RESUMEN

Recombinant adeno-associated virus (AAV) vectors are transforming therapies for rare human monogenic deficiency diseases. However, adaptive immune responses to AAV and its limited DNA insert capacity, restrict their therapeutic potential. HEDGES (high-level extended duration gene expression system), a nonviral DNA- and liposome-based gene delivery platform, overcomes these limitations in immunocompetent mice. Specifically, one systemic HEDGES injection durably produces therapeutic levels of transgene-encoded human proteins, including FDA-approved cytokines and monoclonal antibodies, without detectable integration into genomic DNA. HEDGES also controls protein production duration from <3 weeks to >1.5 years, does not induce anti-vector immune responses, is reexpressed for prolonged periods following reinjection, and produces only transient minimal toxicity. HEDGES can produce extended therapeutic levels of multiple transgene-encoded therapeutic human proteins from DNA inserts >1.5-fold larger than AAV-based therapeutics, thus creating combinatorial interventions to effectively treat common polygenic diseases driven by multigenic abnormalities.

Asunto(s)

ADN/genética , Técnicas de Transferencia de Gen , Transgenes , Animales , Línea Celular , ADN/farmacología , Femenino , Humanos , Ratones , Ratones Endogámicos ICR

RESUMEN

Neutralizing anti-drug antibodies (NAbs) can adversely impact efficacy and safety of biologic therapeutics. However, current assay formats to detect NAbs are limited in their use during the dosing phase due to interference by circulating drug, resulting in low drug tolerance. To improve the drug tolerance for NAb detection, an alternative approach for indirect NAb (iNAb) assessment was developed and qualified that uses a combination of pharmacokinetic (PK) assays to measure the serum concentrations of free and total drug. It is demonstrated that the ratio of free to total drug concentrations, referred to as F/T ratio, is a novel PK parameter that can indicate neutralizing activity in test samples. The iNAb assessment correctly identified NAb-positive samples with high drug concentrations that led to false negative results in a conventional NAb assay. Moreover, iNAb reliably distinguished between NAbs and non-neutralizing anti-drug antibodies over a wide range of concentrations. A proposal on how to deploy iNAb assessment within a broader immunogenicity testing strategy is presented.

Asunto(s)

Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Antagonismo de Drogas , Preparaciones Farmacéuticas/sangre , Farmacocinética , Humanos

RESUMEN

The analgesic efficacy of liposomal hydromorphone (LE-hydro) was tested in dogs undergoing limb amputation. The positive controls (n = 10) received subcutaneous (SQ) hydromorphone (0.2 mg/kg) and 1.5 mL of blank liposomes before surgery; fentanyl continuous rate infusion (CRI), 5-10 µg/kg/hr IV, during and for 24 hr after surgery; and a fentanyl patch at extubation. The negative controls (n = 7) received SQ hydromorphone (0.2 mg/kg) and 1.5 mLs of blank liposomes SQ before surgery, fentanyl CRI (5-10 µg/kg/hr IV) during surgery but stopped at extubation, and a fentanyl patch at extubation. The test group (n = 11) received 3 mg/kg of LE-hydro and 1.5 mL of saline SQ before surgery, 1.5 mL of saline SQ, and a saline CRI during surgery. All groups received a bupivacaine block in the limb prior to amputation and carprofen prior to surgery. Treatment failures, pain scores, opioid side effects, heart rate, respiratory rate, temperature, and client-reported pain and side effects were evaluated. There were three treatment failures in the positive control (3/10) and test groups (3/11). Negative controls had seven treatment failures (7/7). Side effects for all three groups were within expected limits. LE-hydro provides postoperative analgesia equivalent to fentanyl CRI in dogs undergoing limb amputation.

Asunto(s)

Amputación Quirúrgica/veterinaria , Analgésicos Opioides/administración & dosificación , Enfermedades de los Perros/cirugía , Hidromorfona/administración & dosificación , Dolor Postoperatorio/veterinaria , Analgésicos Opioides/efectos adversos , Analgésicos Opioides/uso terapéutico , Animales , Neoplasias Óseas/cirugía , Neoplasias Óseas/veterinaria , Perros , Femenino , Hidromorfona/efectos adversos , Hidromorfona/uso terapéutico , Liposomas , Masculino , Dolor Postoperatorio/tratamiento farmacológico , Sarcoma/cirugía , Sarcoma/veterinaria

