SDS-coated atovaquone nanosuspensions show improved therapeutic efficacy against experimental acquired and reactivated toxoplasmosis by improving passage of gastrointestinal and blood-brain barriers.

Toxoplasmic encephalitis (TE) is the most common clinical manifestation of reactivated infection with Toxoplasma gondii in immunocompromised patients that is lethal if untreated. The combination of pyrimethamine plus sulfadiazine or clindamycin is the standard therapy for the treatment of TE, but these combinations are associated with hematologic toxicity and/or life-threatening allergic reactions. Therefore, alternative treatment options are needed. Atovaquone is safe and highly effective against T. gondii in vitro, but the oral micronized solution shows poor bioavailability. We synthesized atovaquone nanosuspensions (ANSs) coated with poloxamer 188 (P188) and sodium dodecyl sulfate (SDS) to improve oral bioavailability and passage through the blood-brain barrier (BBB). Coating of ANSs with SDS resulted in enhanced oral bioavailability and enhanced brain uptake of atovaquone compared to Wellvone(®) in murine models of acute and reactivated toxoplasmosis as measured by high performance liquid chromatography (HPLC). Parasite loads and inflammatory changes in brains of mice treated with SDS-coated ANS were significantly reduced compared to untreated controls and to Wellvone(®)-treated mice. In conclusion, nanosuspensions coated with SDS may ultimately lead to improvements in the treatment of TE and other cerebral diseases.

The role of apolipoprotein E in uptake of atovaquone into the brain in murine acute and reactivated toxoplasmosis.

We investigated whether coating of atovaquone nanosuspensions (ANSs) with apolipoprotein E (apoE) peptides improves the uptake of atovaquone into the brain. The passage across the blood-brain barrier (BBB) of ANSs stabilized by polysorbate 80 (Tween 80), poloxamer 184 (P184), or poloxamer 338 (P338) and the same formulations coated with apoE peptides were analyzed in vitro and in vivo. Passage through a rat coculture model of the BBB did not differ between individual atovaquone formulations, and the addition of apoE peptides did not enhance the transport. Following the induction of toxoplasmic encephalitis (TE) in mice, treatment with all atovaquone formulations reduced the number of parasites and inflammatory foci compared with untreated mice. Uptake of atovaquone into the brain did not depend on coating with apoE. Finally, incubation of apoE peptide-coated ANSs with brain endothelial cells for 30 min did result in the accumulation of nanoparticles on the cell surface but not in their uptake into the cells. In conclusion, ANSs coated with Tween 80 or poloxamers showed therapeutic efficacy in murine toxoplasmosis. ApoE- and apoE-derived peptides do not induce the uptake of ANSs into the brain. Alternative mechanisms seem to be in operation, thereby mediating the passage of atovaquone across the BBB.

Glucarpidase (carboxypeptidase g2) intervention in adult and elderly cancer patients with renal dysfunction and delayed methotrexate elimination after high-dose methotrexate therapy.

OBJECTIVE: Leucovorin and extracorporeal removal of methotrexate (MTX) have limited efficacy in delayed MTX elimination after high-dose methotrexate (HD-MTX) therapy. Glucarpidase (carboxypeptidase G2) cleaves MTX into nontoxic metabolites, but experience with this enzyme is limited in adult patients. We evaluated the effects of glucarpidase intervention in adult and elderly patients with delayed MTX elimination. PATIENTS AND METHODS: Forty-three patients (age, 18-78 years) with MTX serum concentrations (sMTX) of 1-1,187 micromol/l received glucarpidase, leucovorin rescue guided by MTX immunoassay, and standard supportive care. MTX and MTX metabolites were quantified in serum (24 patients) and urine (8 patients) by high-performance liquid chromatography. Contributory risk factors, toxicities, and survival were recorded in all patients. RESULTS: Glucarpidase was well tolerated and resulted in an immediate >97% reduction in sMTX, with a 0.2%-35% urinary recovery of the total MTX dose as inactive MTX metabolites. Forty (93%) of 43 patients had normalization (n = 25) or improvement (n = 15) of their serum creatinine. Frequent grade III-IV MTX toxicities were hematological (60%) and mucositis (35%); only eight (19%) patients developed grade III-IV nephrotoxicity. Ten (23%) of 43 patients experienced fatal complications associated with HD-MTX therapy. Patients with three or more contributory risk factors for delayed MTX elimination had a significantly poorer survival than patients with fewer than three risk factors (hazard ratio, 3.64; confidence interval, 1.14-17.54). CONCLUSIONS: Glucarpidase is well tolerated and produces a rapid inactivation of substantial amounts of MTX. However, overall results are still unsatisfactory in adult and elderly patients, suggesting that earlier recognition of delayed MTX elimination and more rapid intervention are needed.

