Intracellular time course, pharmacokinetics, and biodistribution of isoniazid and rifabutin following pulmonary delivery of inhalable microparticles to mice

Intracellular concentrations of isoniazid and rifabutin resulting from administration of inhalable microparticles of these drugs to phorbol-differentiated THP-1 cells and the pharmacokinetics and biodistribution of these drugs upon inhalation of microparticles or intravenous administration of free drugs to mice were investigated. In cultured cells, both microparticles and dissolved drugs established peak concentrations of isoniazid (~1.4 and 1.1 μg/10⁶ cells) and rifabutin (~2 μg/ml and ~1.4 μg/10⁶ cells) within 10 min. Microparticles maintained the intracellular concentration of isoniazid for 24 h and rifabutin for 96 h, whereas dissolved drugs did not. The following pharmacokinetic parameters were calculated using WinNonlin from samples obtained after inhalation using an in-house apparatus (figures in parentheses refer to parameters obtained after intravenous administration of an equivalent amount, i.e., 100 μg of either drug, to parallel groups): isoniazid, serum half-life (t_1/2) = 18.63 ± 5.89 h (3.91 ± 1.06 h), maximum concentration in serum (C_max) = 2.37 ± 0.23 μg·ml⁻¹ (3.24 ± 0.57 μg·ml⁻¹), area under the concentration-time curve from 0 to 24 h (AUC_0-24) = 55.34 ± 13.72 μg/ml⁻¹ h⁻¹ (16.64 ± 1.80 μg/ml⁻¹ h⁻¹), and clearance (CL) = 63.90 ± 13.32 ml·h⁻¹ (4.43 ± 1.85 ml·h⁻¹); rifabutin, t_1/2 = 119.49 ± 29.62 h (20.18 ± 4.02 h), C_max = 1.59 ± 0.01 μg·ml⁻¹ (3.47 ± 0.33 μg·ml⁻¹), AUC_0-96 = 109.35 ± 14.78 μg/ml⁻¹ h⁻¹ (90.82 ± 7.46 μg/ml⁻¹ h⁻¹), and CL = 11.68 ± 7.00 ml·h⁻¹ (1.03 ± 0.11 ml·h⁻¹). Drug targeting to the lungs in general and alveolar macrophages in particular was observed. It was concluded that inhaled microparticles can reduce dose frequency and improve the pharmacologic index of the drug combination.     

Reversal of Secondary Hyperparathyroidism by Cimetidine in Chronically Uremic Dogs

Chronic cimetidine therapy has been shown to suppress circulating concentrations of immunoreactive parathyroid hormone (iPTH) in hemodialysis patients. To evaluate the long-term metabolic effects of cimetidine treatment, we studied seven chronically uremic dogs for 20 wk. The dogs were studied under metabolic conditions before, during, and after cimetidine therapy. iPTH fell progressively in the five treated dogs from 536±70 μleq/ml (mean±SE) (nl < 100 μleq/ml) before treatment to 291±25 μleq/ml at 12 wk (P < 0.001) and 157±32 μleq/ml at 20 wk (P < 0.001). The control dogs showed no consistent change in iPTH. The fall in iPTH was not associated with a change in serum ionized calcium. However, serum phosphorus decreased from 5.7±0.9 mg/dl to 3.4±0.2 mg/dl by the 20th wk (P < 0.05). By contrast, the serum concentration of 1,25-dihydroxycholecalciferol increased in all treated dogs from 33.4±4.3 pg/ml to 51.8±2.4 pg/ml during treatment (P < 0.01). Calcium balance was negative in all seven dogs before cimetidine (−347±84 mg/72 h) and remained so in the control dogs; it became positive in the five treated dogs after 12 wk (1,141±409 mg/72 h) (P < 0.05). Phosphorus balance, 24-h fractional phosphate excretion, and creatinine clearance remained unchanged. Pooled samples of serum obtained during the control and 20th wk of therapy were fractionated by gel filtration and the eluates assayed for immunoreactivity. The decrease in iPTH was associated with a decrease in all the immunoreactive species, indicating suppression of parathyroid gland secretion.     

Renal Sodium- and Potassium-Activated Adenosine Triphosphatase and Sodium Reabsorption in the Hypothyroid Rat

The relationship between net tubular reabsorption of sodium and renal microsomal sodium- and potassium-activated adenosine triphosphatase (Na-K-ATPase) was evaluated in hypothyroid and hyperthyroid rats and in age-matched euthyroid controls. Tubular sodium reabsorption per gram of kidney was lower in thyroidectomized rats than in controls (186±14 vs. 246±12 μeq/min; P < 0.005) and was accompanied by a quantitatively similar reduction in Na-K-ATPase specific activity (49.4±2.4 vs. 65.8±2.3 μmol inorganic phosphate (P_t)/mg protein per h; P < 0.001). This decrement was present in both cortex and outer medulla, and was limited to Na-K-ATPase since other representative enzymes not involved in sodium transport (magnesium-dependent adenosine triphosphatase [Mg-ATPase], glucose-6-phosphatase, 5′-nucleotidase) remained unchanged or increased in the hypothyroid animals. Conversely, Na-K-ATPase rose when sodium reabsorption increased in euthyroid rats treated with triiodothyronine.     