RESUMEN

Doxycycline (doxy) is used in treating intracellular and extracellular infections. Liposomal (LE) antibiotics allow low-frequency dosing and extended efficacy compared with standard (STD) formulations. We developed a novel sulfuric acid-loading method for doxycycline liposomes (LE-doxy). We hypothesized that a single s.c. injection of LE-doxy would be detectable in serum for at least 2 weeks at concentrations equal to or better than STD-doxy and would be bactericidal in an in vitro Mycobacterium smegmatis infection of J774A.1 macrophage cells. Liposomes were encapsulated by sulfuric acid gradient loading, and release kinetics were performed in vitro and in vivo. LE-doxy made using 8.25 mg/ml doxycycline loaded for 24 hours achieved 97.77% capture in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 43.87% in sphingomyelin (sphing). Rats were injected s.c. with 50 mg/kg LE-doxy or 5 mg/kg STD-doxy, and serial blood samples were collected. Pharmacokinetics were analyzed using high-performance liquid chromatography. Liver and injection site skin samples were collected at euthanasia (4 weeks postinjection). Minimal histologic tissue reactions occurred after injection of STD (nonliposomal), DPPC, or sphing-doxy. DPPC-doxy had slightly faster in vitro leakage than sphing liposomes, although both were detectable at 264 hours. The mean residence time for DPPC was the highest (111.78 hours), followed by sphing (56.00 hours) and STD (6.86 hours). DPPC and sphing-doxy were detectable at 0.2 µg/ml in serum at 336 hours postadministration. LE-doxy was not toxic to J774A.1 cells in vitro and produced inhibition of viable Mycobacterium smegmatis at 24 and 48 hours. LE-doxy will require further testing in in vivo infection models.

Asunto(s)

Antibacterianos/administración & dosificación , Doxiciclina/administración & dosificación , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , 1,2-Dipalmitoilfosfatidilcolina/química , Animales , Antibacterianos/química , Antibacterianos/uso terapéutico , Línea Celular , Química Farmacéutica , Doxiciclina/química , Doxiciclina/uso terapéutico , Composición de Medicamentos , Sistemas de Liberación de Medicamentos , Femenino , Inyecciones Subcutáneas , Liposomas , Masculino , Pruebas de Sensibilidad Microbiana , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Mycobacterium smegmatis/efectos de los fármacos , Tamaño de la Partícula , Ratas , Esfingomielinas/química , Ácidos Sulfúricos/química

RESUMEN

OBJECTIVE: To evaluate the pharmacokinetics, in dogs, of liposome-encapsulated oxymorphone and hydromorphone made by the ammonium sulfate gradient loading technique (ASG). ANIMALS: Four healthy purpose-bred Beagles aged 9.5 ± 3.2 months and weighing 13.4 ± 2.3 kg. STUDY DESIGN: Randomized cross-over design. METHODS: Each dog was given either 4.0 mg kg(-1) of ASG-oxymorphone or 8.0 mg kg(-1) of ASG-hydromorphone SC on separate occasions with a 3-month washout period. Blood was collected at baseline and at serial time points up to 1032 hours (43 days) after injection for determination of serum opioid concentrations. Serum opioid concentrations were measured with HPLC-MS and pharmacokinetic parameters were calculated using commercial software and non-compartmental methods. RESULTS: Serum concentrations of oxymorphone remained above the limit of quantification for 21 days, while those for hydromorphone remained above the limit of quantification for 29 days. Cmax for ASG-oxymorphone was 7.5 ng mL(-1) ; Cmax for ASG-hydromorphone was 5.7 ng mL(-1) . CONCLUSIONS AND CLINICAL RELEVANCE: Oxymorphone and hydromorphone, when encapsulated into liposomes using the ammonium sulfate gradient loading technique, result in measureable serum concentrations for between 3 to 4 weeks. This formulation may have promise in the convenient use of opioids for clinical treatment of chronically painful conditions in dogs.

Asunto(s)

Sulfato de Amonio/química , Perros/sangre , Hidromorfona/farmacocinética , Liposomas , Oximorfona/farmacocinética , Analgésicos Opioides/administración & dosificación , Analgésicos Opioides/sangre , Analgésicos Opioides/química , Analgésicos Opioides/farmacocinética , Animales , Área Bajo la Curva , Semivida , Hidromorfona/administración & dosificación , Hidromorfona/sangre , Hidromorfona/química , Masculino , Oximorfona/administración & dosificación , Oximorfona/sangre , Oximorfona/química

