The importance of measuring cotinine levels to identify smokers in clinical trials.

First-time-in-humans studies of drugs (phase I) typically exclude unsuitable volunteers by testing for recreational drugs. However, volunteers are usually not screened for cotinine, a metabolite of nicotine, even though tobacco products may alter pharmacokinetic and pharmacodynamic parameters and withdrawal from tobacco may cause additional adverse events. The accuracy of personal histories as a means of excluding smokers was examined prospectively in three phase I units in the northeastern, midwestern, and southwestern United States. In studies intended for nonsmokers, 45 of 282 purported nonsmokers screened before enrollment tested positive for cotinine. This suggests that personal histories are unreliable in determining tobacco use in clinical trials designated for nonsmokers.

Suppression of experimental autoimmune encephalomyelitis by gallium nitrate.

We examined the effect of gallium (Ga) nitrate on the development of the development of experimental autoimmune encephalomyelitis (EAE). Weekly subcutaneous injections of 10-30 mg/kg prevented clinical signs as well as histopathological changes of EAE. The optimal timing of a single injection of Ga was 6 days after induction of EAE, with amelioration also apparent following a single injection on day 3 or 9 but not day 12. Ga administered in vivo suppressed myelin basic protein (MBP) and purified protein derivative-specific lymphocyte proliferative responses in vitro. Addition of Ga to MBP-specific T lymphocyte line cultures at various times after initiation of culture revealed that Ga exerts an effect at an early stage of cellular activation.

Prevention of murine cardiac allograft rejection with gallium nitrate. Comparison with anti-CD4 monoclonal antibody.

Gallium nitrate (GN) was evaluated for its ability to interfere with a cute rejection of DBA/2-->C57BL/6 heterotopic cardiac allografts, in comparison with the depleting anti-CD4 mAb, GK1.5. The administration of GN for 30 days (s.c. 30 mg/kg elemental gallium on days 0 and 3, 10 mg/kg every third day) resulted in >60-day graft survival in 78% (25 of 32) of the graft recipients, whereas 2 perioperative injections of anti-CD4 monoclonal antibody (mAb) resulted in >60-day graft survival in 58% (24 of 41) of the graft recipients. Serum gallium levels peaked at about 2000 ng/ml after 2-3 weeks of treatment and decreased to about 300 ng/ml by day 60, a level that was maintained for at least 30 more days. During the early posttransplant period, 25% of GN-treated grafts, but not anti-CD4 mAb-treated grafts, exhibited an unusual, transient reduction in graft impulse strength, suggesting a transient rejection response. Macroscopically, the long-surviving (>60 days) grafts from either treatment group exhibited none of the features of rejecting allografts. Histologically, they exhibited minor edema and rare epicardial inflammation but no tissue necrosis. However, there were vascular changes in allografts from GN-treated mice, including altered endothelial morphology, associated with moderate intimal hyperplasia and mild perivascular leukocytic infiltration. Allografts from anti-CD4 mAb-treated mice exhibited prominent neointimal hyperplasia associated with endothelial morphologic changes and prominent vascular and perivascular leukocytic infiltration. In general, both GN and anti-CD4 mAb promoted long-term allograft survival, but these allografts displayed the histopathologic signs of ongoing inflammation and chronic allograft rejection.

Effect of sertraline on protein binding of warfarin.

The effect of sertraline on the plasma protein binding of warfarin was investigated in a nonblinded randomised placebo-controlled parallel trial in 12 healthy male volunteers. The study participants received single doses of warfarin before administration of sertraline or placebo and again after sertraline or placebo had been administered for 22 days. Treatment with sertraline for 26 days increased the area under the mean prothrombin time vs time curve by 145 sec *h (7.9%), compared with a decrease of 17 sec *h (-1.0%) in the placebo group. Although statistically significant (p = 0.02), this difference was not felt to be clinically meaningful. There appeared to be a slight delay in the normalisation of the prothrombin time in the sertraline-treated group after the second dose of warfarin, which also would not be expected to be clinically significant. After 22 days, a statistically significant (p = 0.02) increase in unbound warfarin was observed in the sertraline group compared with the placebo-treated individuals. Neither the change in prothrombin time nor the change in plasma protein binding were considered to have any clinical relevance; however, good clinical practice dictates that prothrombin time should be monitored in patients treated concurrently with warfarin and sertraline to ensure that the integrity of coagulation response is maintained. The metabolism of warfarin is principally mediated by the cytochrome P450 (CYP) isoenzyme CYP2C9/10. Thus, sertraline appears to have a minimal effect on the CYP2C9/10 isoenzyme.