Effects of enteral feeding on the oral bioavailability of moxifloxacin in healthy volunteers.

BACKGROUND AND OBJECTIVE: Moxifloxacin is a new generation fluoroquinolone antimicrobial agent used worldwide. In clinical practice in intensive care units, moxifloxacin may be frequently administered through a nasogastric feeding tube. In the absence of an oral liquid formulation and since the multivalent cations contained in enteral feeds may potentially impair absorption of moxifloxacin administered via this route, we studied the effect of concomitant enteral feeding on the pharmacokinetics and tolerability of moxifloxacin administered as a crushed tablet through the nasogastric tube. PARTICIPANTS AND METHODS: This was a single-centre, open-label, randomised, controlled, nonblinded, three-way crossover study. Twelve young healthy volunteers (nine females and three males) aged 20-42 years were included in the study. Each participant received three separate treatment regimens in a randomised fashion: an intact moxifloxacin 400 mg tablet (regimen A, reference treatment), a crushed moxifloxacin 400 mg tablet as a suspension through a nasogastric tube with water (regimen B) and a crushed moxifloxacin 400 mg tablet as a suspension through a nasogastric tube with enteral feeding (regimen C). A washout period of 1-week followed each treatment. Concentrations of moxifloxacin in serum were measured by a validated high-performance liquid chromatography method. Pharmacokinetic parameters were calculated by noncompartmental methods. Additionally, the primary parameters indicative for changes in absorption (area under the serum concentration-time curve from time zero to infinity [AUC(infinity)] and peak serum concentration [C(max)]), were tested for bioequivalence, assuming log-normally distributed data using ANOVA. RESULTS: All moxifloxacin treatment regimens were well tolerated. The AUC(infinity) was slightly, but not statistically significantly, decreased in treatments with regimens B and C. AUC(infinity) (geometric means 39.6 [regimen A] vs 36.1 [regimen B] vs 36.1 mg.h/L [regimen C] and point estimates 91% for B : A and C : A) indicated bioequivalence of the treatments. Bioequivalence criteria of AUC(infinity) and C(max) were met upon retrospective statistical analysis. Likewise C(max) after moxifloxacin administration through nasogastric tube with water (regimen B) and with tube feed (regimen C) were slightly decreased (geometric means 3.20 [regimen A] vs 3.05 [regimen B] vs 2.83 mg/L [regimen C]; point estimates 88% for B : A, and 95% for C : A). They were within the range seen in other studies conducted with oral administration of the drug. No statistically significant differences were observed in time to reach C(max) (t(max); median 1.75 [regimen A] vs 1.00 [regimen B] vs 1.75 hours [regimen C]). Thus, the rate of absorption of moxifloxacin was not affected by administration through a nasogastric tube. This route of ingestion seems to be associated with a slight loss of bioavailability independent of the carrier medium used (water vs enteral feed); no clinically relevant interaction with the multivalent cations contained in the enteral feed was observed, indicating that moxifloxacin and enteral nutrition can be administered concomitantly. CONCLUSION: There was no clinically relevant effect of enteral feeding on the pharmacokinetics of oral moxifloxacin in healthy volunteers. This result has to be evaluated in patients, particularly those from the intensive care unit, who are characterised by severe infectious and/or concomitant diseases that might influence absorption of moxifloxacin.

Penetration of ertapenem into different pulmonary compartments of patients undergoing lung surgery.

Ertapenem is approved for the treatment of community-acquired pneumonia (CAP), but its in vivo penetration into lung tissue (LT), epithelial lining fluid (ELF), and alveolar cells (AC) is unknown. Fifteen patients undergoing thoracotomy were treated with 1 g intravenously for perioperative prophylaxis. Bronchoalveolar lavage was performed 1, 3, and 5 hours after ertapenem infusion. Normal LT was sampled at the time of lung extraction. Blood was collected before and at different time points up to 24 hours after infusion. Mean concentrations of ertapenem in plasma, ELF, and AC were at 1.0 hour, 63.1, 4.06, 0.004 mg/L; at 3.0 hours, 39.7, 2.59, 0.003 mg/L; and at 5.0 hours, 27.2, 2.83, 0.007 mg/L. Mean (range) concentration in LT was 7.60 (2.5-19.4) mg/kg tissue 1.5 to 4.5 hours after infusion. In plasma, ertapenem exhibited a Cmax of 94.7 +/- 23.3 mg/L and an AUC(0-last) of 501.1 +/- 266.3 mg x h/L. These results, combined with the reported (MIC)90 of most CAP bacteria, support the previously observed clinical efficacy of ertapenem in the treatment of CAP.