RESUMEN

OBJECTIVE: To evaluate the pharmacokinetics of nalbuphine decanoate after IM administration to Hispaniolan Amazon parrots (Amazona ventralis). ANIMALS: 9 healthy adult Hispaniolan Amazon parrots of unknown sex. PROCEDURES: Nalbuphine decanoate (37.5 mg/kg) was administered IM to all birds. Plasma samples were obtained from blood collected before (time 0) and 0.25, 1, 2, 3, 6, 12, 24, 48, and 96 hours after drug administration. Plasma samples were used for measurement of nalbuphine concentrations via liquid chromatography-tandem mass spectrometry. Pharmacokinetic parameters were estimated with computer software. RESULTS: Plasma concentrations of nalbuphine increased rapidly after IM administration, with a mean concentration of 46.1 ng/mL at 0.25 hours after administration. Plasma concentrations of nalbuphine remained > 20 ng/mL for at least 24 hours in all birds. The maximum plasma concentration was 109.4 ng/mL at 2.15 hours. The mean terminal half-life was 20.4 hours. CONCLUSIONS AND CLINICAL RELEVANCE: In Hispaniolan Amazon parrots, plasma concentrations of nalbuphine were prolonged after IM administration of nalbuphine decanoate, compared with previously reported results after administration of nalbuphine hydrochloride. Plasma concentrations that could be associated with antinociception were maintained for 24 hours after IM administration of 37.5 mg of nalbuphine decanoate/kg. Safety and analgesic efficacy of nalbuphine treatments in this species require further investigation to determine the potential for clinical use in pain management in psittacine species.

Asunto(s)

Amazona/fisiología , Analgésicos Opioides/farmacocinética , Nalbufina/farmacocinética , Analgésicos/administración & dosificación , Analgésicos/sangre , Analgésicos/farmacocinética , Analgésicos Opioides/administración & dosificación , Analgésicos Opioides/sangre , Animales , Área Bajo la Curva , Cromatografía Liquida/veterinaria , Semivida , Inyecciones Intramusculares/veterinaria , Nalbufina/administración & dosificación , Nalbufina/sangre , Espectrometría de Masa por Ionización de Electrospray/veterinaria , Espectrometría de Masas en Tándem/veterinaria

RESUMEN

OBJECTIVE: To evaluate the thermal antinociceptive effects and duration of action of nalbuphine decanoate after IM administration to Hispaniolan Amazon parrots (Amazona ventralis). ANIMALS: 10 healthy adult Hispaniolan Amazon parrots of unknown sex. PROCEDURES: Nalbuphine decanoate (33.7 mg/kg) or saline (0.9% NaCl) solution was administered IM in a randomized complete crossover experimental design (periods 1 and 2). Foot withdrawal threshold to a noxious thermal stimulus was used to evaluate responses. Baseline thermal withdrawal threshold was recorded 1 hour before drug or saline solution administration, and thermal foot withdrawal threshold measurements were repeated 1, 2, 3, 6, 12, 24, 48, and 72 hours after drug administration. RESULTS: Nalbuphine decanoate administered IM at a dose of 33.7 mg/kg significantly increased thermal foot withdrawal threshold, compared with results after administration of saline solution during period 2, and also caused a significant change in withdrawal threshold for up to 12 hours, compared with baseline values. CONCLUSIONS AND CLINICAL RELEVANCE: Nalbuphine decanoate increased the foot withdrawal threshold to a noxious thermal stimulus in Hispaniolan Amazon parrots for up to 12 hours and provided a longer duration of action than has been reported for other nalbuphine formulations. Further studies with other types of nociceptive stimulation, dosages, and dosing intervals as well as clinical trials are needed to fully evaluate the analgesic effects of nalbuphine decanoate in psittacine birds.

Asunto(s)

Amazona/fisiología , Hipnóticos y Sedantes/farmacocinética , Nalbufina/farmacocinética , Analgésicos/administración & dosificación , Analgésicos/sangre , Analgésicos/farmacocinética , Animales , Área Bajo la Curva , Cromatografía Liquida/veterinaria , Estudios Cruzados , Semivida , Hipnóticos y Sedantes/administración & dosificación , Hipnóticos y Sedantes/sangre , Inyecciones Intramusculares/veterinaria , Nalbufina/administración & dosificación , Nalbufina/sangre , Espectrometría de Masa por Ionización de Electrospray/veterinaria , Espectrometría de Masas en Tándem/veterinaria

RESUMEN

Liposome encapsulation of opioids by using an ammonium-sulfate-gradient loading technique significantly slows the release time of the drug. This study evaluated the duration of analgesia in a rodent model of monoarthritis after epidural administration of liposome-encapsulated hydromorphone (LE-hydromorphone; prepared by ammonium-sulfate-gradient loading) compared with standard hydromorphone and a negative control of blank liposomes. Analgesia was assessed by changes in thermal withdrawal latency, relative weight-bearing, and subjective behavioral scoring. Analgesia in arthritic rats was short-lived after epidural hydromorphone; increases in pain threshold were observed only at 2 h after administration. In contrast, thermal pain thresholds after epidural LE-hydromorphone were increased for as long as 72 h, and subjective lameness scores were lower for as long as 96 h after epidural administration. Injection of LE-hydromorphone epidurally was associated with various mild changes in CNS behavior, and 2 rats succumbed to respiratory depression and death. In conclusion, LE-hydromorphone prolonged the duration of epidural analgesia compared with the standard formulation of hydromorphone, but CNS side effects warrant careful administration of this LE-hydromorphone in future studies.

