Improvement of lipophilicity and membrane transport of cefuroxime using in vitro models.

Most beta-lactam antibiotics cannot be absorbed orally and, therefore, must be administered intravenously (i.v.) or intramuscularly (i.m.). Because of the obvious drawbacks of drug delivery by injection, the development of alternatives with enhanced oral bioavailability is receiving much attention in pharmaceutical research. Cefuroxime exhibiting significant advantages in the parental treatment of common infections, was used as model drug in the present study. The effect of the cationic absorption enhancers (four quaternary ammonium salts) on the lipophilicity of cefuroxime was investigated by means of the n-octanol/water system. The results on partitioning coefficients in the n-octanol/buffer system were confirmed using an in vitro transport model with artificial (dodecanol collodium membrane) and biological membranes (Charles-River guinea pig).     

Cefodizime. A review of its antibacterial activity, pharmacokinetic properties and therapeutic use.

Cefodizime is a third generation cephalosporin with a broad spectrum of antibacterial activity. Administered intravenously or intramuscularly, cefodizime 1 to 4 g daily for an average of 7 to 10 days produced clinical cure in 80 to 100% of patients (adults, elderly or children) with upper or lower respiratory tract infections or urinary tract infections, and in comparative trials cefodizime was as effective as other third generation cephalosporins. A single dose of cefodizime 1 or 2 g is also useful in treating lower urinary tract infections, particularly uncomplicated infections, with a rate of clinical success of 72 to 88%. Urogenital gonorrhoea, whether caused by beta-lactamase producing or non-beta-lactamase producing Neisseria gonorrhoeae, is very effectively treated by single dose therapy with intramuscular cefodizime 0.25 to 1 g (virtually 100% cured). Preliminary data from a small number of patients indicate that cefodizime may also be useful in the treatment of otitis media, sinusitis and gynaecological infections, and for the prophylaxis or treatment of surgical infections. The clinical efficacy of cefodizime in comparison with other third generation cephalosporins is superior to that predicted from in vitro results. This superior activity of cefodizime may be related to the relatively long elimination half-life of the drug or its ability to modify some functions of the immune system--a potentially important finding awaiting further investigation. Cefodizime is well tolerated and has a tolerability profile similar to other members of its class with systemic adverse events being primarily gastrointestinal or dermatological. Thus, limited comparative studies indicate cefodizime has the potential to become a useful alternative to current antimicrobial therapy for the treatment of a variety of infections. Cefodizime may be more convenient to administer than some other agents of its class as it may be given once or twice daily. While there are no trials comparing cefodizime to other third generation cephalosporins in immunosuppressed populations, preliminary information indicates cefodizime may be useful in this group.     

Progress in absorption enhancers based on tight junction

Absorption enhancers have been investigated since the 1960s, in order to assist the transfer of drugs across the paracellular space in the intestinal epithelium. However, few absorption enhancers are presently used clinically, due to the difficulty of developing enhancers with high specificity and low toxicity. Using high-throughput genomic techniques, new drug candidates such as, non-Lipinski molecules, peptides, antibodies and nucleic acids, are being discovered, so the need for oral drug delivery strategies using absorption enhancers is gaining importance. The key to addressing this issue is to understand the molecular mechanism of the paracellular route in epithelial cell sheets. Towards this end, basic research in cell biology has revealed the components that regulate the paracellular route, and how the transport of substances is regulated. Based on these findings, novel strategies for enhancing drug absorption have been proposed. In this article, the authors first survey the development of absorption enhancers, then outline recent progress in the cell biology of tight junctions, and finally discuss novel approaches for absorption enhancers based on these advances.     

Progress in absorption enhancers based on tight junction

Absorption enhancers have been investigated since the 1960s, in order to assist the transfer of drugs across the paracellular space in the intestinal epithelium. However, few absorption enhancers are presently used clinically, due to the difficulty of developing enhancers with high specificity and low toxicity. Using high-throughput genomic techniques, new drug candidates such as, non-Lipinski molecules, peptides, antibodies and nucleic acids, are being discovered, so the need for oral drug delivery strategies using absorption enhancers is gaining importance. The key to addressing this issue is to understand the molecular mechanism of the paracellular route in epithelial cell sheets. Towards this end, basic research in cell biology has revealed the components that regulate the paracellular route, and how the transport of substances is regulated. Based on these findings, novel strategies for enhancing drug absorption have been proposed. In this article, the authors first survey the development of absorption enhancers, then outline recent progress in the cell biology of tight junctions, and finally discuss novel approaches for absorption enhancers based on these advances.     