Metabolism of phenytoin and covalent binding of reactive intermediates in activated human neutrophils.

ontivation of neutrophils by phorbol-12-myristate-13-acetate (PMA) causes rapid production of superoxide radical (O2-), leading to the formation of additional reactive oxygen species, including hydrogen peroxide (H2O2), hypochlorous acid (HOCl), and possibly hydroxyl radical (.OH). These reactive oxygen species have been associated with the oxidation of some drugs. We investigated the metabolism of phenytoin (5,5-diphenylhydantoin) and the covalent binding of reactive intermediates to cellular macromolecules in activated neutrophils. In incubations with 100 microM phenytoin, PMA-stimulated neutrophils from six human subjects produced p-, m-, and o-isomers of 5-(hydroxyphenyl)-5-phenylhydantoin (HPPH) in a ratio of 1.0:2.1:2.8, respectively, as well as unidentified polar products. Analysis of cell pellets demonstrated that phenytoin was bioactivated to reactive intermediates that bound irreversibly to macromolecules in neutrophils. Glutathione, catalase, superoxide dismutase, azide, and indomethacin all diminished the metabolism of phenytoin and the covalent binding of its reactive intermediates. The iron-inactivating chelators desferrioxamine and diethylenetriaminepentaacetic acid had little or no effect on the metabolism of phenytoin by neutrophils, demonstrating that adventitious iron was not contributing via Fenton chemistry. In an .OH-generating system containing H2O2 and Fe2+ chelated with ADP, phenytoin was oxidized rapidly to unidentified polar products and to p-, m-, and o-HPPH (ratio 1.0:1.7:1.5, respectively). Reagent HOCl and human myeloperoxidase (MPO), in the presence of Cl- and H2O2, both formed the reactive dichlorophenytoin but no HPPH. However, no chlorinated phenytoin was detected in activated neutrophils, possibly because of its high reactivity. These findings, which demonstrated that activated neutrophils biotransform phenytoin in vitro to hydroxylated products and reactive intermediates that bind irreversibly to tissue macromolecules, are consistent with phenytoin hydroxylation by .OH generated by a transition metal-independent process, chlorination by HOCl generated by MPO, and possibly cooxidation by neutrophil hydroperoxidases. Neutrophils activated in vivo may similarly convert phenytoin to reactive intermediates, which could contribute to some of the previously unexplained adverse effects of the drug.

The effect of azithromycin on the pharmacokinetics of indinavir.

This study was performed to examine the effect of the coadministration of azithromycin on the pharmacokinetics of the protease inhibitor indinavir (Crixivan). In an open-label, parallel-design study, 32 healthy male and female volunteers were given indinavir (800 mg tid) for 5 days. One hour prior to the first dose of indinavir on day 5, 18 subjects received 1200 mg azithromycin (Zithromax), and 14 subjects received matching placebo. Serial samples of plasma were obtained for 8 hours following the morning dose of indinavir on days 4 and 5 and assayed for indinavir by HPLC/UV. Twenty-seven subjects completed the study. Following coadministration of azithromycin with indinavir, there was no significant change between day 5 and day 4 in AUC (20.7 mg.hr/ml and 23.1 mg.hr/ml; 90% CI on the ratio 81%-100%) or Cmax (9.88 mg/ml and 10.3 mg/ml; 90% CI 86%-108%). The day 5 to day 4 difference in indinavir concentrations following coadministration with azithromycin was not significantly different from the day 5 to day 4 difference with placebo (AUC p = 0.68; Cmax p = 0.074). Therefore, azithromycin does not significantly alter the kinetics of indinavir.