Effect of Biliary Diversion on Rat Mesenteric Lymph Apolipoprotein-I and High Density Lipoprotein

The effect of biliary diversion on intestinal apolipoprotein (apoA)-I and high density lipoprotein formation was studied in mesenteric lymph fistula rats. Bile diversion was produced by an exteriorized catheter that allowed interruption and reconstitution of the enterohepatic circulation. Bile diversion reduced lymph cholesterol output from 0.47±0.05 μmol/h to 0.17±0.03 μmol/h (P < 0.025), and lymph triglyceride output from 3.6±0.3μmol/h to 0.6±0.05 μmol/h (P < 0.025) after 24 h. This was due to depletion of lymph chylomicrons and very low density lipoprotein (VLDL). Despite the reduced lipid outputs, lymph apoA-I output was maintained during biliary diversion (basal: 119±15 μg/h; diverted 140±20 μg/h, n = 12). During biliary diversion, high density lipoprotein (HDL) were maintained in mesenteric lymph as shown by lipoprotein and immunoelectrophoresis. Bile diversion altered the lipid composition of lymph HDL. Bile-diverted lymph HDL was depleted in total cholesterol and has a greater phospholipid/cholesterol ester ratio than basal lymph HDL. Lymph HDL contained discoidal particles when examined by negative stain electron microscopy. Bile diversion was associated with a reduction in the size of discoidal HDL particles (basal, nondiverted, 165±7Å (n = 112) compared with diverted 126±5Å (n = 98, P < 0.025). Experiments were then carried out to determine the source of the apoA-I and HDL found in lymph from bile-diverted animals. The transfer of HDL from plasma into lymph was determined by the intravenous infusion of ¹²⁵I-apoA-I labeled HDL into lymph fistula rats. In both nonbile-diverted and diverted rats, the specific activity of apoA-I in the HDL fraction of lymph was 23% of the specific activity of apoA-I in plasma HDL, indicating that the major portion (75%) of mesenteric lymph apoA-I did not come from plasma filtration. In other experiments the intraduodenal infusion of [³H]leucine to bile fistula, lymph fistula rats resulted in relative fivefold increase in the specific activity in apoA-I in lymph HDL when compared with the specific activity of apoA-I in plasma HDL from the same animal. We conclude that intestinal apoA-I secretion is maintained during biliary diversion and that synthesis of this apoprotein occurs in the absence of chylomicron formation. We also conclude that discoidal HDL are present in mesenteric lymph despite reduced triglyceride absorption and secretion into lymph.     

Abnormal Carbohydrate Metabolism in Chronic Renal Failure: THE POTENTIAL ROLE OF ACCELERATED GLUCOSE PRODUCTION, INCREASED GLUCONEOGENESIS, AND IMPAIRED GLUCOSE DISPOSAL

To delineate the potential role of disordered glucose and glucose-precursor kinetics in the abnormal carbohydrate metabolism of chronic renal failure, alanine and glucose production and utilization and gluconeogenesis from alanine were studied in patients with chronic compensated renal insufficiency and in normal volunteers. With simultaneous primed injection-continuous infusions of radiolabeled alanine and glucose, rates of metabolite turnover and precursor-product interrelationships were calculated from the plateau portion of the appropriate specific activity curves. All subjects were studied in the postabsorption state. In 13 patients with chronic renal failure (creatinine = 10.7±1.2 mg/100 ml; mean±SEM), glucose turnover was found to be 1,035±99.3 μmol/min. This rate was increased 56% (P = 0.003) over that observed in control subjects (664±33.5 μmol/min). Alanine turnover was 474±96.0 μmol/min in azotemic patients. This rate was 191% greater (P = 0.007) than the rate determined in control subjects (163±19.4 μmol/min). Gluconeogenesis from alanine and the percent of glucose production contributed by gluconeogenesis from alanine were increased in patients with chronic renal failure (192% and 169%, respectively) as compared to controls (P < 0.05 for each). Alanine utilization for gluconeogenesis was increased from 40.2±3.86 μmol/min in control subjects to 143±39.0 μmol/min in azotemic patients (P < 0.05). The percent of alanine utilization accounted for by gluconeogenesis was not altered in chronic renal insufficiency. In nondiabetic azotemic subjects, mean fasting glucose and immunoreactive insulin levels were increased 24.3% (P = 0.005) and 130% (P = 0.046), respectively.     

