol pharmacokinetic variables were estimated after two single doses on days 1 and 29 using a non-compartmental method. Pharmacokinetic variables were calculated using the intended sampling time. All samples were collected within 10% of the intended sampling time. Maximum plasma concentration (Cmax) and time to reach Cmax (Tmax) were obtained directly from individual concentration versus time history. The area under the plasma concentration-time curve (AUC∞) is calculated as the sum of AUCt, the area up to the terminal measurement concentration is calculated using the linear trapezoidal formula, and the extrapolation to infinity is the terminal measurement possible concentration (Ct ) And terminal excretion rate constant (β). Terminal excretion rate constants were calculated as the negative slope of the logarithm of plasma concentration versus time starting 18 hours after administration. The terminal half-life (t1 / 2) was obtained by dividing the natural logarithm of 2 by β.

  The two ethinylestradiol concentrations were reported as lost and replaced by linear interpolation (Subject # 101, Day 29, Day 6 and Subject # 124, Day 29, Day 4). One ritonavir concentration was reported as lost (subject number 124, day 29, day 4) but not replaced.

  The serum concentrations of ethinyl estradiol and the plasma concentration of ritonavir at each sampling time were tabulated and summarized statistics were calculated. The pharmacokinetic variables (Tmax, Cmax, AUC∞ and β) of ethinylestradiol measured on the first day when the oral contraceptive was administered alone are the variables on the 29th day when the oral contraceptive was administered during the concurrent administration of ritonavir. And a paired t test for differences. A paired t-test was performed using SAS PROC UNIVARIATE version 6.06 on a UNIX operating system. A p value ≦ 0.050 was considered statistically significant.

  For both Cmax and AUC∞, a 95% confidence interval was obtained for the ratio between the average of 29 days when oral contraceptives were administered during concurrent ritonavir and the average of day 1 when oral contraceptives were administered alone. Relationship between ethinyl estradiol AUC ratio (day 29: day 1) and ritonavir concentration on day 29 and ethinyl estradiol AUC ratio (day 29: day 1) and natural logarithm (ln) converted triglyceride ratio (28 The relationship between day 1 day) or natural log-transformed gamma-glutamyl transpeptidase (GGT) ratio (day 28 day 1) was examined by single regression analysis.

  The following table summarizes the results of ethinyl estradiol pharmacokinetic variables measured after oral contraceptives alone and after ritonavir was administered every 12 hours for 2 weeks.

  After administration of a single dose of oral contraceptive, the serum ethinyl estradiol concentration peaked approximately 4 hours after administration and then decreased, generally terminal elimination half-life was 17 hours, FIG. A second dose of ethinylestradiol was performed on day 29 after ritonavir 500 mg was administered every 12 hours for about 2 weeks. Administration of ethinylestradiol with ritonavir resulted in a 32% decrease in mean Cmax for ethinylestradiol (p <0.001) and a 42% decrease in average AUC (p <0.001) compared to oral contraceptives alone. It was. Furthermore, when ritonavir was used in combination, the average terminal elimination rate constant increased by 31% (p <0.001), and the harmonic mean half-life was 13 hours. Changes in individual and average ethinyl estradiol pharmacokinetic variables, Cmax, AUC and β, respectively, after oral contraceptives alone and with ritonavir are represented in FIGS.

  The impact on AUC is consistent across individuals as evidenced by the relatively narrow (0.506-0.694) 95% confidence interval for the ratio of AUC averages. In all but one subject, ritonavir administration reduced AUC. There was no statistically significant change in Tmax (p = 0.387).

  In the ethinyl estradiol pharmacokinetic variables measured after oral contraceptives alone and during ritonavir 500 mg every 12 hours, statistically significant differences were observed except for Tmax. When combined with ritonavir, the mean Cmax of ethinylestradiol was reduced by 32% and the mean AUC was reduced by 41%, but the mean terminal excretion rate constant was increased (+ 31%). The harmonic mean half-life of ethinylestradiol decreased from 17.0 hours after oral contraceptive alone to 13.0 hours after concurrent oral contraceptive and ritonavir.