Asunto(s)

Analgesia Epidural/métodos , Artritis/complicaciones , Sistemas de Liberación de Medicamentos/métodos , Hidromorfona/uso terapéutico , Liposomas/uso terapéutico , Dolor/tratamiento farmacológico , Rodilla de Cuadrúpedos/patología , Análisis de Varianza , Animales , Artritis/patología , Preparaciones de Acción Retardada , Hidromorfona/administración & dosificación , Hidromorfona/efectos adversos , Actividad Motora/efectos de los fármacos , Dolor/etiología , Dimensión del Dolor , Ratas , Médula Espinal/patología

RESUMEN

The purpose of this study was to determine the experimental side effects of liposome-encapsulated hydromorphone (LE-Hydro) in beagles and to evaluate LE-Hydro analgesia in dogs undergoing ovariohysterectomies (OVH). Beagles were injected subcutaneously with 1-3 mg/kg LE-Hydro or 0.1 mg/kg hydromorphone. Dogs were evaluated for sedation, temperature, respiratory rate, and heart rate. OVH dogs were injected with 2 mg/kg LE-Hydro subcutaneously or 0.2 mg/kg morphine and 0.05 mg/kg acepromazine intramuscularly. Side effects of LE-Hydro were within clinically acceptable limits. The analgesic efficacy was superior in dogs administered LE-Hydro at 12 hr postsurgically. LE-Hydro provided adequate, durable analgesia in dogs undergoing OVH.

Asunto(s)

Analgésicos Opioides/farmacología , Perros/fisiología , Hidromorfona/farmacología , Analgésicos Opioides/administración & dosificación , Analgésicos Opioides/farmacocinética , Animales , Área Bajo la Curva , Química Farmacéutica , Relación Dosis-Respuesta a Droga , Femenino , Semivida , Hidromorfona/administración & dosificación , Hidromorfona/farmacocinética , Histerectomía/veterinaria , Inyecciones Subcutáneas/veterinaria , Tasa de Depuración Metabólica , Ovariectomía/veterinaria , Dimensión del Dolor/veterinaria , Cuidados Posoperatorios/veterinaria , Resultado del Tratamiento

RESUMEN

INTRODUCTION: This study aims to evaluate the pharmacokinetic, behavioral, and motor effects of a liposomal preparation of hydromorphone hydrochloride (LE-hydro) in rhesus monkeys. We administered either 2 mg/kg of LE-hydro (n = 8) subcutaneous (s.c.) or 0.1 mg/kg of standard pharmaceutical hydromorphone HCl (hydro) preparation either intravenous (i.v.; n = 4) or s.c. (n = 5). MATERIALS AND METHODS: Serial blood samples were drawn after injection and analyzed for serum hydro concentration by liquid chromatography/mass spectrometry. Following s.c. injection of 0.1 mg/kg hydro or 2 mg/kg LE-hydro, behavioral evaluations were conducted in groups of rhesus monkeys (n = 10/group) in the presence of a compatible stimulus animal and motor skills were also evaluated (n = 10/group). The motor skills test consisted of removing a food reward (carrot ring) from either a straight peg (simple task) or a curved peg (difficult task). RESULTS: LE-hydro (MRT(0-INF) = 105.9 h) demonstrated extended-release pharmacokinetics compared to hydro when administered by either i.v. (MRT(0-INF) =1.1 h) or s.c. (MRT(0-INF) =1.3 h) routes. Hydro did not affect motor performance of the simpler task, but the monkeys' performance deteriorated on the more difficult task at 0.5 and 1 h after injection. LE-hydro had no effect on motor skills in either the simpler or more difficult task. CONCLUSIONS: The results of these studies indicate that LE-hydro has a pharmacokinetic and behavioral side effects profile consistent with an analgesic that could be tested for surgical use in animals. Our studies also expand the use of rhesus monkeys as a translational behavioral pharmacodynamics model for testing extended-release opioid medication.

Asunto(s)

Analgésicos Opioides/farmacología , Conducta Animal/efectos de los fármacos , Hidromorfona/farmacología , Destreza Motora/efectos de los fármacos , Analgésicos Opioides/administración & dosificación , Analgésicos Opioides/farmacocinética , Animales , Cromatografía Liquida , Preparaciones de Acción Retardada , Relación Dosis-Respuesta a Droga , Femenino , Hidromorfona/administración & dosificación , Hidromorfona/farmacocinética , Inyecciones Subcutáneas , Liposomas , Macaca mulatta , Masculino , Espectrometría de Masas , Recompensa