Distribution of cefodizime to exudate in the croton oil-induced rat granuloma pouch and its therapeutic effects on experimental infections in the pouch

Cefodizime was compared with cefotaxime (CTX) in regard to its distribution to an inflammatory site (exudate in croton oil-induced granuloma pouch) of rats and its therapeutic effects on experimental infections in such pouches after intravenous injection. Cefodizime levels in rat serum and pouch exudate were higher than those of CTX, and the former compound disappeared from the serum and pouch exudate far more slowly than the latter. In the tests for therapeutic effects, cefodizime showed almost the same degree of inhibitory activity as CTX against growth in pouch exudate of Escherichia coli Ec-7, Proteus mirabilis Pm-428, and Serratia marcescens Sm-390 for which the minimum inhibitory concentrations (MICs) of cefodizime were equal to or greater than the CTX values, and such activity of cefodizime lasted for a longer period than that of CTX. These results suggest that the above pharmacokinetic features of cefodizime compensate for its MICs against organisms displaying lower values for CTX.     

Effect of 1-alkyl- or 1-alkenylazacycloalkanone derivatives on the penetration of drugs with different lipophilicities through guinea pig skin

The percutaneous penetration-enhancing effects of 1-dodecyl- (azone), l-geranyl-, and 1-farnesylazacycloheptan-2-one were investigated using seven penetrants having a wide range of n-octanol-water partition coefficients. The penetration of the drugs from water vehicle (aqueous system) and ethanol vehicle (ethanolic system) through excised guinea pig skin was increased by pretreatment with the enhancers. Large enhancement was observed for the drugs, such as 5-fluorouracil and 6-mercaptopurine, with n-octanol-water partition coefficients of approximately unity. The penetration profiles were analyzed based on a one-layer skin model. Two parameters corresponding to the drug diffusivity and partitioning into the skin were obtained. In the aqueous system, the partitioning of drugs into the skin was increased by pretreatment with the enhancers. This led to an increase in drug penetration and accumulation in the skin; diffusivities were little affected. From these parameters, the drug amounts in the vehicle and the skin were well estimated for drugs having partition coefficients of less than 1. In the ethanolic system, the enhancement was far less than that observed in the aqueous system.     

In-vitro and in-vivo studies of cefpirom using bile salts as absorption enhancers

Cephalosporins have to be administered by injection because of the poor intestinal absorption of the orally delivered drugs. Because of the obvious drawbacks of drug delivery by injection, the development of alternatives with enhanced oral bioavailability is receiving much attention in pharmaceutical research. Cefpirom (Cp) is a new semi-synthetic amino-2-thiazolyl-methoxyimino cephalosporin that has been substituted in position 3 with a cyclopenteno-pyridinium group in order to create a zwitterionic compound. It exhibits highly hydrophilic properties, as shown from its extremely low partition coefficient, and therefore its lipophilicity was increased using bile salts. The effect of this on the partition coefficients determined in the n-octanol/buffer system was confirmed using an in-vitro transport model with artificial and biological membranes. The pharmacokinetic properties of Cp were investigated in rabbits after intraduodenal administration with and without bile salts. Furthermore, the physiological compatibility of the bile salts was investigated using active D-glucose transport.     

Effect of cefodizime (HR 221) on immunological defects induced by surgical stress.