Atovaquone maintenance therapy prevents reactivation of toxoplasmic encephalitis in a murine model of reactivated toxoplasmosis.

Acute therapy with pyrimethamine plus sulfadiazine is the treatment of choice for reactivated toxoplasmic encephalitis (TE). Acute therapy is followed by lifelong maintenance therapy (secondary prophylaxis) with the same drugs at lower dosages. The use of pyrimethamine plus sulfadiazine is hampered by severe side effects including allergic reactions and hematotoxicity. Alternative treatment regimens with pyrimethamine plus clindamycin or other antiparasitic drugs are less efficacious. Atovaquone nanosuspensions show excellent therapeutic effects for "acute" intravenous (i.v.) treatment of reactivated TE in a murine model. In the present study, the therapeutic efficacy of atovaquone for oral "maintenance" therapy was investigated. Mice with a targeted mutation in the interferon regulatory factor 8 gene were latently infected with Toxoplasma gondii, developed reactivated TE, and received acute i.v. therapy with atovaquone nanosuspensions. Mice were then treated orally with atovaquone suspension or other antiparasitic drugs to prevent relapse of TE. Maintenance therapy with atovaquone at daily doses of 50 or 100 mg/kg (body weight) protected mice against reactivated TE and death. This maintenance treatment was superior to standard therapy with pyrimethamine plus sulfadiazine. The latter combination was superior to the combination of pyrimethamine plus clindamycin. Inflammatory changes in the brain parenchyma and meninges, as well as parasite numbers, in the brains of mice confirmed the therapeutic efficacy of atovaquone for maintenance therapy. Atovaquone was detectable in sera, brains, livers, and lungs of infected mice by high-performance liquid chromatography and/or mass spectrometry. In conclusion, atovaquone appears to be superior to the standard maintenance therapy regimens in a murine model of reactivated TE. The therapeutic efficacy of atovaquone for maintenance therapy against TE should be further investigated in clinical trials.

Blockade of the intermediate-conductance calcium-activated potassium channel as a new therapeutic strategy for restenosis.

BACKGROUND: Angioplasty stimulates proliferation and migration of vascular smooth muscle cells (VSMC), leading to neointimal thickening and vascular restenosis. In a rat model of balloon catheter injury (BCI), we investigated whether alterations in expression of Ca2+-activated K+ channels (KCa) contribute to intimal hyperplasia and vascular restenosis. METHODS AND RESULTS: Function and expression of KCa in mature medial and neointimal VSMC were characterized in situ by combined single-cell RT-PCR and patch-clamp analysis. Mature medial VSMC exclusively expressed large-conductance KCa (BKCa) channels. Two weeks after BCI, expression of BKCa was significantly reduced in neointimal VSMC, whereas expression of intermediate-conductance KCa (IKCa1) channels was upregulated. In the aortic VSMC cell line, A7r5 epidermal growth factor (EGF) induced IKCa1 upregulation and EGF-stimulated proliferation was suppressed by the selective IKCa1 blocker TRAM-34. Daily in vivo administration of TRAM-34 to rats significantly reduced intimal hyperplasia by approximately 40% at 1, 2, and 6 weeks after BCI. Two weeks of treatment with the related compound clotrimazole was equally effective. Reduction of intimal hyperplasia was accompanied by decreased neointimal cell content, with no change in the rate of apoptosis or collagen content. CONCLUSIONS: The switch toward IKCa1 expression may promote excessive neointimal VSMC proliferation. Blockade of IKCa1 could therefore represent a new therapeutic strategy to prevent restenosis after angioplasty.

Single- and multiple-dose pharmacokinetics of linezolid and co-amoxiclav in healthy human volunteers.