  The foregoing is merely illustrative of the invention and is not intended to limit the invention to the disclosed methods and compositions. Modifications and variations apparent to those skilled in the art are intended to be included within the scope and characteristics of the invention as defined by the appended claims.

FIG. 3 shows mean (± SD) serum bilirubin over time in 3 groups of Gunn rats, where group I receives atazanavir / ritonavir 500/100 mg / kg / day and group II receives ritonavir 100 mg / kg / day. And Group III received vehicle alone. FIG. 2 shows mean (± SD) serum bilirubin over time in 3 groups of Gunn rats, group I administered indinavir / ritonavir 500/100 mg / kg / day and group II administered ritonavir 100 mg / kg / day. And Group III received vehicle alone. FIG. 2 shows mean (± SD) ethinyl estradiol concentration after single administration of ethinyl estradiol and after administration with ritonavir. FIG. 6 shows mean (± SD) ethinyl estradiol Cmax after single administration of ethinyl estradiol and after administration with ritonavir. FIG. 2 shows mean (± SD) ethinyl estradiol AUC∞ after single administration of ethinyl estradiol and after administration with ritonavir. FIG. 2 shows mean (± SD) ethinyl estradiol excretion rate constants after administration of ethinyl estradiol alone and after administration with ritonavir.

Claims (18)

  A method of inducing UGT1A1 isoform expression to treat a disease or disorder or adverse effect resulting from increased serum concentration of UGT1A1 substrate, comprising administering to a patient an effective amount of ritonavir.   2. The method of claim 1, wherein the disease or disorder is unconjugated hyperbilirubinemia.   The method of claim 1, wherein the UGT1A1 substrate is bilirubin.   The method of claim 1, wherein the effective amount of ritonavir ranges from about 25 mg to about 1200 mg per day.   A method of treating unconjugated hyperbilirubinemia comprising administering an effective amount of ritonavir to a patient in need of treatment for unconjugated hyperbilirubinemia.   6. The method of claim 5, wherein the effective amount of ritonavir ranges from about 25 mg to about 1200 mg per day.   Increased serum concentration of UGT1A1 substrate by administering an active pharmaceutical ingredient, including co-administering an effective amount of ritonavir to a patient in need of treatment for a disease, disorder or adverse effect caused by increased serum concentration of UGT1A1 substrate A method of treating a disease, disorder or adverse effect caused by the disease.   8. The method of claim 7, wherein the effective amount of ritonavir ranges from about 25 mg to about 1200 mg.   The active pharmaceutical ingredients are indinavir, atazanavir, amphotericin B / cholesteryl sulfate complex, testosterone, interferon beta-1b, bicalutamide, ciprofloxacin, oxaliplatin, floxlidine, gemcitabine hydrochloride, salgramostim, gemtuzumab ozogamicin, vinorelbine tartrate, 8. The method of claim 7, wherein the method is selected from the group consisting essentially of carboplatin, pegylated interferon alpha-2B, tacrolimus, aldesleukin, darhopristin / quinupristin, didanosine and capecitabine.   8. The method of claim 7, wherein the active pharmaceutical ingredient is indinavir.   8. The method of claim 7, wherein the active pharmaceutical ingredient is atazanavir.   8. The method according to claim 7, wherein the disease, disorder or adverse effect resulting from increased serum concentration of UGT1A1 substrate is unconjugated hyperbilirubinemia.   A method of increasing glucuronidation of a UGT1A1 substrate comprising administering an effective amount of ritonavir.   14. The method of claim 13, wherein the UGT1A1 substrate is bilirubin.   14. The method of claim 13, wherein the effective amount of ritonavir ranges from about 25 mg to about 1200 mg per day.   A method of increasing excretion of a UGT1A1 substrate comprising administering an effective amount of ritonavir.   17. The method of claim 16, wherein the UGT1A1 substrate is bilirubin.   17. The method of claim 16, wherein the effective amount of ritonavir ranges from about 25 mg to about 1200 mg per day.

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