The relationship between in vivo nasal drug clearance and in vitro nasal mucociliary clearance: Application to the prediction of nasal drug absorption.

Drug absorption after nasal application is dependent on drug clearance from the nasal cavity, which is determined by nasal mucociliary clearance (MC). We previously developed an in vitro method to evaluate MC via the translocation velocity of fluorescent microspheres (VFMS) applied to excised rat nasal mucosa. In the present study, the relationship between in vivo nasal MC and in vitro VFMS was examined to optimize our PK model for the prediction of nasal drug absorption. Appropriate inhibitors (propranolol and atropine) and enhancers (terbutaline and acetylcholine chloride) of MC were utilized to modify MC. In vivo clearance of drug from the nasal cavity was determined from the disappearance of fluorescent microspheres (FMS) from the nasal cavity following nasal application to rats. The first order elimination rate constant, kmc, was determined from the disappearance profiles of FMS. kmc was decreased to 35.8% by propranolol and 52.6% by atropine, but increased to 117% by terbutaline and 168% by acetylcholine chloride. A significant linear correlation was observed between kmc and VFMS (r2 = 0.9745, p < 0.001). These results indicate that in vivo kmc can be estimated from the in vitro parameter, VFMS. By introducing linear correlation into our PK model, nasal drug absorption may be precisely estimated, even with changes in MC.

Development of Safe and Potent Oil-in-Water Emulsion of Paclitaxel to Treat Peritoneal Dissemination.

To develop a safer and more potent paclitaxel (PTX) formulation, we prepared various oil-in-water emulsions by using egg phosphatidylcholine, Tween 80, and a mixture of triglycerides with different fatty acid chain lengths as the cosurfactant, surfactant, and oil phase component, respectively. The mean particle diameters of the PTX-emulsions prepared were around 100 nm. The PTX-emulsions did not provoke histamine release from rat mast cells and did not show any significant hemolytic activity, suggesting that PTX-emulsions are biocompatible. In vivo antitumor activity after single intraperitoneal injection of PTX-emulsions to ascites tumor-bearing mice revealed that the formulation containing tricaproin and triacetin (3:1, wt/wt, PTX-emulsion B) significantly prolonged the survival time and suppressed the accumulation of ascites fluid. Two distinct in vitro release studies showed that the release of PTX from emulsion B was significantly faster than those from other preparations. These results indicate that the adequately sustained PTX release would lead to potent in vivo antitumor activity and that PTX-emulsion B would offer an alternative approach to treat peritoneal dissemination.

In-vivo disposition characteristics of PEG niosome and its interaction with serum proteins.

Three different nonionic surfactants (Brij 72, Span 20 and Tween 60) were used to prepare various naked and PEG niosomes. In-vivo study demonstrated that PEGylation dramatically increased the AUC and decreased the affinity to the liver of Brij 72 and Span 20 niosomes in rats. Liver perfusion experiments suggested that the hepatic uptake of naked Brij 72 and Span 20 niosomes could mainly be ascribed to the receptor-mediated uptake, while PEGylation of these niosomes could diminish the receptor-mediated hepatic disposition. Evaluation of serum proteins associated with niosomes revealed that PEGylation of these niosomes significantly reduced the association of serum proteins with them, including typical opsonins such as IgG and C3. On the other hand, in the case of Tween 60 niosomes, naked Tween 60 niosome showed large AUC and its PEGylation did not show any additional effect on the in-vivo pharmacokinetics. Furthermore, PEGylation of Tween 60 niosome did not significantly affect the hepatic disposition or the association of serum proteins with Tween 60 niosome. These results demonstrated that niosomes would exhibit distinct in-vivo disposition characteristics depending on the physicochemical properties of surfactants used and that PEGylation of niosomes with adequate compositions would be a powerful tool to improve their in-vivo behavior.

Determinants for in vivo antitumor effect of angiogenesis inhibitor SU5416 formulated in PEGylated emulsion.