In an open, randomized, two-period crossover study the pharmacokinetics of linezolid and co-amoxiclav were investigated after single- and multiple-dose administration in 12 healthy volunteers (six females and six males). Linezolid was given in tablets of 600 mg twice a day for 7 days and co-amoxiclav in tablets of 1000 mg (875 + 125 mg) once a day for 7 days. The wash-out period was 4 weeks between the administration of the two antibacterial agents. Blood and urine samples were collected on days 1 and 7 before and at different time points up to 24 h after medication. The concentrations of the antibiotics in serum and urine were measured by validated high-performance liquid chromatography methods. Linezolid exhibited a mean C(max) of 14.5 +/- 4.6 mg/L after T(max) of 47.5 +/- 20.1 min on day 1, with a significant increase to 24.0 +/- 6.9 mg/L on day 7 (P < 0.01). The AUD(tot) (total area under the data) revealed a significant increase from 140.5 +/- 28.3 mg.h/L on day 1 to 220.2 +/- 42.6 mg.h/L on day 7 (P < 0.01). There were no significant differences in terminal elimination half-life between days 1 and 7 (9.53 +/- 2.87 versus 7.97 +/- 3.08 h) or in total clearance (71.6 +/- 17.6 versus 81.5 +/- 14.7 ml/min.1.73 m(2)). Results are in agreement with the assumption of a limited accumulation of linezolid under the dosage regimen given. Serum linezolid concentrations in females were always higher than those in males. The volume of distribution V(ss)/f differed significantly between females and males (41.6 +/- 4.2 versus 52.2 +/- 3.3 L/70 kg; P < 0.01). Pharmacokinetic parameters of amoxicillin and clavulanic acid found in this study were similar to previously published data. No accumulation was found with co-amoxiclav. No serious adverse event was observed with the study drugs.

Pharmacokinetics of lansoprazole, amoxicillin and clarithromycin after simultaneous and single administration.

In a randomized, double-blind, placebo-controlled, four-way crossover study, possible influences of the triple therapy with amoxicillin, clarithromycin and the proton pump inhibitor lansoprazole on the pharmacokinetics of each of the drugs and the active 14-OH-clarithromycin metabolite were assessed. Twelve Helicobacter pylori-negative healthy male volunteers (age 27 +/- 4.3 years; creatinine clearance 7.0 +/- 2.0 L/h) were given lansoprazole 30 mg, amoxicillin 1 g and clarithromycin 500 mg, alone and in triple combination. Drug elimination intervals were at least 9 days between the dosing periods. The study medication was administered twice daily for 4 days. On the fifth day of each period, drugs were only given once in the morning, and blood and urine samples were collected for 12 h. The concentrations of the three substances administered, and 14-OH-clarithromycin, were determined by validated HPLC methods. Alterations in the serum kinetics were found for lansoprazole and the active 14-OH-clarithromycin metabolite (all data expressed as mean +/- S.D.). For lansoprazole, the elimination half-life (t(1/2)) was significantly prolonged (1.46 versus 1.7 h, P < 0.05) and the area under the concentration-time curve from 0 to 8 h (AUC(0-8)) was significantly increased (3.65 versus 4.59 mg.h/L, P < 0.05) by combination of the drugs. For 14-OH-clarithromycin, the peak concentration (C(max)) was 0.95 versus 1.18 mg/L and the AUC from 0 to 12 h (AUC(0-12)) was 8.3 versus 10.5 mg.h/L (augmented significantly, P < 0.05). The amoxicillin concentrations were slightly elevated by concomitant administration of lansoprazole and clarithromycin but without statistical significance (11.1 versus 12.6 mg/L). For clarithromycin, the time to maximum concentration of drug in serum (T(max)) was increased (2.73 versus 3.31 h, P < 0.05), whereas AUC and C(max) remained unchanged. Simultaneous administration of lansoprazole, amoxicillin and clarithromycin increases the serum concentrations of lansoprazole and the active 14-OH-clarithromycin metabolite significantly. These effects were not so pronounced as to have any therapeutic influence, making dosage adjustment unnecessary.

Single- and multiple-dose pharmacokinetics of oral moxifloxacin and clarithromycin, and concentrations in serum, saliva and faeces.

Moxifloxacin and clarithromycin are important antibacterial drugs in the treatment of community-acquired respiratory tract infections. In a double-blind, randomized, 2-period cross-over study the pharmacokinetics of moxifloxacin versus clarithromycin were determined after single and multiple doses in 12 healthy male volunteers. The concentrations of the antibiotics in serum, saliva and faeces were measured by validated high-performance liquid chromatographic methods. In serum, moxifloxacin exhibited a mean peak concentration of 3.1 +/- 0.6 mg/l after a time to peak concentration of 1.67 +/- 0.96 h on day 1, with a significant increase to 3.98 +/- 1.10 mg/l on day 7 (p < 0.05). The area under the curve-12 revealed a highly significant increase from 28.2 + 4.1 mg*h/l on day 1 to 39.5 +/- 6.6 mg*h/l on day 7 (p < 0.01). There were also significant differences in terminal half-life between day 1 and day 7 [10.6 h (range 9.0-12.8) vs 14.9 h (range 12.6-28.1); p < 0.01] and in mean residence time (15.1 +/- 1.9 vs 18.2 +/- 2.4 h; p < 0.01). The concentrations of moxifloxacin in saliva were well equilibrated with serum at a relatively constant saliva-serum ratio of about 0.8. Pharmacokinetic parameters of clarithromycin and its metabolite, 14-hydroxy-clarithromycin, were similar to previously published data. Accumulation was found. No serious adverse events were observed with either study drug.