RESUMEN

We have studied the loading of the opioid hydromorphone into liposomes using ammonium sulfate gradients. Unlike other drugs loaded with this technique, hydromorphone is freely soluble as the sulfate salt, and, consequently, does not precipitate in the liposomes after loading. We have derived a mathematical relationship that can predict the extent of loading based on the ammonium ion content of the liposomes and the amount of drug added for loading. We have adapted and used the Berthelot indophenol assay to measure the amount of ammonium ions in the liposomes. Plots of the inverse of the fraction of hydromorphone loaded versus the amount of hydromorphone added are linear, and the slope should be the inverse of the amount of ammonium ions present in the liposomes. The inverse of the slopes obtained closely correspond to the amount of ammonium ions in the liposomes measured with the Berthelot indophenol assay. We also show that loading can be less than optimal under conditions where osmotically driven loss of ammonium ions or leakage of drug after loading may occur.

Asunto(s)

Sulfato de Amonio/química , Liposomas/química , Estructuras Celulares , Hidromorfona , Membranas , Preparaciones Farmacéuticas , Compuestos de Amonio Cuaternario/química

RESUMEN

We have used a murine model of Acetaminophen induced hepatoxicity to determine if S-adenosyl methionine 1,4 butanedisulfonate (SD4) in liposomes can prevent liver injury when administered immediately prior to acetaminophen, as judged by serum aspartate aminotransferase and alanine aminotransferase levels, and histological evidence of liver necrosis. No protection was observed when mice received 1 g/kg unencapsulated SD4. Partial protection was observed with 5 or 0.5 mg/kg SD4 in unextruded distearoylphosphatidylglycerol (DSPG) liposomes. Protection comparable to that seen in mice receiving encapsulated SD4 is achieved when mice received lipid alone in equivalent amounts, suggesting that the contribution of encapsulated SD4 to the efficacy of the liposomes may be minimal. Unextruded distearoylphosphatidylcholine (DSPC) liposomes show only slight effects even at 50 mg/kg SD4. This is likely caused by the size of unextruded DSPC lipsomes, because extruded DSPC liposomes, whose size is smaller, are of comparable efficacy to unextruded DSPG liposomes.

Asunto(s)

Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Liposomas/química , S-Adenosilmetionina/análogos & derivados , Animales , Hígado/efectos de los fármacos , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Fosfatidilgliceroles/química , S-Adenosilmetionina/administración & dosificación , S-Adenosilmetionina/uso terapéutico

RESUMEN

OBJECTIVE: To evaluate the microcrystalline sodium urate (MSU) method for inducing arthritis in parrots and to compare the analgesic efficacy of long-acting liposome-encapsulated butorphanol (LEBT), carprofen, or a combination of both. ANIMALS: 20 Hispaniolan parrots. PROCEDURES: MSU was injected into a tibiotarsal-tarsometatarsal (intertarsal) joint to induce arthritis (time 0). Four treatments were compared (LEBT [15 mg/kg, SC] administered once at time 0; injections of carprofen [3 mg/kg, IM, q 12 h] starting at time 0; administration of LEBT plus carprofen; and a control treatment of saline [0.9% NaCl] solution). Weight load testing and behavioral scoring were conducted at 0, 2, 6, 26, and 30 hours. RESULTS: Injection of MSU into the intertarsal joint induced arthritis, which resolved within 30 hours. Treatment with LEBT or LEBT plus carprofen resulted in significantly greater weight-bearing load on the limb with induced arthritis, compared with the control treatment. Treatment with carprofen alone caused a slight but nonsignificant improvement in weight-bearing load on the arthritic limb, compared with the control treatment. Behaviors associated with motor activity and weight bearing differed between the control and analgesic treatments. CONCLUSIONS AND CLINICAL RELEVANCE: Butorphanol was an effective treatment for pain associated with arthritis, but carprofen administered every 12 hours was insufficient. Injection of MSU to induce arthritis in a single joint was a good method for evaluating tonic pain in parrots, and measurement of the weight-bearing load was accurate for assessment of arthritic pain; however, behavioral changes associated with pain were subtle.

Asunto(s)

Analgésicos Opioides/uso terapéutico , Antiinflamatorios no Esteroideos/uso terapéutico , Artritis/veterinaria , Enfermedades de las Aves/tratamiento farmacológico , Butorfanol/uso terapéutico , Carbazoles/uso terapéutico , Amazona , Analgésicos Opioides/administración & dosificación , Animales , Artritis/inducido químicamente , Butorfanol/administración & dosificación , Estudios Cruzados , Formas de Dosificación , Liposomas , Ácido Úrico/toxicidad