Dopexamine can attenuate the inflammatory response and protect against organ injury in the absence of significant effects on hemodynamics or regional microvascular flow

Introduction

The effects of dopexamine, a β2-agonist, on perioperative and sepsis-related hemodynamic, microvascular, immune, and organ dysfunction are controversial and poorly understood. We investigated these effects in a rodent model of laparotomy and endotoxemia.

Methods

In two experiments, 80 male Wistar rats underwent laparotomy. In 64 rats, this was followed by administration of endotoxin; the remainder (16) underwent sham endotoxemia. Endotoxemic animals received either dopexamine at 0.5, 1, or 2 μg/kg/min or 0.9% saline vehicle (controls) as resuscitation fluid. The effects of dopexamine on global hemodynamics, mesenteric regional microvascular flow, renal and hepatic function and immune activation were evaluated.

Results

Endotoxin administration was associated with a systemic inflammatory response (increased plasma levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-10, as well as cell-adhesion molecules CD11a and CD11b), and increased pulmonary myeloperoxidase (MPO) activity (indicating pulmonary leukocyte infiltration), whereas biochemical changes demonstrated lactic acidosis with significant renal and hepatic injury. Dopexamine administration was associated with less-severe lactic acidosis (pooled dopexamine versus controls, (lactate, 2.2 mM ± 0.2 mM versus 4.0 mM ± 0.5 mM; P < 0.001) and reductions in the systemic inflammatory response (pooled dopexamine versus control, 4 hour (TNF-α): 324 pg/ml ± 93 pg/ml versus 97 pg/ml ± 14 pg/ml, p < 0.01), pulmonary myeloperoxidase (MPO) activity, and hepatic and renal injury (pooled dopexamine versus control (ALT): 81 IU/L ± 4 IU/L versus 138 IU/L ± 25 IU/L; P < 0.05; (creatinine): 49.4 μM ± 3.9 μM versus 76.2 μM ± 9.8 μM; P < 0.005). However, in this study, clinically relevant doses of dopexamine were not associated with clinically significant changes in MAP, CI, or gut regional microvascular flow.

Conclusions

In this model, dopexamine can attenuate the systemic inflammatory response, reduce tissue leukocyte infiltration, and protect against organ injury at doses that do not alter global hemodynamics or regional microvascular flow. These findings suggest that immunomodulatory effects of catecholamines may be clinically significant when used in critically ill surgical patients and are independent of their hemodynamic actions.     

Role of Thromboxane and Prostacyclin in Pulmonary Vasomotor Changes after Endotoxin in Dogs

Cyclooxygenase inhibitors prevent the pulmonary vasomotor changes in response to low-dose endotoxin. We, therefore, explored the role of two highly vasoactive prostanoids, thromboxane A₂, a vasoconstrictor, and prostacyclin, a vasodilator, in the transient pulmonary vasoconstriction and subsequent loss of alveolar hypoxis vasoconstriction (AHPV) that follows endotoxin. AHPV was tested in the dog with a double-lumened endotracheal tube allowing ventilation of one lung with nitrogen as a hypoxic challenge while the other lung was ventilated with oxygen to maintain systemic oxygenation. Relative distribution of perfusion to the two lungs was assessed with intravenous ¹³³Xe and external scintillation detectors. The stable metabolites of thromboxane and prostacyclin, i.e., thromboxane B₂ and 6-keto-prostaglandin F_1α were measured in plasma with radioimmunoassay. 15 μg/kg i.v. of endotoxin induced no rise in pulmonary vascular resistance (PVR), but prevented AHPV so that the initial 33% (±2 SEM) decrease in perfusion to the hypoxic lung became only a 2% (±1) decrease. Circulating levels of thromboxane and prostacyclin concurrently rose (P < 0.01) from nondetectable levels to 380 pg/ml (±40) and 360 pg/ml (±130). 150 μg/kg of endotoxin induced a transient rise in PVR from 4.09 to 9.00 mm Hg/liter per min in association (r = 0.89, P < 0.01) with a sharp rise in thromboxane levels to 4,460 pg/ml (±1,350) whereas prostacyclin levels were elevated less markedly to 550 pg/ml (±400). Prostaglandin F_2α, another vasoconstrictor, was not elevated. 30 min after endotoxin when PVR was again base line and AHPV lost, thromboxane fell significantly (P < 0.01) to 2,200 pg/ml (±1,100) whereas prostacyclin remained elevated at 360 pg/ml (±135), a level similar to that seen when 15 μg/kg of endotoxin induced loss of AHPV. Indomethacin prevented the rise in thromboxane and prostacyclin after endotoxin as well as the changes in pulmonary vasomotor tone. Thus, a complex interaction between thromboxane and prostacyclin is involved in the pulmonary vasomotor response to low-dose endotoxin.     