Changes in fecal flora and comparative multiple-dose pharmacokinetics of ceftibuten, cefpodoxime proxetil and amoxycillin/clavulanate.

OBJECTIVE: To investigate changes in fecal flora and multiple-dose pharmacokinetics with the oral antibiotics ceftibuten 400 mg daily and cefpodoxime proxetil (CPX) 200 mg every 12 h, compared to amoxycillin/clavulanate 500/125 mg every 8 h during and following 1 week of medication. METHODS: In an open randomized triple crossover design, 18 (nine female, nine male) healthy volunteers received each drug for 7 days, followed by a 'washout' period of 4 weeks. Serum and urine levels of the substances were determined by bioassay, and for ceftibuten isomers by high-pressure liquid chromatography. Statistical analysis of quantitative aerobic and anaerobic cultures of feces was performed, and beta-lactamase activity was determined. RESULTS: Ceftibuten showed a mean Cmax of 18.9 (SD 3.0) mg/L, a terminal half-life of 2.89 h, and an AUCtot of 100 (21.8) mg.h/L; protein binding was 63.7 (5.1)%, and accumulation was marginal. Cefpodoxime proxetil had a Cmax of 1.92 (0.61) mg/L, a terminal half-life of 1.97 (0.42) h and an AUCtot of 10.8 (3.3) mg.h/L; no accumulation was seen. Amoxycillin and clavulanate had Cmax values of 7.15 (2.16) mg/L and 3.39 (1.31) mg/L, terminal half-life values of 1.03 (0.15) h and 0.93 (0.17) h, AUCtot values of 20.0 (4.2) mg.h/L and 8.87 (3.10) mg.h/L, and there was no accumulation. Statistical analysis for ech microorganism in fecal samples showed significant differences between amoxycillin/clavulanate and the two third-generation cephalosporins, but virtually no differences between ceftibuten and cefpodoxime proxetil. Eleven of 12 volunteers reported loose stools (days 2-7, mean duration 4.4 (SD 2.7) days) with amoxycillin/clavulanate, but nobody during ceftibuten administration and one volunteer during cefpodoxime proxetil administration. CONCLUSIONS: Ceftibuten showed excellent and cefpodoxime favorable pharmacokinetic properties, with significantly less pronounced fecal flora changes and intestinal side effects compared to amoxycillin/clavulanate. The multiple crossover design allows powerful microbiological statistical analysis and pharmacokinetic parameter comparisons.

Comparative pharmacokinetics of macrolide antibiotics and concentrations achieved in polymorphonuclear leukocytes and saliva.

OBJECTIVES: The pharmacokinetics of macrolide antibiotics --- erythromycin (ER), clarithromycin (CL), roxithromycin (RO), azithromycin (AZ), dirithromycin (DI) and the concentrations achieved in polymorphonuclear neutrophils (PMNs) and saliva were investigated. METHODS: In a four-way crossover trial, 10 healthy volunteers received 1000 mg ER twice a day, 500 mg CL twice a day, 150 mg RO twice a day and 500 mg AZ every day over a period of 3 days. In a second trial, 10 healthy volunteers received 500 mg DI every day over a period of 5 days. Concentrations of these antibiotics were measured in serum, urine, saliva and PMNs by high-performance liquid chromatography (HPLC) on days 1 and 3 in the first trial and on days 1 and 5 in the second trial. RESULTS: We found considerable differences in the pharmacokinetics, not only in serum, but also in PMNs and saliva. All substances except RO exhibited higher concentrations in PMNs than in serum, indicating excellent intraphagocytic distribution. In contrast, concentrations in saliva were lower than those measured in serum, with the exception of AZ. ER is characterized by low serum concentrations and moderate concentrations in saliva and PMNs. CL reached considerable concentrations in serum, saliva and PMNs. RO achieved the highest serum levels, but concentrations in saliva and in PMNs were below the detection limit. In contrast, AZ and DI yielded the lowest serum concentrations and the highest saliva and PMN concentrations. CONCLUSIONS: Our data emphasize the importance of tissue distribution, in addition to serum kinetics, in evaluating the pharmacokinetic profiles of antibiotics.