RESUMEN

OBJECTIVE: To evaluate injection of microcrystalline sodium urate (MSU) for inducing articular pain in green-cheeked conures (Pyrrhura molinae) and the analgesic efficacy of liposome-encapsulated butorphanol tartrate (LEBT) by use of weight load data, behavioral scores, and fecal corticosterone concentration. ANIMALS: 8 conures. PROCEDURES: In a crossover study, conures were randomly assigned to receive LEBT (15 mg/kg) or liposomal vehicle subsequent to experimental induction of arthritis or sham injection. The MSU was injected into 1 tibiotarsal-tarsometatarsal (intertarsal) joint to induce arthritis (time 0). weight-bearing load and behavioral scores were determined at 0, 2, 6, 26, and 30 hours. RESULTS: MSU injection into 1 intertarsal joint caused a temporary decrease in weight bearing on the affected limb. Treatment of arthritic conures with LEBT resulted in significantly more weight bearing on the arthritic limb than treatment with vehicle. Administration of vehicle to arthritic conures caused a decrease in activity and feeding behaviors during the induction phase of arthritis, but as the arthritis resolved, there was a significant increase in voluntary activity at 30 hours and feeding behaviors at 26 and 30 hours, compared with results for LEBT treatment of arthritic birds. Treatment with LEBT or vehicle in conures without arthritis resulted in similar measurements for weight bearing and voluntary and motivated behaviors. CONCLUSIONS AND CLINICAL RELEVANCE: Experimental induction of arthritis in conures was a good method for evaluating tonic pain. Weight-bearing load was the most sensitive measure of pain associated with induced arthritis. Pain associated with MSU-induced arthritis was alleviated by administration of LEBT.

Asunto(s)

Artritis/veterinaria , Enfermedades de las Aves/tratamiento farmacológico , Butorfanol/uso terapéutico , Carbazoles/uso terapéutico , Analgésicos Opioides/administración & dosificación , Analgésicos Opioides/uso terapéutico , Animales , Antiinflamatorios no Esteroideos/uso terapéutico , Artritis/inducido químicamente , Butorfanol/administración & dosificación , Estudios Cruzados , Formas de Dosificación , Liposomas , Masculino , Loros , Ácido Úrico/toxicidad

RESUMEN

A sensitive and selective liquid chromatography tandem mass spectrometry (LC\MS\MS) method has been developed for the simultaneous quantification in human plasma of the endocannabinoid anandamide (AEA) and three other related ethanolamides, linoleoyl ethanolamide (LEA), oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA). The analytical methodology requires 50 microL of human plasma which is processed via protein precipitation using a 96-well protein precipitation plate. Chromatographic separation of plasma extract was achieved with a Phenomenex Gemini C6-Phenyl HPLC column (2.1 mm x 50 mm, 5 microm) at a flow rate of 0.30 mL/min using gradient elution and a mobile phase consisting of acetonitrile and 5 mM ammonium formate. All four fatty acid ethanolamides were quantified by positive ion electrospray ionization tandem mass spectrometry, with the detection of ion current signal generated from the selected reaction monitoring (SRM) transition of [M+H](+)-->m/z 62. Deuterated anandamide (AEA-d8) was used as an internal standard for all four ethanolamides. The lower limit of quantitation was 0.05 ng/mL for AEA and LEA, 0.5 ng/mL for OEA and 1.0 ng/mL for PEA. Inter-assay precision and accuracy were typically within 12% for the four endogenous analytes and overall extraction recoveries ranged between 40% and 100%.

Asunto(s)

Ácidos Araquidónicos/sangre , Moduladores de Receptores de Cannabinoides/sangre , Cromatografía Líquida de Alta Presión/métodos , Ácidos Linoleicos/sangre , Alcamidas Poliinsaturadas/sangre , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Endocannabinoides , Humanos

RESUMEN

In humans and rats, a synergistic blood pressure reduction was observed when the fibrate gemcabene (CI-1027) was coadministered with the angiotensin-converting enzyme inhibitor quinapril. In a quinapril (3 mg/kg) pharmacokinetic rat study, there was a 40% decrease in urinary excretion and a 53% increase in plasma area under the curve from 0 to 24 h of the active metabolite quinaprilat when coadministered with gemcabene (30 mg/kg). This observation revealed a possible transporter-mediated drug-drug interaction (DDI) between gemcabene and quinapril. This led to a series of studies investigating the underlying clearance mechanisms associated with these compounds intended to elucidate renal transporter interactions between quinapril and gemcabene. In vitro transporter studies using human embryonic kidney 293 cells transfected with human or rat organic anion transporter 3 (hOAT3, rOat3) revealed that quinaprilat is a substrate in both species, with a K(m) value of 13.4 microM for hOAT3. Subsequent studies discovered that gemcabene inhibited quinaprilat uptake by hOAT3 and rOat3 at IC(50) values of 35 and 48 microM, respectively. Moreover, gemcabene acylglucuronide, the major metabolite of gemcabene glucuronidation, also inhibited hOAT3- and rOat3-mediated uptake of quinaprilat at IC(50) values of 197 and 133 microM, respectively. High plasma concentrations of gemcabene (>100 microM) achieved in humans and rats upon oral dosing corroborate with gemcabene inhibition of renal OAT3-mediated secretion of quinaprilat in vitro. This investigation established that a DDI between gemcabene and quinapril involving inhibition of renal transporters and subsequent elevation in plasma concentrations of quinaprilat is responsible for the apparent synergistic blood pressure reduction observed with these compounds.