Angiogenesis, the sprouting of capillaries from preexisting ones, is essential for the sustained growth of solid tumors. In this study, we used SU5416, a hydrophobic molecule with potent tyrosine kinase inhibitor of type 2 receptor for vascular endothelial growth factor (VEGF), as PEGylated emulsion (SU5416-PE), and evaluated the antitumor potency of this formulation in Lewis lung cancer (LLC), Colon-26 (C26), and B16BL6 melanoma (B16) tumor-bearing mice. Intravenous injection of SU5416-PE into tumor-bearing mice significantly suppressed the growth of C26 and B16 tumors, but had no effect on the growth of LLC tumors. MTT assay revealed that SU5416 inhibited the proliferation of human umbilical vein endothelial cells in a concentration-dependent manner but did not show such an inhibitory effect on all types of tumor cells examined, demonstrating the specificity of SU5416 for endothelial cells. Considering that VEGF levels within C26 and B16 tumors were found to be about 10-fold and 20-fold higher than that in LLC tumors, respectively, it was suggested that SU5416-PE would inhibit angiogenesis in certain types of tumor tissue such as C26 and B16 where VEGF plays a major role for promoting angiogenesis, leading to the suppression of in vivo tumor growth.

A novel approach to overcome multidrug resistance: utilization of P-gp mediated efflux of paclitaxel to attack neighboring vascular endothelial cells in tumors.

We tried to overcome the paclitaxel (PTX) resistance of cancer cells due to P-glycoprotein (P-gp) overexpression in the in vivo anti-tumor chemotherapy by utilizing polyethylene glycol-modified liposomal paclitaxel (PL-PTX). First of all, established were PTX-resistant Colon-26 cancer cells (C26/PTX) overexpressing P-gp, which provided IC50 value of PTX solution about 30 times larger than that obtained for control C26 (C26/control) in the in vitro MTT assay. Western blot analysis confirmed P-gp expression in C26/PTX 10 times higher than that in C26/control, indicating that the resistance acquisition of C26/PTX to PTX would be ascribed to the enhanced efflux of PTX by P-gp overexpressed in C26/PTX. However, the in vivo anti-tumor effect of PL-PTX in C26/PTX-bearing mice was similar to that in C26/control-bearing mice. Double immunohistochemical staining of vascular endothelial cells and apoptotic cells within tumor tissues demonstrated that the apoptotic cell death was preferentially observed in vascular endothelial cells in C26/PTX tumors after intravenous administration of PL-PTX, while that was in tumor cells in C26/control tumors. These results suggest that the in vivo anti-tumor effect of PL-PTX in C26/PTX-bearing mice would be ascribed to the cytotoxic action of PTX pumped out of tumor cells by overexpressed P-gp to vascular endothelial cells in tumor tissues.

Development of transdermal therapeutic formulation of CNS5161, a novel NMDA receptor antagonist, by utilizing pressure-sensitive adhesives II: improved transdermal absorption and evaluation of efficacy and safety.

The aim of this study was to prepare a transdermal therapeutic formulation of CNS5161, an NMDA receptor antagonist developed as a drug for neuropathic pain. Since a silicone pressure-sensitive adhesive (PSA) was found to be the best PSA for CNS5161 among six different PSAs examined in our previous study, the effects of the loading concentration of CNS5161 on release and rat skin permeability were investigated using silicone PSAs. The release of CNS5161 was elevated with an increase in the drug concentration from 1% to 14%. The transdermal flux at the steady state reached a plateau at 8% and over, while crystallization of CNS5161 was not observed for any formulation even at high drug concentrations. The drug concentration in rat skin at the steady state was also saturated at 8% and over, which correlated well with the transdermal flux at the steady state. Therefore, skin permeation clearance defined to the skin concentration at the steady state was almost constant at 0.21/h from 2% to 14% of CNS5161, which suggests that drug concentrations in the skin would be a driving force for transport of the drug to the receptor side. Since increasing the concentration of CNS5161 in the PSA patch was not able to elevate the transdermal flux, 12 formulations containing several permeation enhancers were examined to improve the transdermal transport of CNS5161. Among them, the formulation containing propylene glycol, diisopropyl adipate, and polyvinylpyrrolidone significantly increased the transdermal flux by approximately 1.8-fold by improving the diffusivity of CNS5161 in the skin, and also significantly enhanced the analgesic effect of CNS5161. This formulation caused only slight skin irritation, which indicated that it would be a promising transdermal therapeutic system for CNS5161.