Comparison of azithromycin and clarithromycin in their interactions with rifabutin in healthy volunteers.

A 14-day, randomized, open, phase I clinical trial was designed to examine possible pharmacokinetic interactions between rifabutin and two other antibiotics, azithromycin and clarithromycin, used in the treatment of Mycobacterium avium complex infections. Thirty healthy male and female volunteers were divided into five groups of six participants each: 18 received 300 mg/day of rifabutin, 12 in combination with therapeutic doses of either azithromycin or clarithromycin; the remaining 12 received azithromycin or clarithromycin alone. On day 10 the study was terminated because of adverse events, including severe neutropenia. Fourteen participants who received rifabutin developed neutropenia, including all 12 participants who received azithromycin or clarithromycin concomitantly. Analyses of serum revealed no apparent pharmacokinetic interaction between azithromycin and rifabutin. However, the mean concentrations of rifabutin and 25-O-desacetyl-rifabutin (an active metabolite) in participants who received clarithromycin and rifabutin concomitantly were more than 400% and 3,700%, respectively, of concentrations in those who received rifabutin alone. Physicians should be aware that recommended prophylactic doses of rifabutin may be associated with severe neutropenia within 2 weeks after initiation of therapy, and all patients receiving rifabutin, especially with clarithromycin, should be monitored carefully for neutropenia.

Pharmacokinetics and safety of oral eletriptan during different phases of the menstrual cycle in healthy volunteers.

The purpose of this study was to determine the pharmacokinetics and safety of eletriptan in different phases of the menstrual cycle. Female volunteers (n = 16) with a regular menstrual cycle (28 +/- 4 days) received a single oral dose of 80 mg eletriptan during each of the four cycle phases: phase 1 (menses), days 1 to 4; phase 2 (follicular), days 6 to 10; phase 3 (ovulatory), days 11 to 13; and phase 4 (luteal), days 21 to 24. Eletriptan plasma concentrations were determined from serial plasma samples taken during a 24-hourperiod after dosing. Blood pressure, pulse rate, and ECG measurements were performed at baseline, 1 and 24 hours after dosing. No significant differences between phases were observed for maximum plasma concentration (cmax, range of means = 188-234 ng/ml), time to maximum concentration (tmax, range of means = 1.8-2.5 h), or systemic exposure (area under the curve [AUC], range of means = 1194-1514 ng x h/ml). Although there was a statistically significant difference in the terminal phase elimination rate constant (kel) between phases 1 and2 (0.175/h vs. 0.158/h, p = 0.044), the corresponding difference in terminal phase half-life (t 1/2) (4.0 h vs. 4.4 h) was not considered to be clinicallyrelevant. No clinically relevant differences in blood pressure, pulse rate, or ECG were observed, and the incidence, nature, and severity of adverse events were similar in all phases. The different phases of the menstrual cycle had no clinically significant effect on the pharmacokinetics, safety, or tolerability of oral 80 mg eletriptan in healthy females.

Induction of fluconazole metabolism by rifampin: in vivo study in humans.

The effects of rifampin on the pharmacokinetics of fluconazole were analyzed in an open-label, placebo-controlled, parallel study. Sixteen healthy male volunteers, randomized into two groups, received 200 mg of oral fluconazole on days 1 and 22. On days 8 through 27, group I received oral rifampin, 600 mg/d, and group II received placebo. Fluconazole in serum was analyzed by HPLC. On days 1 and 22, respectively, the AUC (micrograms.hr/mL) (mean +/- SD) was 160.5 +/- 19.5 and 124 +/- 22.2 in group I, 152 +/- 25 and 152.8 +/- 33.9 in group II; the Kel (hr-1) was .0211 +/- .0030 and .0264 +/- .0040 in group I, .0219 +/- .0036 and .0216 +/- .0053 in group II. Cmax and Tmax did not change significantly in either group. Urinary 6 beta-hydroxycortisol/cortisol increased from 3.47 +/- 1.04 to 15.2 +/- 5.07 in group I, but was unchanged (3.54 +/- 1.33-4.26 +/- 2.36) in group II on days 1 and 22, respectively. The findings in this study indicate that rifampin induces the metabolism of fluconazole.