Cefodizime, a new aminothiazolylcephalosporin, has been shown to possess immunomodulating activity in many experimental models in vivo and in vitro. The in-vivo effect of the drug was evaluated in a model represented by the surgical patient, being surgical practices usually associated with an immunological impairment involving many aspects of the immune response. Two groups of ten subjects were treated respectively with cefodizime (2 g i.v. daily) and another cephalosporin (ceftriaxone) at the same dosage. Aspecific immunity (complement activity, neutrophil phagocytosis, chemiluminescence and superoxide anion production) and cell-mediated reactivity (lymphocyte subpopulations and E-rosette-forming cells) were evaluated before, and at predetermined intervals after, surgery and antibiotic treatment. In the control group an important immunological derangement is observed, involving both lymphocytes and neutrophil functions and complement system. The treatment with cefodizime displays a positive effect with a significant improvement of impaired functions. The effect of the drug particularly influences neutrophil phagocytosis, explored with both the NBT test and determinations of chemiluminescence, and the complement system, through both the classic and the alternative pathways. A slight effect is observed on lymphocyte functions.     

Cefodizime, an aminothiazolylcephalosporin. I. In vitro activity

Cefodizime possesses a broad antibacterial spectrum including staphylococci, streptococci and Enterobacteriaceae. Neisseria gonorrhoeae and Haemophilus influenzae are also highly susceptible to cefodizime. Because of its beta-lactamase stability cefodizime is active against bacterial strains producing especially plasmid-coded enzymes. The MICs of cefodizime are slightly higher than those of cefotaxime, but with most Gram-negative bacteria they are lower than those of cefazolin, cefotiam and piperacillin. The in vitro activity of cefodizime is not dependent on inoculum size, or on the pH and composition of the test medium. Cefodizime did not induce in vitro resistance of Staphylococcus aureus or Escherichia coli. Because of its binding properties to PBPs 1A/B and 3, cefodizime leads to filamentation of Gram-negative rods and, at only slightly higher concentrations, to bacteriolysis.     

The effects of some antibiotics on polymorphonuclear leukocyte functions of elderly patients in vitro before and after zinc supplementation

The effects of ciprofloxacin, cefodizime, rifampicine, doxycycline and cefodizime + rifampicine combination on polymorphonuclear leukocyte (PMN) functions (phagocytosis and intracellular killing activity) were investigated in vitro in elderly patients and compared with those of healthy young volunteers before and after zinc supplementation. PMNs of 13 elderly hypertensive patients and 10 healthy young volunteers were isolated by Ficoll-Hypaque gradient centrifugation method from venous blood with EDTA. The subjects were given 22 mg/daily/oral zinc supplementation for 1 month. Serum zinc levels before and after supplementation were measured by flame atomic absorbtion spectrophotometer and the effects of each drug on PMN functions at therapeutic concentrations were investigated. Ciprofloxacin significantly increased the PMN's phagocytic activity of elderly patients (p = 0.002) before zinc supplementation and significantly increased both PMN functions of elderly patients (p = 0.002) after zinc supplementation. The same antibiotic significantly increased both PMN functions of healthy young volunteers (p = 0.005 and p<0.05, respectively) before and after zinc supplementation when compared with the control (drug-free). Cefodizime significantly increased the PMN's phagocytic activity of elderly patients (p = 0.003, p = 0.002) before and after zinc supplementation when compared with the control (drug-free). It also significantly increased both PMN functions of healthy young volunteers (p = 0.005 and p<0.05, respectively) before and after zinc supplementation when compared with the control (drug-free). Doxycycline significantly increased PMN's intracellular killing activity of healthy young volunteers before zinc supplementation (p<0.05) when compared with the control (drug-free) values. Rifampicine significantly decreased PMN's phagocytic activity of elderly patients (p<0.05) after zinc supplementation. Cefodizime+rifampicine combination significantly increased PMN's phagocytic activity at therapeutic concentrations of healthy young volunteers (p = 0.005) before zinc supplementation and PMN's phagocytic activity of elderly patients (p<0.05) after zinc supplementation when compared with the control (drug-free). Consequently, in the present study from the antibiotics ciprofloxacin, cefodizime and cefodizime + rifampicine combination, which are accepted as biological response modifiers have demonstrated stimulatory effects by significantly increasing polymorphonuclear leucocyte functions (phagocytosis and/or intracellular killing activity) of elderly patients and healthy young volunteers in vitro before and after zinc supplementation. Additionally zinc supplementation has more immunostimulatory effects on PMN functions of healthy young volunteers than elderly patients.     