Stimulation of Monovalent Cation Active Transport by Low Concentrations of Cardiac Glycosides: ROLE OF CATECHOLAMINES

The stimulatory effect of low concentrations of ouabain on the Na-K pump in isolated guinea pig left atria was studied in vitro by assessing active transport of the K⁺ analog Rb⁺. Active transport of Rb⁺ was stimulated 20±8% (SEM, P < 0.05) above control values by 3 nM ouabain, but was inhibited by concentrations >10 nM. Preincubation with the β-adrenergic antagonist propranolol (1 μM) completely blocked stimulation of active transport of Rb⁺ by 3 nM ouabain. Norepinephrine, 10 nM, increased Rb⁺ active transport 29±10% (P < 0.02) above control values. The β-adrenergic agonist l-isoproterenol, 10 nM, increased active transport of Rb⁺ by 33±10% (P < 0.01) above control levels. This stimulatory effect was abolished if tissues were first exposed to propranolol. Tyramine (0.1 μM), a stimulator of endogenous catecholamine release, increased active transport of Rb⁺ 26±12% (P < 0.05) above control values. Rb⁺ active transport was not significantly changed when left atrial tissues were incubated with α-adrenergic agonists or antagonists. Ouabain stimulation of Rb⁺ active transport was prevented by in vivo depletion of myocardial endogenous catecholamines by either reserpine or 6-hydroxydopamine. These findings indicated that in myocardial tissue, Na-K pump stimulation by low concentrations of ouabain is mediated at least in part through β-adrenergic effects of endogenous catecholamines.     

Levosimendan attenuates multiple organ injury and improves survival in peritonitis-induced septic shock: studies in a rat model

Introduction

The aim of this study was to investigate the effects of levosimendan on rodent septic shock induced by cecal ligation and puncture (CLP).

Methods

Three hours after peritonitis-induced sepsis, male Wistar rats were randomly assigned to receive an intravenous infusion of levosimendan (1.2 μg/kg/min for 10 min and then 0.3 μg/kg/min for 6 h) or an equivalent volume of saline and vehicle (5% dextrose) solution.

Results

The levosimendan-treated CLP animals had significantly higher arterial pressure and lower biochemical indices of liver and kidney dysfunction compared to the CLP animals (P < 0.05). Plasma interleukin-1β, nitric oxide and organ superoxide levels in the levosimendan-treated CLP group were less than those in CLP rats treated with vehicle (P < 0.05). In addition, the inducible nitric oxide synthase (iNOS) in lung and caspase-3 expressions in spleen were significantly lower in the levosimendan-treated CLP group (P < 0.05). The administration of CLP rats with levosimendan was associated with significantly higher survival (61.9% vs. 40% at 18 h after CLP, P < 0.05). At postmortem examination, the histological changes and neutrophil filtration index in liver and lung were significantly attenuated in the levosimendan-treated CLP group (vs. CLP group, P < 0.05).

Conclusions

In this clinically relevant model of septic shock induced by fecal peritonitis, the administration of levosimendan had beneficial effects on haemodynamic variables, liver and kidney dysfunction, and metabolic acidosis. (1) Lower levels of interleukin-1β, nitric oxide and superoxide, (2) attenuation of iNOS and caspase-3 expressions, and (3) decreases of neutrophil infiltration by levosimendan in peritonitis-induced sepsis animals suggest that anti-inflammation and anti-apoptosis effects of levosimendan contribute to prolonged survival.     

Evidence for a Role of Opioid Peptides in the Release of Arginine Vasopressin in the Conscious Rat

Recent reports have suggested that opioid peptides may be involved in renal water excretion. The present in vivo experiments, therefore, were undertaken to determine the effect of opioid peptides on the osmotic and nonosmotic release of arginine vasopressin (AVP) in the conscious rat. Experimental animals were infused intravenously with naloxone (20 μg/kg per min) or oxilorphan (40 μg/kg per min), chemically dissimilar opioid antagonists. Control rats were infused with normal saline, the vehicle for the opioid antagonists. In all three groups the osmotic release of AVP was examined during an acute hypertonic saline (3%) infusion (2 ml/100 g body wt). The antidiuresis following the hypertonic saline infusion was significantly attenuated in naloxone- and oxilorphan-treated rats, as the peak urinary osmolality (Uosm) rose to 581.4±22.4 and 558.2±27.6 mosmol/kg H₂O in naloxone- and oxilorphan-treated rats as compared with the value in control rats of 735.3±24.2 mosmol/kg H₂O (both P < 0.001 vs. control). At the same time the plasma AVP levels of 5.4±1.3 and 5.2±1.1 pg/ml in naloxone- and oxilorphan-treated rats, respectively, were significantly lower than the plasma AVP in control rats of 16.9±2.5 pg/ml (P < 0.001). In another three groups of rats the nonosmotic release of AVP was examined during hypovolemia induced by intraperitoneal 6% dextran (1.8 ml/100 g body wt). Following intraperitoneal administration of dextran the peak Uosm of 703.0±87.8 and 734.8±99.1 mosmol/kg H₂O in naloxone- and oxilorphan-treated rats, respectively, was significantly less than the value in control rats of 1,169.3±135.5 mosmol/kg H₂O (both P < 0.02 vs. control). A comparable decrease in blood volume of 13% occurred in all three groups of animals. During the dextran administration plasma AVP levels in naloxone- and oxilorphan-treated rats increased to 4.3±1.0 and 6.0±2.0 pg/ml, respectively; both of these values were significantly lower than the plasma AVP of 12.9±1.4 pg/ml in control rats (P < 0.02). The effect of opioid antagonists to impair the osmotic and nonosmotic release of AVP occurred in the absence of differences in mean arterial pressure, glomerular filtration rate and the renal response to AVP. These results, therefore, indicate that opioid peptides are involved in renal water excretion primarily by modulating the central release of AVP.     