Asunto(s)

Caproatos/metabolismo , Riñón/metabolismo , Transportadores de Anión Orgánico/fisiología , Tetrahidroisoquinolinas/metabolismo , Animales , Caproatos/sangre , Caproatos/farmacocinética , Línea Celular , Interacciones Farmacológicas/fisiología , Quimioterapia Combinada , Humanos , Masculino , Transportadores de Anión Orgánico/antagonistas & inhibidores , Transportadores de Anión Orgánico/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/antagonistas & inhibidores , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/fisiología , Quinapril , Ratas , Ratas Endogámicas SHR , Tetrahidroisoquinolinas/sangre , Tetrahidroisoquinolinas/farmacocinética

RESUMEN

The objectives of the study were to determine the pharmacokinetics of oxymorphone (oxy) and of ammonium sulfate-loaded, liposome-encapsulated oxymorphone (LE-ASG oxy) and to evaluate the behavioral effects of both opioid preparations by using ethographic evaluation specific to rhesus monkeys. Rhesus monkeys (n = 8) were injected with 2.0 mg/kg LE-ASG oxy s.c.. Blood samples were collected at serial time points up to 144 h in six monkeys and up to 456 h in two monkeys. Separate groups of monkeys were injected with 0.1 mg/kg oxy s.c. (n = 4) or i.v. (n = 5). Blood samples were collected at serial time points up to 24 h after injection. Pharmacokinetic parameters were calculated by using commercially available software. Behavior was recorded in a different group of 10 monkeys administered LE-ASG oxy (2.0 mg/kg s.c.) or oxy (0.1 mg/kg s.c.) on separate occasions. Behavioral evaluations were made at serial time points while monkeys were in an extended cage with a compatible stimulus animal. Oxymorphone was rapidly eliminated from the serum in the oxy group. Measurable drug was present in serum for up to 4 h after oxy was administered subcutaneously or intravenously. LE-ASG oxy was present in serum in measurable concentrations for more than 2 weeks. Neither oxy nor LE-ASG oxy produced observable sedation. LE-ASG oxy decreased some environmentally directed behaviors, but this drug formulation increased watchfulness, decreased self-directed and elimination behaviors, increased nonspecific social contact, and decreased threat behaviors. LE-ASG oxy persisted for an extended period in rhesus monkey serum and produced behavioral changes consistent with this opioid.

Asunto(s)

Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Oximorfona/administración & dosificación , Oximorfona/farmacocinética , Animales , Cápsulas , Preparaciones de Acción Retardada/administración & dosificación , Preparaciones de Acción Retardada/farmacocinética , Femenino , Liposomas/farmacocinética , Macaca mulatta , Masculino

RESUMEN

The primary objective of this study was to demonstrate the use of stable isotope (SI)-labeled compound as an approach for pharmacokinetic analysis such as fraction absorbed, hepatic extraction ratio, and fraction metabolized from the parent drug to a metabolite. (S,S)-3-[3-(Methylsulfonyl)phenyl]-1-propylpiperidine hydrochloride (PNU96391) was selected as the model compound because of its simple biotransformation pathway, i.e., the predominant metabolic pathway to the N-despropyl metabolite (M1), which makes it a suitable candidate. The second objective was to fully characterize the pharmacokinetics of PNU96391 in rats using the SI coadministration approach with quantitative analysis by liquid chromatography-tandem mass spectrometry. Overall the present study showed that 1) absorption of PNU96391 from the gastrointestinal tract was near complete (>90% of the dose), 2) PNU96391 was predominantly metabolized to M1 (approximately 70% of the dose), and 3) M1 was exclusively eliminated into urine with negligible biotransformation (ratio of renal clearance to plasma clearance approximately 0.9). Therefore, the present study demonstrated the utility of the SI methodology for characterizing the pharmacokinetics of a compound within the drug discovery and development process. Furthermore, the compartmental pharmacokinetic modeling provided insights into the disposition and biotransformation rates of PNU96391 and M1, suggesting that the modeling could add further advantages to the SI coadministration approach. Despite the greater availability of SI-labeled compounds, absorption, distribution, metabolism, and excretion (ADME) scientists have yet to take full advantage of the potential use of these analogs for mechanistic ADME studies. These SI-labeled compounds can be used more widely to gain a better understanding of ADME properties in drug discovery and development.