Stimulatory effect of cefodizime on macrophage-mediated phagocytosis

We evaluated the ingestion of anti-sheep erythrocyte (anti-E) IgG- and IgM-coated sheep erythrocytes by murine peritoneal macrophages exposed to cefodizime, a new semisynthetic cephalosporin, and other antibiotics. Cefodizime enhanced the ingestion of anti-E IgG-coated erythrocytes by peritoneal macrophages from CD-1 and BALB/c mice in a dose-dependent manner, but had no effect on uncoated or IgM-coated erythrocytes. Similar enhancement was observed only in the case of cefpimizole (AC-1370), among the other antibiotics examined. These results suggest that the favorable in vivo activity of cefodizime and cefpimizole may result from their phagocytosis-enhancing as well as antimicrobial properties.     

Spiral progression in the development of absorption enhancers based on the biology of tight junctions

Epithelium covers the body and, therefore, separates the inner body from the outside environment. Passage across the epithelium is the first step in drug absorption. Tight junctions (TJs) seal the space between adjacent epithelial cells and prevent the free movement of solutes through the paracellular space. Modulation of the epithelial barrier is the most important strategy for enhancing drug absorption. Development of the strategy has accelerated with progress in understanding of the biology of the TJ seal. The first-generation absorption enhancers were screened on the basis of their absorption-enhancing activity in vivo. However, TJs were not well understood initially. The identification of TJ components, including those based on occludin and claudins, has led to the development of new strategies for drug absorption. Accumulation of knowledge of claudins has provided new insights into the paracellular transport of drugs. This review examines the relationship between advances in understanding of TJ biology and paracellular transport of drugs and discusses progress in the development of mucosal absorption enhancers.     

Effects of a new lipid-based drug delivery system on the absorption of low molecular weight heparin (Fragmin) through monolayers of human intestinal epithelial Caco-2 cells and after rectal administration to rabbits

The effects of a novel drug delivery system, consisting of a lipid matrix and a drug, on the permeability, morphology and drug transport across monolayers of human intestinal epithelial cells were investigated. A 1:3 mixture (w/w) of chromatographically purified soybean phosphatidylcholine and medium chain monoacylglycerol was chosen as lipid matrix (PC:MG) and mannitol (mol. wt. 182) and Fragmin (low molecular weight heparin; mean mol. wt. 5000) were chosen as hydrophilic model drugs. PC:MG had an immediate and dose-dependent effect on the permeability of the cell monolayers, and on epithelial morphology (as seen under transmission electron microscopy). PC:MG (4–10 mM) dose-dependently enhanced the transport rate of mannitol and Fragmin, causing an approximately 10-fold increase in the transport rate of Fragmin at concentrations of 6–8 mM. The increase was independent of the dose of Fragmin and was reversible in concentrations up to 6 mM. At higher doses, clear effects on the apical cell membranes were observed although the tight junctions remained intact. PC:MG also enhanced Fragmin absorption in vivo after rectal administration to New Zealand White rabbits. Co-administration of Fragmin with PC:MG (7 mM) resulted in an increase in the relative bioavailability (compared with s.c. administration) from < 1% (without PC:MG) to 21 ± 12%. A maximal increase in relative bioavailability to 90 ± 19% was obtained at a PC:MG concentration of 35 mM. Thus, PC:MG functions as an absorption enhancer both in the cell monolayer model and in vivo, in the same concentration range. The results indicate that as well as providing mechanistic information, studies of absorption enhancers in Caco-2 monolayers also provide information on suitable dosage regimens for in vivo studies.     

Biomembrane permeability of peptides: strategies to improve their mucosal uptake

In order to gain a therapeutic response after mucosal administration peptide drugs have to permeate the absorption membrane based on the mucus layer (I) and the epithelial tissue (II) in significant quantities. The peptide drug transport across the membrane can be improved by the use of mucolytic agents and the permeation enhancers. The generation of novel, more potent permeation enhancers, based on an improved knowledge of the absorption membrane in combination with the appropriate delivery systems will strongly improve the bioavailability of mucosally applied peptide drugs.     