Allergic Eosinophil-rich Inflammation Develops in Lungs and Airways of B Cell–deficient Mice

Immunoglobulins (Ig), particularly IgE, are believed to be crucially involved in the pathogenesis of asthma and, equally, in allergic models of the disease. To validate this paradigm we examined homozygous mutant C57BL/6 mice, which are B cell deficient, lacking all Ig. Mice were immunized intraperitoneally with 10 μg ovalbumin (OVA) plus alum, followed by daily (day 14–20) 30 min exposures to OVA aerosol (OVA/OVA group). Three control groups were run: OVA intraperitoneally plus saline (SAL) aerosol (OVA/SAL group); saline intraperitoneally plus saline aerosol; saline intraperitoneally plus OVA aerosol (n = 6–7). Lung and large airway tissues obtained 24 h after the last OVA or SAL exposure were examined by light microscopy and transmission electron microscopy (TEM). The Ig-deficient mice receiving OVA/ OVA treatment had swollen and discolored lungs and exhibited marked eosinophilia both in large airway subepithelial tissue (49.2 ± 12.0 cells/mm basement membrane [BM] versus OVA/ SAL control 1.2 ± 0.3 cells/mm BM; P <0.001), and perivascularly and peribronchially in the lung (49.3 ± 9.0 cells/unit area versus OVA/SAL control 2.6 ± 0.6 cells/unit area; P <0.001). The eosinophilia extended to the regional lymph nodes. TEM confirmed the subepithelial and perivascular localization of eosinophils. Mucus cells in large airway epithelium increased from 1.5 ± 0.8 (OVA/SAL mice) to 39.5 ± 5.7 cells/mm BM in OVA/OVA treated mice (P <0.001). OVA/SAL mice never differed from the other control groups. Corresponding experiments in wild-type mice (n = 6–7 in each group) showed qualitatively similar but less pronounced eosinophil and mucus cell changes. Macrophages and CD4⁺ T cells increased in lungs of all OVA/OVA-treated mice. Mast cell number did not differ but degranulation was detected only in OVA/OVA-treated wild-type mice. Immunization to OVA followed by OVA challenges thus cause eosinophil-rich inflammation in airways and lungs of mice without involvement of B cells and Ig.     

Indomethacin is a Placental Vasodilator in the Dog: THE EFFECT OF PROSTAGLANDIN INHIBITION

The effect of 8 mg/kg of indomethacin on uterine blood flow, prostaglandin production, and intraamniotic fluid pressure was examined in late pregnant dogs. Uterine blood flow was measured with 15 μm radiolabeled microspheres. Because we found that a significant percentage of the microspheres shunted through the placental circulation into the lungs, we calculated placental blood flow by adding the shunted microspheres through the placenta to the nonshunted microspheres in the placenta. Total uterine blood flow significantly increased from 271±69 ml/min during control period to 371±72 ml/min (P < 0.01) 30 min after indomethacin. This increase was attributable to the change in blood flow to the placental circulation (222±58 to 325±63 ml/min; P < 0.01). Associated with these hemodynamic changes we found an almost complete suppression of uterine prostaglandin E₂ production (1,654±305 to 51±25 pg/ml; P < 0.01) as measured by gas chromatography-mass spectrometry. In addition, we found that indomethacin treatment resulted in uterine relaxation as measured by intraamniotic fluid pressure changes (11.2±1.3 mm Hg to 8.5±1.2 mm Hg; P < 0.001).     

In Vitro Activities of Cefminox against Anaerobic Bacteria Compared with Those of Nine Other Compounds