Sertraline does not alter steady-state concentrations or renal clearance of lithium in healthy volunteers.

An open-label, placebo-controlled study was conducted to determine the effects of sertraline on the steady-state levels and renal clearance of lithium in 20 healthy volunteers. Subjects received 600 mg of lithium twice daily for 9 days. On the evening of day 8, subjects received orally either placebo or 100 mg of sertraline; these were administered twice, 8 hours apart, beginning 2 hours after the evening dose of lithium. In a comparison of day 8 with day 9 (before administration of the morning doses of lithium), sertraline was associated with only a 0.01 mEq/L (1.4%) decrease in steady-state levels and a 0.11 L/hour (6.9%) increase in the renal clearance of lithium. Neither change was statistically significant relative to placebo. Four subjects were excluded from analysis because of protocol violations or laboratory abnormalities unrelated to sertraline. Seven subjects who received lithium plus sertraline experienced side effects, mainly tremors, possibly related to treatment, whereas none of those administered lithium plus placebo experienced side effects. No sertraline-related laboratory abnormalities were observed.

Gallium nitrate suppresses lupus in MRL/lpr mice.

Gallium (Ga) nitrate, a drug which prevents a variety of experimental autoimmune diseases, was investigated in a murine model of systemic lupus erythematosus (SLE). In one experiment, female MRL/Mp lpr/lpr (MRL/lpr) mice were randomized into 2 groups of 6: 1) vehicle (trisodium citrate) and 2) Ga. Subcutaneous injections began at 3 weeks of age and continued weekly until the mice were euthanized a week after the thirteenth injection. The loading dose of Ga (calculated as elemental Ga) was 45 mg/kg, followed by 15 mg/kg/week. In another experiment (n = 18) with 3 males and 3 females per group, mice received 1) vehicle, 2) Ga x 1 (one 45 mg/kg dose), and 3) Ga x 13. In the experiment with 12 mice, axillary lymph nodes from Ga-treated mice were significantly smaller than those from vehicle-treated mice (91+/-42 and 360+/-358 mg respectively, mean+/-SD), and spleens as well as lymph nodes from the former showed significantly less lymphoid infiltrate. In the experiment with 18 mice, prescapular lymph nodes weighed 312+/-98, 217+/-52, and 42+/-34 mg, and spleens weighed 732+/-492, 409+/-164, and 192+/-93 mg in the groups which received vehicle, Ga x 1, and Ga x 13 respectively. Control mice had significantly more lymphoid infiltrates in the lungs, spleen, and lymph nodes and, unlike Ga x 13 mice, exhibited glomerulitis and renal vasculitis. Within groups, females developed more severe disease than males. The Ga x 13 group had increased percentages of CD4-bearing and CD8-bearing lymphocytes in lymph nodes and increased CD4-bearing lymphocytes in the spleen, with an increased proliferative response to mitogen stimulation in vitro in lymph nodes, although not in the spleen. The Ga x 13 group also gained less weight and developed osteosclerosis. Although preliminary, our findings suggest that clinical trials with Ga in SLE are merited.

Gallium nitrate suppresses the production of nitric oxide and liver damage in a murine model of LPS-induced septic shock.