In vitro evaluation of the ciliary beat frequency of the rat nasal epithelium using a high-speed digital imaging system.

Mucociliary clearance (MC) is an important factor in determining nasal drug absorption and the ciliary beat of ciliated epithelial cells of the nasal mucosa is the driving force of MC. However, the relationship between MC and ciliary beat frequency (CBF) is still ambiguous. The purpose of this study was to establish an evaluation method of CBF as an index of mucociliary function and examine the relationship between MC and CBF. A sequence of images of ciliary beating of an excised rat nasal septum was captured using a high-speed digital video camera. CBF (beats per second, Hz) was determined from periodic changes in the contrast value of a specific location in a sequence of images. CBF under control conditions was 8.49±0.38 Hz, which is similar to values reported for cultured human nasal epithelial cells and rat tracheal cells. ß-Adrenergic and cholinergic antagonists decreased CBF, while ß-adrenergic agonists and acetylcholine increased CBF. These results were similar with those observed for MC in our previous study. It was found that CBFs were significantly and linearly correlated with MC, indicating that MC is directly regulated by CBF and that this evaluation system allows the quantitative determination of nasal mucociliary function.

Nanoparticle-based passive drug targeting to tumors: considerations and implications for optimization.

There are many potential barriers to the effective delivery of small-molecule drugs to solid tumors. Most small-molecule chemotherapeutic drugs have a large volume of distribution upon intravenous administration, which is often associated with a narrow therapeutic index due to their high level of toxicity in healthy tissues. Nanoparticle-based therapeutics for tumor targeting have emerged as one of the promising approaches to overcome the lack of tissue specificity of conventional chemotherapeutic drugs. Various different concepts have been envisioned for nanoparticle-mediated drug targeting. Among them, the passive drug targeting strategy has been the most widely investigated, and numerous preclinical studies have provided insights into the validity of the strategy. This review article briefly introduces our recent findings related to the passive drug targeting strategy including its application in anti-angiogenic therapy, along with considerations to be taken into account and implications for the rational design of a passive drug targeting strategy.

Deeper penetration into tumor tissues and enhanced in vivo antitumor activity of liposomal paclitaxel by pretreatment with angiogenesis inhibitor SU5416.

The recently emerged concept of "vessel normalization" implies that judicious blockade of vascular endothelial growth factor (VEGF) signaling may transiently "normalize" the tumor vasculature, making it more suitable for tumor disposition of subsequently administered drugs. In this study, therefore, the effect of pretreatment with SU5416, a selective VEGF receptor-2 inhibitor, on tumor disposition and in vivo antitumor activity of polyethylene glycol (PEG)-modified liposomal paclitaxel (PL-PTX) was evaluated in Colon-26 solid tumor-bearing mice. To improve the solubility and in vivo disposition characteristics of SU5416, the inhibitor was formulated in PEGylated O/W emulsion (PE-SU5416). Pretreatment with PE-SU5416 significantly enhanced the in vivo antitumor effect of PL-PTX, although PE-SU5416 administration alone did not show any antitumor effect. Immunostaining for endothelial cells and pericytes demonstrated that the pretreatment with PE-SU5416 enhanced the pericyte coverage of the tumor vasculature. In addition, tumors treated with PE-SU5416 contained significantly smaller hypoxic regions compared with the nontreated control group, demonstrating that structural normalization of the tumor vasculature resulted in an improvement in tumor vessel functions, including oxygen supply. Furthermore, the pretreatment with PE-SU5416 increased the distribution of PEG liposomes and included PTX in the core region of the tumor, as well as conversely decreasing the ratio of their peripheral distribution. These results suggest that the structural and functional normalization of the tumor vasculature by the pretreatment with PE-SU5416 enabled liposomes to reach the deeper regions within tumor tissues, leading to more potent antitumor activity of PL-PTX.