Cefodizime, an aminothiazolyl cephalosporin. III. Therapeutic activity against experimentally induced pneumonia in mice

The activity of the aminothiazolyliminomethoxy cephalosporin cefodizime (HR 221) was compared to that of cefotaxime, cefuroxime and cefazolin in experimental pneumonia caused by Klebsiella pneumoniae DT-S in mice. Cefodizime exhibited high and long-acting levels in the blood and lung homogenates of infected mice; the blood and tissue concentrations obtained with the other cephalosporins tested were low by comparison. In the treatment of experimental Klebsiella pneumonia, cefodizime was superior to cefotaxime and cefuroxime. Counts of the number of viable bacteria present in the infected tissue showed that cefodizime exerted a more marked bactericidal effect than cefotaxime or cefuroxime. Hardly any therapeutic activity was seen with cefazolin.     

The influence of absorption enhancers on nasal absorption of acyclovir.

The objective of this work was to increase the nasal absorption of acyclovir by using absorption enhancers. Acyclovir was selected as a model drug. A rat in situ nasal perfusion technique was utilized in the investigation to examine the rate and extent of absorption of acyclovir. In vitro enzymatic drug degradation study was carried out with rat nasal washings. Various experimental conditions such as nasal perfusion rate, pH of the perfusion medium and concentrations of absorption enhancers such as sodium deoxycholate, hydroxypropyl beta-cyclodextrin, sodium caprate, sodium tauroglycocholate and EDTA were optimized. Nasal absorption of acyclovir was pH dependent. Initial absorption rate constants were determined by the plot of log% remaining amount of drug in perfusate vs time. It was found maximum at pH 7.4 and decreased at lower and higher pH conditions. In in vitro enzymatic degradation study, no measurable degradation was observed during first week. The extent of drug absorption was increased via absorption enhancers. In vivo studies were carried out for the optimized formulation in rabbits and the pharmacokinetics parameters of nasal solution were compared with oral solution. Hydroxypropyl beta-cyclodextrin appeared to be more effective for enhancing the nasal absorption of acyclovir than the other absorption enhancers. The order of increasing absorption of acyclovir caused by the enhancers was hydroxypropyl beta-cyclodextrin>sodium deoxycholate>sodium caprate>sodium tauroglycocholate>EDTA.     

Effect of cefodizime on parameters of cell-mediated immunity in vitro.

A positive effect of cefodizime (CAS 69739-16-8), a new aminothiazolyl cephalosporin, on a number of immunological variables, and in particular phagocytosis, was demonstrated in several test systems. The aim of the present investigation was to establish whether clinically relevant concentrations of cefodizime affect cell-mediated immune variables. Peripheral lymphocytes from healthy subjects were isolated and incubated with cefodizime in increasing concentrations from 0 to 200 mg/l. The effects of cefodizime on membrane-bound antigenic determinants of the lymphocytes were determined in the rosette inhibition test, and its effects on the proliferative capacity of lymphocytes after stimulation with phytohaemagglutinin, concanavalin A and pokeweed mitogen were determined in the lymphocyte transformation test. Cefodizime inhibited rosette formation in a concentration dependent manner. A direct inhibitory effect on proliferation was not, however, demonstrated in the lymphocyte transformation test. Indeed, stimulation of mitogen-induced lymphocyte transformation, particularly of concanavalin A-sensitive cells was observed at concentrations higher than 100 mg/l. The findings in healthy volunteers were reproduced in samples from three female patients with impaired host-defence. These results may suggest a positive effect of cefodizime on the proliferative capacity of the cellular immune system. However, no conclusions can be drawn on the clinical relevance of these findings until the results of in vivo investigations are available.     