The efficacy of gallium (Ga) nitrate was examined in a murine model of sepsis. Male Balb/c mice (6-8 weeks) were randomized into 3 groups: 1) vehicle-treated controls 2) mice with sepsis induced by treatment with 0.3 mg i.v. of Propionibacterium acnes followed one week later by 0.01 microg lipopolysaccharide (LPS) and 10 mg of D-galactosamine (GalN) 3) mice with sepsis injected with 45 mg/kg s.c. of gallium nitrate (calculated as elemental Ga) 24 hours prior to LPS/GalN. Two hours after LPS/GalN or vehicle, plasma concentrations of tumor necrosis factor (TNF-alpha) in groups 1, 2 and 3 were 54+/-31 (n=6), 21,390+/-5139 (n=4), and 21,909+/-943 (n=5) pg/ml, respectively. After 6 hours, plasma concentrations of gamma interferon (IFN-gamma) were <10 (n=8), 4771+/-1078 (n=6), and 1622+/-531 (n=15) pg/ml, respectively, and of nitrate/nitrite (products of nitric oxide) were 64+/-8 (n=7), 146+/-18 (n=8), and 57+/-8 (n=15) microM. At 18 hours, serum chemistries were; SGOT 171+/-46 (n=13), 10,986+/-3062 (n=7), and 1078+/-549 (n=8) IU/L; SGPT 165+/-59, 17,214+/-4340, and 2088+/-1097 IU/L; and total bilirubin 0.2+/-0.0, 0.9+/-0.4, and 0.2+/-0.0 mg/dl for groups 1, 2, and 3 respectively. Blinded histologic evaluation of livers at 18 hours revealed inflammatory infiltrate scores (x [range], 0=none, 1=minimal, 2=mild, 3=moderate, and 4=severe) of 0.1 [0-1] (n=8), 3.0 [2-4] (n=15), and 2.0 [0-3] (n=10), and necrosis scores of 0.0, 2.8 [0-4], and 0.9 [0-4]. Although Ga did not affect production of TNF-alpha, it ameliorated hepatocellular injury and protected against necrosis. Based on this model of sepsis, Ga may have a role in treating the human disease.

Lack of effect of gallium nitrate on bone density in a rat model of simulated microgravity.

The effect of gallium (Ga) nitrate upon bone density was studied in 24 male Sprague-Dawley rats (275-335 g) in an established model of simulated microgravity. Rats, tail-suspended for 14 d from a system of double pulleys, were allowed free mobility with their hind limbs unloaded. Animals were randomized into four groups of six: 1) unsuspended, saline; 2) suspended, saline; 3) unsuspended, Ga; and 4) suspended, Ga. On day 0, 1 d prior to suspension, rats received equal volumes of a single subcutaneous injection of either saline or 30 mg/kg of elemental Ga in the form of Ga nitrate. On day 15, all animals were euthanized under anesthesia, and their hind limbs analyzed in vitro using single-photon absorptiometry. Although we previously demonstrated that Ga inhibits bone resorption in patients with Paget's disease of bone, and although Ga exhibits many apparent similarities in efficacy to diphosphonates, which prevent bone loss in ovariectomized, paralyzed, and single-limb immobilized rats, Ga failed to prevent bone loss in the tail-suspended rats.

Therapeutic uses of gallium nitrate: past, present, and future.

Injectable gallium (Ga) nitrate, approved in the United States for the treatment of hypercalcemia of malignancy, has been known for more than 2 decades to have immunosuppressive properties. At therapeutic doses, it has few adverse effects, although high-dose infusions may result in severe nephrotoxicity, particularly in patients who are not adequately hydrated, and severe anemia. In animal models, Ga has been shown to have efficacy in the treatment of adjuvant arthritis, type 1 diabetes, experimental autoimmune encephalomyelitis, experimental pulmonary inflammation, cardiac allograft rejection, experimental autoimmune uveitis, endotoxic shock, and systemic lupus erythematosus. Clinical trials have demonstrated efficacy in Paget's disease of bone and activity against some malignancies, including epithelial ovarian carcinoma, non-squamous cell carcinoma of the cervix, bladder cancer, and non-Hodgkin's lymphoma. Other clinical trials underway include studies of sarcoidosis and rheumatoid arthritis. Future studies should be conducted not only in other autoimmune diseases, such as multiple sclerosis, but also in graft-versus-host disease, leprosy, and acquired immunodeficiency syndrome (AIDS).

Death from a morphine infusion during a sickle cell crisis.

A 15-year-old boy died during a vaso-occlusive sickle cell crisis after having received a prolonged infusion of morphine. Even in therapeutic doses, narcotics may cause significant respiratory acidosis and hypoxemia, enhancing polymerization of hemoglobin SS and thereby promoting sickling and vaso-occlusion. When narcotics are used during a sickle cell crisis, the best method is pharmacokinetically based patient-controlled analgesia.