Formulation and evaluation of paclitaxel-loaded polymeric nanoparticles composed of polyethylene glycol and polylactic acid block copolymer.

To develop potent paclitaxel (PTX) formulations for cancer chemotherapy, we formulated PTX into polymeric nanoparticles composed of polyethylene glycol (PEG) and polylactic acid (PLA) block copolymer (PN-PTX). First, the physicochemical properties of PN-PTX prepared were assessed; the mean particle size was around 80 nm and the zeta potential was found to be almost neutral. Next, the in vitro PTX release property was assessed by a dialysis method. Although rapid release of PTX was observed just after dosing, around 70% of PTX was stably incorporated in polymeric nanoparticles for a long time in the presence of serum. Then, the in vivo pharmacokinetics of PN-PTX after intravenous administration was investigated in Colon-26 (C26) tumor-bearing mice. Both polymeric nanoparticles and PTX incorporated exhibited a long blood circulating property, leading to enhanced permeability and retention (EPR) effect-driven, time-dependent tumor disposition of PTX. Tumor distribution increased gradually for 24 h, and tissue uptake clearance of polymeric nanoparticles in the liver and spleen was lower than that of PEG liposomes. Since these results indicated that the in vivo disposition characteristics of PN-PTX were very favorable, we then evaluated the anti-tumor effect of PN-PTX in C26 tumor-bearing mice. However, PN-PTX did not exhibit any significant anti-tumor effect, presumably due to the poor PTX release from polymeric nanoparticles. From these results, it is considered that the favorable pharmacokinetic properties of nanoparticles and the drug incorporated do not always lead to its potent in vivo pharmacological activity, suggesting the importance of PTX release properties within tumor tissues.

In vitro evaluation of nasal mucociliary clearance using excised rat nasal septum.

Mucus on the nasal mucosa is translocated to the pharynx by ciliary beating, which is an important nonspecific defense mechanism called mucociliary clearance (MC). MC is one of the important factors determining the rate and extent of drug absorption after nasal application. The purpose of this study is to evaluate MC using rat nasal septum under physiological condition in an in vitro system. The nasal septum was excised from rats anesthetized with urethane and the movement of fluorescent microspheres (FMS) applied on the nasal septum was observed with a fluorescence microscope. FMS were transported at a constant velocity in the same direction for a few minutes, but addition of 4% mucin solution on the nasal septum maintained MC for at least 90 min after excision. With our evaluation system established by modifying the method of Saldiva, MC was determined to be around 1 mm/min. Furthermore, the ciliostatic effect of benzalkonium chloride was observed, and it was confirmed that ß-adrenergic antagonists and a cholinergic antagonist decreased MC, and that ß-adrenergic agonists and a cholinergic agonist tended to increase MC, indicating that our system is valid and useful for evaluating MC function and the effect of drugs and pharmaceutical additives for nasal application on MC.

Development of transdermal therapeutic formulation of CNS5161, a novel N-methyl-D-aspartate receptor antagonist, by utilizing pressure-sensitive adhesives I.

The aim of this study was to investigate the feasibility of percutaneous absorption of CNS5161, a novel N-methyl-D-aspartate (NMDA) receptor antagonist developed as a potential treatment for neuropathic pain and other neurological disorders. Six pressure-sensitive adhesives (PSA) with different physicochemical properties, namely, styrene-isoprene-styrene (1) (SIS(1)), styrene-isoprene-styrene (2) (SIS(2)), silicone, acrylate with a hydroxyl group (acrylate(OH)), acrylate without a functional group (acrylate(none)) and acrylate with a carboxyl group (acrylate(COOH)), were investigated for their release of CNS5161 and its subsequent skin permeability. Among the adhesives examined, silicone PSA provided the highest value of transdermal flux of CNS5161, which could be attributable to the highest release rate from it due to its very high thermodynamic activity. Although CNS5161 was also in the supersaturated state in SIS(1) and SIS(2) PSAs, the release and transdermal permeation from these adhesives were slower than those from silicone PSA. As for the acrylic PSAs, the highest release rate and permeability of CNS5161 were observed for acrylate(OH) PSA, followed by acrylate(none) and acrylate(COOH) PSAs, but none of them was better in terms of either the release or the permeability of CNS5161 than silicone PSA. These results clearly indicated that silicone PSA would be the most suitable for transdermal delivery of CNS5161 and silicone PSA containing 10% CNS5161 would be suitable for clinical use in humans.