Cefodizime penetration into skin suction blister fluid following a single intravenous dose

Summary Cefodizime pharmacokinetics was investigated, evaluating drug concentrations in serum, skin suction blister fluid (SBF), saliva and urine in six healthy male subjects who were administered a 1-g dose intravenously. Serum levels in five subjects can be described according to a two-compartment open model; terminal half-life is 181±14 min. Volume of distribution (V_d) amounts to 15.3±1.61, serum clearance to 59±6 ml/min, renal clearance to 33±3 ml/min. Of the administered dose, 54% is renally excreted unchanged within 27 h. Unbound drug fraction in serum is 19.0% and in SBF 38.4%. Thus renal clearance of free cefodizime amounts to 172 ml/min, V_dss to 68.91 (free drug). Whereas cefodizime has not been detected in saliva samples, SBF concentration 3–9 h post administration parallel serum levels, amounting to 40% of the respective serum concentration. At 9 h, unbound cefodizime concentrations in SBF amount to 1.4±0.4 µg/ml, this value being well above the MIC_90% values of many clinically relevant bacteria.     

注射用头孢地嗪钠细菌内毒素检查法方法学确证

目的建立注射用头孢地嗪钠的细菌内毒素检查方法。方法采用《中华人民共和国药典》2005年版二部附录XI E收载的细菌内毒素检查法。结果注射用头孢地嗪钠稀释成质量浓度为1.25 g.L-1供试液,用浓度为0.125 EU.mL-1的鲎试剂对细菌内毒素检查无干扰。结论采用细菌内毒素检查法检查注射用头孢地嗪钠的细菌内毒素是可行的。     

Transcellular and Lipophilic Complex-Enhanced Intestinal Absorption of Human Growth Hormone

Purpose. To evaluate the transcellular mechanism of novel enhancers absorption enhancement of human growth hormone (hGH), by examining the involvement of a P-glycoprotein-like efflux system, changes in membrane fluidity, and membrane damage. Methods. Caco-2 cell monolayers were grown on Snapwell^® filter supports and placed in a side-by-side diffusion apparatus. Transport in both the apical to basolateral (AP to BL) and basolateral to apical (BL to AP) direction was measured at different temperatures and in the presence of potential inhibitors. Fluorescence anisotropy measurement was used to measure membrane fluidity. The fluorescence anisotropy of DPH- and TMA-DPH-labeled cell suspensions was measured at room temperature. LDH (a measure of cytosolic lactate dehydrogenase) leakage assay was used to evaluate cytotoxicity. Results. The bi-directional transepithelial fluxes of hGH in the presence of these novel enhancers across Caco-2 cells showed marked asymmetry. Average permeability coefficient values obtained in the apical to basolateral (AP to BL) direction were lower than those of the reverse (BL to AP) direction. On the other hand, the fluxes for hGH alone were symmetric. When P-gp-like efflux inhibitors were included in the transport medium, the permeability coefficient value of BL to AP direction was significantly decreased while the transport was increased in the reverse direction in the presence of novel enhancers. In addition, lowering the temperature to 25°C completely eliminated the asymmetry of hGH transport in the presence of novel enhancers. It was also shown by fluorescence anisotropy that these novel enhancers alone only slightly increased membrane fluidity. On the other hand, upon addition of hGH to the novel enhancers, the cell membrane showed a dramatic change as compared to treatment with novel enhancers alone. The results from the LDH assay showed that the novel enhancers and/or hGH did not cause cell damage, at least up to 1 hour, and the damage seen at the 2 hour point is also much lower than other known enhancers. Conclusions. This study shows that human growth hormone alone cannot be transported across Caco-2 cells, except in small quantities, by passive diffusion, but in the presence of novel enhancers, human growth hormone permeation is substantial. In addition, the asymmetry of transport of the complexed hGH appears to be due to a P-gp-like efflux system. Because it is a lack of definitive evidence that the efflux system presented in this paper is a P-glycoprotein, the authors will address the efflux system as a P-gp-like efflux system throughout. Assuming that the present substrate specificity of the P-gp-like efflux system shows the same preference for hydrophobic molecules as p-gp, the present work also indirectly shows that human growth hormone has become more lipophilic in the presence of these novel enhancers. Furthermore, membrane fluidity data also supports the premise that these novel enhancers interact and stabilize hGH, to make them more hydrophobic and easier to be transported through cell membranes.