The agar dilution MIC method was used to test the activity of cefminox, a β-lactamase-stable cephamycin, compared with those of cefoxitin, cefotetan, moxalactam, ceftizoxime, cefotiam, cefamandole, cefoperazone, clindamycin, and metronidazole against 357 anaerobes. Overall, cefminox was the most active β-lactam, with an MIC at which 50% of isolates are inhibited (MIC₅₀) of 1.0 μg/ml and an MIC₉₀ of 16.0 μg/ml. Other β-lactams were less active, with respective MIC₅₀s and MIC₉₀s of 2.0 and 64.0 μg/ml for cefoxitin, 2.0 and 128.0 μg/ml for cefotetan, 2.0 and 64.0 μg/ml for moxalactam, 4.0 and >128.0 μg/ml for ceftizoxime, 16.0 and >128.0 μg/ml for cefotiam, 8.0 and >128.0 μg/ml for cefamandole, and 4.0 and 128.0 μg/ml for cefoperazone. The clindamycin MIC₅₀ and MIC₉₀ were 0.5 and 8.0 μg/ml, respectively, and the metronidazole MIC₅₀ and MIC₉₀ were 1.0 and 4.0 μg/ml, respectively. Cefminox was especially active against Bacteroides fragilis (MIC₉₀, 2.0 μg/ml), Bacteroides thetaiotaomicron (MIC₉₀, 4.0 μg/ml), fusobacteria (MIC₉₀, 1.0 μg/ml), peptostreptococci (MIC₉₀, 2.0 μg/ml), and clostridia, including Clostridium difficile (MIC₉₀, 2.0 μg/ml). Time-kill studies performed with six representative anaerobic species revealed that at the MIC all compounds except ceftizoxime were bactericidal (99.9% killing) against all strains after 48 h. At 24 h, only cefminox and cefoxitin at 4× the MIC and cefoperazone at 8× the MIC were bactericidal against all strains. After 12 h, at the MIC all compounds except moxalactam, ceftizoxime, cefotiam, cefamandole, clindamycin, and metronidazole gave 90% killing of all strains. After 3 h, cefminox at 2× the MIC produced the most rapid effect, with 90% killing of all strains.     

Chronic Hyperglycemia Reduces Surface Active Material Flux in Tracheal Fluid of Fetal Lambs

I tested the hypothesis that chronic hyperglycemia alters fetal lung maturation by continuous infusion of glucose (14±2 mg/kg per min, mean±SE) from 112 up to 145 d gestation into six chronically catheterized fetal lambs from which tracheal fluid could be collected. Serum glucose levels (32±2 mg/dl) and serum insulin levels (38±4 μU/ml) in these glucose-treated fetuses were significantly higher than serum glucose levels (18±2 mg/dl, P < 0.001) and serum insulin levels (12±3 μU/ml, P < 0.001) in six chronically catheterized control fetuses of the same gestational ages. Glucose infusion to the fetuses did not alter maternal serum glucose (60±3 mg/dl) or serum insulin levels (35±5 μU/ml). Arterial blood gases (pH 7.34±0.01, Po₂ 24.3±0.5 mmHg, Pco₂ 41.5±0.9 mmHg), oxygen saturation (73±2%), hematocrit (31±1%), and tracheal fluid flow (2.4±0.1 ml/g per h) in the glucose-treated fetuses were not significantly different from controls. Among the control fetuses, surface active material (SAM) began to appear in tracheal fluid at 123 d gestation and was present in all six fetuses by 129 d gestation, whereas SAM did not appear at all in tracheal fluid of four of the glucose-treated fetuses, and appeared in two at low levels after 142 d gestation. SAM flux in the glucose-treated fetuses (<1 μg/g per h) was statistically lower than SAM flux in the control fetuses (60±9 μg/kg per h, P < 0.001). Between 130 and 140 d gestation, tracheal fluid phospholipid content rose fourfold, mixed lecithin content rose ninefold, disaturated phosphatidylcholine content rose fourfold in the control fetuses, whereas little or no increase in these measurements occurred in the glucose-treated fetuses (all differences significant). I conclude that chronic hyperglycemia with secondary hyperinsulinemia reduces SAM flux in tracheal fluid of fetal lambs. The reduction in SAM flux is attributed to low surface active phospholipid content of the SAM. A similar mechanism may operate in utero to cause respiratory distress in infants of diabetic mothers whose maternal glucose homeostasis is poorly controlled.     

Epinephrine-induced Insulin Resistance in Man

Endogenous release of epinephrine after stress as well as exogenous epinephrine infusion are known to result in impaired glucose tolerance. Previous studies of man and animals have demonstrated that this effect of epinephrine results from inhibition of insulin secretion and augmentation of hepatic glucose production. However, the effect of epinephrine on tissue sensitivity to insulin, and the relative contributions of peripheral vs. hepatic resistance to impaired insulin action, have not been defined. Nine young normal-weight subjects were studied with the insulin clamp technique. Plasma insulin was raised by ~100 μU/ml while plasma glucose concentration was maintained at basal levels by a variable glucose infusion. Under these conditions of euglycemia, the amount of glucose metabolized equals the glucose infusion rate and is a measure of tissue sensitivity to insulin. Subjects received four studies: (a) insulin (42.6 mU/m²·min), (b) insulin plus epinephrine (0.05 μg/kg·min), (c) insulin plus epinephrine plus propranolol (1.43 μg/kg·min), and (d) insulin plus propranolol. During insulin administration alone, glucose metabolism averaged 5.49±0.58 mg/kg·min. When epinephrine was infused with insulin, glucose metabolism fell by 41% to 3.26 mg/kg·min (P < 0.001). After insulin alone, hepatic glucose production declined by 92% to 0.16±0.08 mg/kg·min. Addition of epinephrine was associated with a delayed and incomplete suppression of glucose production (P < 0.01) despite plasma insulin levels >100 μU/ml. When propranolol was administered with epinephrine, total glucose metabolism was restored to control values and hepatic glucose production suppressed normally. Propranolol alone had no effect on insulin-mediated glucose metabolism. These results indicate that epinephrine, acting primarily through a β-adrenergic receptor, markedly impairs tissue sensitivity to an increase in plasma insulin levels, and that this effect results from both peripheral and hepatic resistance to the action of insulin.     