Sex differences in pharmacokinetics of cilostazol in rats.

The pharmacokinetics of cilostazol was investigated after oral and intravenous administration in both male and female rats. After oral administration, area under serum concentration-time curve (AUC) was about 35-fold higher in female rats than in male rats, and absolute bioavailability was about 5.8-fold higher in female rats than in male rats. Total body clearance (CL(total)) for female rats was around one-sixth of that for male rats. In vivo hepatic clearance (CL(h)) calculated based on isolated liver perfusion studies was even higher than or around 90% of the in vivo CL(total) of cilostazol for female and male rats, respectively, indicating that cilostazol is mainly eliminated by the liver in both male and female rats. In vitro metabolism studies utilizing hepatic microsomes and recombinant cytochrome (CYP) isoforms clearly indicated that major metabolites of cilostazol were generated extensively with hepatic microsomes of male rats and that male-predominant CYP3A2 and male-specific CYP2C11 were mainly responsible for the hepatic metabolism of cilostazol. Therefore, the great sex differences in the pharmacokinetics of cilostazol were mainly attributed to the large difference in hepatic metabolism. Our experimental results also suggested that the substantial metabolism of cilostazol in the small intestine and its possible saturation would be responsible for dose-dependent bioavailability in both male and female rats.

PEG liposomalization of paclitaxel improved its in vivo disposition and anti-tumor efficacy.

To find out potent paclitaxel (PTX) formulations for cancer chemotherapy, we formulated PTX in O/W emulsion and liposome selected as candidates of nanocarriers for PTX. Surface modification of these nanoparticles with polyethylene glycol (PEG) improved their in vivo behavior, but the effect of PEGylation on the pharmacokinetics of emulsion was not so remarkable and the release of PTX from emulsion was found to be very fast in blood circulation, indicating that emulsion would not be an adequate formulation for PTX. On the other hand, AUC of PEG liposome was 3.6 times higher than that of naked liposome after intravenous injection into normal rats due to the lower disposition into the reticuloendothelial system tissues such as liver and spleen. Since PEG liposome was able to stably encapsulate PTX in blood, AUC of PTX was also extensively enhanced after intravenous dosing of PTX-PEG liposome into normal rats. In the in vivo studies utilizing Colon-26 solid tumor-bearing mice, it was confirmed that PTX-PEG liposome delivered significantly larger amount of PTX to tumor tissue and provided more excellent anti-tumor effect than PTX-naked liposome. These results suggest that PEG liposome would serve as a potent PTX delivery vehicle for the future cancer chemotherapy.

Interaction of polyphenolic metabolites with human serum albumin: a circular dichroism study.

Binding sites of polyphenolic compounds on human serum albumin (HSA) were investigated using induced Cotton effects on the circular dichroism (CD) spectra. Polyphenolic compounds used in this study are known to be metabolites from tannins and their related polyphenols in food and medicinal plants. The present investigation revealed that the structural differences markedly affected the binding of the compounds to HSA. Protocatechuic acid, together with its methylated compounds vanillic and isovanillic acids, were assigned to be bound to sites I and II of HSA, based on the competitive relationships with site-I-binding phenylbutazone (PB) and site-II-binding diazepam (DP). 4-O-Methylgallic acid, which is the metabolite from gallic acid, was bound to site I on HSA, while gallic acid did not affect the binding of PB and DP at the concentration examined. Neither ellagic acid nor its metabolite urolithin A was competitive with PB and DP on HSA. The addition of digitoxin did not affect the induced CD of the polyphenolic acids examined.

[Analysis of factors governing drug absorption and their improvement].