Asunto(s)

Antagonistas de los Receptores de Dopamina D2 , Piperidinas/farmacocinética , Sulfonas/farmacocinética , Administración Oral , Animales , Área Bajo la Curva , Biotransformación , Cromatografía Líquida de Alta Presión , Inyecciones Intravenosas , Absorción Intestinal , Masculino , Modelos Estadísticos , Ratas , Ratas Sprague-Dawley , Espectrometría de Masas en Tándem , Distribución Tisular

RESUMEN

The present study demonstrates that the nutritional supplement S-adenosyl methionine (SAMe), the primary methyl donor in mammalian cells, is delivered selectively to cells by anionic liposomes, and is, therefore, a liposome dependent drug. Contrary to our expectations, free SAMe chloride was growth inhibitory in cultured cells. The growth inhibitory potency of SAMe chloride in anionic liposomes composed of distearoylphosphatidylglycerol/cholesterol 2:1 was fivefold greater than that of free SAMe. Neutral liposomes composed of distearoylphosphatidylcholine and cholesterol did not increase the potency of the drug. An improved anionic liposome SAMe formulation was produced by use of the 1,4-butanedisulfonate salt (SD4), adding a metal chelator (EDTA), and lowering the buffer pH from pH 7.0 to pH 4.0. This formulation was 15-fold more potent than free SD4, and was active after more than 28 days at 4 degrees C. SAMe and its potential degradation products were screened for toxicity. Formaldehyde was determined to have potency similar to that of free SAMe chloride in CV1-P cells, suggesting that the growth inhibitory effects of SAMe may partly arise from the formation of formaldehyde. The cytotoxic effects of formaldehyde and the less stable forms of SAMe, (SAMe chloride and SAMe tosylate) were decreased in the presence of 3 mM GSH (IC(50) approximately 0.44 mM). The cytotoxic effects of SD4 were not reduced by GSH, suggesting that this more stable form of SAMe is not toxic through the production of formaldehyde. SD4 in anionic DSPG liposomes stimulated murine IL-6 production in RAW 264 cells at concentrations 25- to 30-fold lower than free drug. This increase in potency for IL-6 production was in keeping with the increase in potency observed in our growth inhibition experiments. These results suggest that SD4 in liposomes may be a potential treatment for acute or chronic liver failure.

Asunto(s)

Proliferación Celular/efectos de los fármacos , Colesterol/química , Suplementos Dietéticos , Liposomas , Macrófagos/efectos de los fármacos , Fosfatidilgliceroles/química , Sustancias Protectoras/farmacología , S-Adenosilmetionina/farmacología , Alcanosulfonatos/química , Animales , Células CHO , Quelantes/química , Química Farmacéutica , Chlorocebus aethiops , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Composición de Medicamentos , Estabilidad de Medicamentos , Ácido Edético/química , Concentración de Iones de Hidrógeno , Interleucina-6/metabolismo , Fallo Hepático/tratamiento farmacológico , Macrófagos/inmunología , Ratones , Sustancias Protectoras/administración & dosificación , Sustancias Protectoras/química , S-Adenosilmetionina/administración & dosificación , S-Adenosilmetionina/química

RESUMEN

The multidrug resistance-associated protein 2 (MRP2/ABCC2) plays an important role in hepatobiliary efflux of many drugs and drug metabolites and has been reported to account for dramatic interspecies differences in the aspects of pharmacokinetics. In the present study, an absolute quantification method was developed to quantitatively measure MRP2/ABCC2 using LC-MS/MS for detection of a selective tryptic peptide. A unique 16-mer tryptic peptide was identified by conducting capillary LC nanospray ESI-Q-TOF analysis of the immunoprecipitation-enriched samples of MRP2/ABCC2 following proteolysis with trypsin. The lower limit of quantification was established to be 31.25pM with the linearity of the standard curve spanned to 2500pM. Both the accuracy (relative error) and the precision (coefficient of variation) of the method were below 15%. Using this method, we successfully determined the absolute amount of MRP2/ABCC2 protein in MRP2/ABCC2 gene-transfected MDCK cells as well as the basal levels of canine Mrp2/Abcc2 protein in MDCK cells. Our findings also demonstrate that the sensitivity of this method exceeds the sensitivity of immunoblotting assay which was not able to detect the basal levels of canine Mrp2/Abcc2 in MDCK cells. The method could be directly applicable to many current research needs related to MRP2/ABCC2 protein.

Asunto(s)

Cromatografía Liquida/métodos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/análisis , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Animales , Calibración , Línea Celular , Perros , Humanos , Inmunoprecipitación , Datos de Secuencia Molecular , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/biosíntesis , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Nanotecnología/métodos , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Reproducibilidad de los Resultados , Alineación de Secuencia , Espectrometría de Masa por Ionización de Electrospray/métodos