Direct Radioimmunoassay of Nuclear 3,5,3′ Triiodothyronine in Rat Anterior Pituitary

Previous tracer studies have suggested that 5′-monodeiodination of l-thyroxine (T₄) in anterior pituitary may contribute a substantial portion of specifically bound nuclear 3,5,3′ l-triiodothyronine (T₃) in this tissue in rats. To evaluate this possibility, a radioimmunoassay for nuclear T₃ in individual anterior pituitaries was developed. Animals received [¹²⁵I]T₃ 60 min before removal of the anterior pituitary and isolation of the nuclei by differential centrifugation. This allowed calculation of the nuclear:serum T₃ ratio and comparison of expected with measured T₃. T₃ was extracted in ethanol, dried, and reconstituted in assay buffer. In untreated hypothyroid rats, anterior pituitary nuclear T₃ was 0.18 ± 0.06 pg/μg DNA which was 0.13 pg/μg DNA greater than expected from the serum T₃ concentration and the pituitary nuclear:serum [¹²⁵I]T₃ ratio. In 10 hypothyroid rats given a single bolus of 400 ng T₃/100 g body wt., the nuclear T₃ by radioimmunoassay was 1.0 ± 0.06 pg/μg DNA, whereas that expected from the T₃ specific activity calculations was 0.85 pg/μg DNA (P < 0.025). Serum T₄ concentrations in these rats were < 0.25 μg/dl but the nuclear T₃ derived from as little as 0.2 μg/dl T₄ could explain a large portion of these small discrepancies between observed and measured nuclear T₃. In 29 normal rats, anterior pituitary nuclear T₃ was 0.63±0.04 pg/μg DNA, whereas that expected from the serum T₃ concentration (55±2 ng/dl) was 0.23±0.02 pg/μg DNA (P < 0.001). Total pituitary T₃ based on this measurement was 92±6 pg. Because the maximal nuclear binding capacity for T₃ in rat anterior pituitary is 0.77 pg/μg DNA, these results suggest there is 82% occupancy of these nuclear receptors. The requirement for normal serum concentrations of both T₄ and T₃ to achieve normal nuclear T₃ saturation in anterior pituitary is in marked contrast to the situation in liver, kidney, and heart muscle which appear to require only a normal serum T₃. As a consequence, the anterior pituitary can monitor both serum T₄ and T₃ and respond appropriately to changes in their concentrations.     

Comparative Pharmacology of Cefaclor and Cephalexin

Two cephalosporin antibiotics, cefaclor and cephalexin, were administered orally to healthy, adult male volunteers for comparison of their pharmacological properties. In doses of 250 mg orally, cefaclor produced a peak serum concentration of 6.01 ± 0.55 (standard deviation [SD]) μg/ml compared with 9.43 ± 2.36 μg/ml for cephalexin (P < 0.01). The half-lives were 0.58 ± 0.07 (SD) h and 0.80 ± 0.12 (SD) h, and elimination constants were 1.22 ± 0.15 and 0.88 ± 0.13 h⁻¹ for cefaclor and cephalexin, respectively (P < 0.001). Neither drug showed accumulation over the dosing period, and both were well tolerated.     

Efficacy of Liposomal Bismuth-Ethanedithiol-Loaded Tobramycin after Intratracheal Administration in Rats with Pulmonary Pseudomonas aeruginosa Infection