Effect of tenidap sodium on the pharmacodynamics and plasma protein binding of warfarin in healthy volunteers.

1. An open-label, randomised study was performed to assess the effect of tenidap sodium on the pharmacodynamics and plasma protein binding of warfarin. 2. Fourteen healthy male volunteers received either a single oral dose of 120 mg tenidap sodium or matching placebo capsules from days 11 to 36. A single oral dose of 0.75 mg kg-1 warfarin was administered on days 1 and 32. 3. The mean prothrombin AUC(1,120h) value between baseline and day 32 increased from 1692.4 +/- 234.5 s h to 1769.3 +/- 218.0 s h in the group given tenidap, and decreased from 1747.6 +/- 289.4 s h to 1708.1 +/- 236.8 s h in the placebo group. 4. Tenidap caused a slight delay in the normalisation of prothrombin times following the second dose of warfarin on day 32 compared with the first dose on day 1. This was significant at 36, 48, 72 and 96 h but not at 120 h after administration of warfarin. 5. The mean percentage of unbound warfarin in the tenidap group (0.08% +/- 0.09) was significantly different (P = 0.047) from that in the placebo group (-0.03% +/- 0.10) but this was not considered to be clinically meaningful. 6. These data indicate that prothrombin times should be monitored during concomitant administration of tenidap and warfarin.

Use of gallium to treat Paget's disease of bone: a pilot study.

The effects of gallium nitrate on bone turnover were evaluated in four patients with active Paget's disease. Treatment with gallium nitrate (100 mg/m2 daily for 5 days, intravenously in 5% glucose) significantly reduced serum calcium, serum phosphate, urinary calcium, and the ratio of maximum tubular reabsorption of phosphate to glomerular filtration rate in each patient. Serum parathyroid hormone levels rose. The findings suggest that the fall in serum calcium caused secondary hyperparathyroidism, resulting in a fall in serum phosphate. Serum alkaline phosphatase and urinary hydroxyproline levels fell substantially, showing that gallium effectively suppressed bone turnover. The fall in hydroxyproline excretion preceded that in serum alkaline phosphatase, suggesting that suppression of bone resorption by osteoclasts preceded that of bone formation by osteoblasts. Alkaline phosphatase levels remained low throughout follow-up (85-141 days), so the effect of gallium seems to be long-lasting.

Practical considerations regarding the influence of the menstrual cycle on leukocyte parameters in clinical trials.

Variations in hormone levels during different phases of the menstrual cycle have been shown to alter the pharmacokinetics of some medications and are known to exert significant effects on seemingly unrelated physiologic parameters. Numerous studies have been undertaken to examine the effects of different phases of the menstrual cycle on leukocyte and differential counts, but results have often been inconclusive and contradictory. This study endeavored to reexamine these parameters, measured by standard laboratory assays, in healthy ovulating females to determine whether the menstrual cycle may have clinically relevant effects on leukocyte counts. Twenty-one women, aged 18 to 35 years and not taking hormonal contraception, were enrolled in an outpatient study within 12 hours after the onset of normal menses. The women reported to the clinical pharmacology unit for a complete blood count with differential on days 1, 2, 7, 10 through 17, 22, and 25 through 32. Blinded duplicate samples were obtained on day 2 to assess variability at the analytic site, and levels of luteinizing hormone and estradiol were measured on days 11 through 16 to determine the day of ovulation. Eighteen women completed the study, with cycle lengths ranging from 24 to 31 days (28.2 +/- 1.9 days, mean +/- SD). Evaluations of the data revealed a trend toward higher leukocyte counts and absolute neutrophil counts at the onset of menses but no significant or clinically relevant effects of different phases of the menstrual cycle on these parameters. Some split samples showed considerable variation in the assays (eg, a 42% increase in absolute neutrophil counts), suggesting that errors at the analytic facility may be a more important consideration than hormonal effects.