We sought to investigate alterations in quorum-sensing signal molecule N-acyl homoserine lactone secretion and in the release of Pseudomonas aeruginosa virulence factors, as well as the in vivo antimicrobial activity of bismuth-ethanedithiol incorporated into a liposome-loaded tobramycin formulation (LipoBiEDT-TOB) administered to rats chronically infected with P. aeruginosa. The quorum-sensing signal molecule N-acyl homoserine lactone was monitored by using a biosensor organism. P. aeruginosa virulence factors were assessed spectrophotometrically. An agar beads model of chronic Pseudomonas lung infection in rats was used to evaluate the efficacy of the liposomal formulation in the reduction of bacterial count. The levels of active tobramycin in the lungs and the kidneys were evaluated by microbiological assay. LipoBiEDT-TOB was effective in disrupting both quorum-sensing signal molecules N-3-oxo-dodeccanoylhomoserine lactone and N-butanoylhomoserine lactone, as well as significantly (P < 0.05) reducing lipase, chitinase, and protease production. At 24 h after 3 treatments, the CFU counts in lungs of animals treated with LipoBiEDT-TOB were of 3 log₁₀ CFU/lung, comparated to 7.4 and 4.7 log₁₀ CFU/lung, respectively, in untreated lungs and in lungs treated with free antibiotic. The antibiotic concentration after the last dose of LipoBiEDT-TOB was 25.1 μg/lung, while no tobramycin was detected in the kidneys. As for the free antibiotic, we found 6.5 μg/kidney but could not detect any tobramycin in the lungs. Taken together, LipoBiEDT-TOB reduced the production of quorum-sensing molecules and virulence factors and could highly improve the management of chronic pulmonary infection in cystic fibrosis patients.     

Relative Inactivation by Staphylococcus aureus of Eight Cephalosporin Antibiotics

These studies extend the recent observation that cefazolin is inactivated to a greater extent than cephaloridine by some strains of penicillinase-producing Staphylococcus aureus, whereas cephalothin undergoes little if any inactivation. In Mueller-Hinton broth (inoculum, 3 × 10⁶) 100 recently isolated strains had minimal inhibitory concentrations (MICs) ≤ 2 μg/ml for cephalothin and cephaloridine, whereas in Trypticase soy broth (TSB) 50% had MICs > 2 μg/ml and 10% (designated “resistant” strains) were >8 μg/ml for cephaloridine but remained ≤2 μg/ml for cephalothin. A large inoculum (3 × 10⁷) of strains with high MICs in TSB almost completely inactivated 50 μg of cefazolin per ml in 6 h, with progressively less inactivation, in the following order, of cephaloridine, cephalexin, cephradine, cephapirin, and cefamandole; cefoxitin and cephalothin underwent little if any inactivation. The greater inactivation in TSB than in Mueller-Hinton broth appeared to be due to a greater production of β-lactamases by each colony-forming unit, since the inoculum size in the two broths was not significantly different. In contrast, “susceptible” strains (MICs ≤ 2 μg/ml in both broths) inactivated cephaloridine more than cefazolin, and equal amounts of powdered bacterial extracts confirmed the fact that qualitatively different β-lactamases were produced by the susceptible and resistant strains. Disk diffusion tests were unreliable in separating the two groups of staphylococci. The clinical significance of inactivation by strains with high MICs is not known but, unless susceptibility can be clearly established, cephalothin appears preferable for severe staphylococcal infections, since it undergoes little if any inactivation by any strains of staphylococci.     

Moxifloxacin (BAY12-8039), a New 8-Methoxyquinolone, Is Active in a Mouse Model of Tuberculosis

Moxifloxacin (BAY12-8039) is a new 8-methoxyquinolone shown to be active against Mycobacterium tuberculosis in vitro. We tested moxifloxacin for activity in mice against M. tuberculosis CSU93, a highly virulent, recently isolated clinical strain. The MIC of moxifloxacin for the CSU93 strain was 0.25 μg/ml. The serum moxifloxacin concentration after oral administration in mice peaked within 0.25 h, reaching 7.8 μg/ml with doses of 100 mg/kg of body weight; the maximum concentration and the analysis of the area under the concentration-time curve revealed dose dependency. When mice were infected with a sublethal inoculum of mycobacteria and then treated with moxifloxacin at 100 mg/kg per day for 8 weeks, the log₁₀ CFU counts in the organs of treated mice were significantly lower than those for the control group (0.6 ± 0.2 versus 5.6 ± 0.3 in the lungs and 1.5 ± 0.7 versus 4.9 ± 0.5 in the spleens, respectively; P < 0.001 in both organs). The effectiveness of moxifloxacin monotherapy was comparable to that seen in mice receiving isoniazid alone. Combination therapy with moxifloxacin plus isoniazid was superior to that with moxifloxacin or with isoniazid alone in reducing bacillary counts in the organs studied. Using a sensitive broth-passage subculture method, we demonstrated that 8 weeks of treatment with moxifloxacin (100 mg/kg per day) or with moxifloxacin plus isoniazid (100 mg/kg and 25 mg/kg, respectively, per day) sterilized the lungs in seven of eight and in eight of eight mice, respectively. Among surviving bacilli isolated from animals infected with a high-titer inoculum and treated for 7 weeks with low-dose moxifloxacin (20 mg/kg per day), breakthrough resistance to moxifloxacin was not observed. These results indicate that moxifloxacin is highly effective in reducing M. tuberculosis infection in mice and has activity comparable to that of isoniazid. Combination therapy with moxifloxacin and isoniazid was highly effective, suggesting that moxifloxacin may be useful in multiple-drug regimens for human tuberculosis.