Drugs have to be absorbed through various mucosal membranes including the gastrointestinal (GI) mucosa to express their systemic effects. However, there are a number of factors governing mucosal absorption of drugs and, for drugs with low bioavailability, the reason for the low bioavailability should be analyzed and clarified to improve the bioavailability. This review deals with the results of our biopharmaceutical studies to analyze factors controlling drug absorption and our trials to improve the absorption, especially from the GI tract and oral cavity.

Amino acids suppress apoptosis induced by sodium laurate, an absorption enhancer.

The formulation containing sodium laurate (C12), an absorption enhancer, and several amino acids such as taurine (Tau) and L-glutamine (L-Gln) is a promising preparation that can safely improve the intestinal absorption of poorly absorbable drugs. The safety for intestinal mucosa is achieved because the amino acids prevent C12 from causing mucosal damages via several mechanisms. In the present study, the possible involvement of apoptosis, programmed cell death, in mucosal damages caused by C12 and cytoprotection by amino acids was examined. C12 induced DNA fragmentation, a typical phenomenon of apoptosis, in rat large-intestinal epithelial cells while the addition of amino acids significantly attenuated it. C12 alone significantly increased the release of cytochrome C, an apoptosis-inducing factor, from mitochondria, which could be via the decrease in the level of Bcl-2, an inhibiting factor of cytochrome C release. The enhancement of cytochrome C release by C12 led to the activation of caspase 9, an initiator enzyme, and the subsequent activation of caspase 3, an effector enzyme. On the other hand, Tau or L-Gln significantly suppressed the release of cytochrome C from mitochondria and attenuated the activities of both caspases, which could be attributed to the maintenance of Bcl-2 expression.

Interaction of polyphenols with proteins: binding of (-)-epigallocatechin gallate to serum albumin, estimated by induced circular dichroism.

The binding of (-)-epigallocatechin gallate (EGCG), a representative natural polyphenol, to human serum albumin (HSA) and bovine serum albumin (BSA) was investigated using induced circular dichroism (CD). The site of the binding EGCG-HSA was analyzed based on the competition with drugs with known binding sites on HSA, such as phenylbutazone (PB) and diazepam (DP). Double-reciprocal plot analyses showed the competitive relations with the site-I- (PB and tolbutamide, TB) and site-II-binding drugs (DP and ibuprofen, IP) indicating the binding of EGCG to sites I and II on HSA, while digitoxin (DG), a site-III-binding drug, did not affect the binding of EGCG. In an analogous way, the competitive relations were observed between EGCG and the site-I- (PB and TB) and site-II-binding (ethacrynic acid, EA) drugs for the binding of EGCG and BSA. The site-III drug DG also showed competitive binding with EGCG to BSA. The binding of EGCG to the albumins indicated its affinity to sites I and II on HSA, while competitive binding for all three sites was observed on BSA.

Effect of adrenergic stimulation on drug absorption via passive diffusion in Caco-2 cells.

It is well known that the enteric nervous system (ENS) regulates the movement and function of the small intestine, but the effects of ENS on drug absorption from the small intestine still remain to be clarified. Focusing on adrenergic effect, we tried to evaluate how adrenergic stimulation influences the drug absorption via passive diffusion using Caco-2 cells as model epithelial cells, a terminal effector of ENS. Adrenaline, an adrenergic agonist, did not affect the transport of small molecules such as antipyrine, phenacetin and mannitol, but decreased the transport of large molecules such as FITC-dextran (FD)-20 and FD-40 without transepithelial electrical resistance (TEER) change. These results suggested that the transport of large molecules via paracellular route would be attenuated by adrenergic stimulation. Only clonidine, an alpha(2)-agonist, among selective adrenoceptor agonists decreased FD-40 transport across Caco-2 cell monolayers and the agonist also decreased intracellular cAMP. Furthermore, H-89, a protein kinase A inhibitor, significantly decreased FD-40 transport and dibutyryl cAMP, a cAMP derivative, increased it. These results suggest that the decrease in FD-40 transport would be mainly attributed to the decrease in intracellular cAMP and subsequent decrease in PKA activity via alpha(2)-receptor stimulation.

Quantitative evaluation of PEPT1 contribution to oral absorption of cephalexin in rats.

PURPOSE: PEPT1 mediates the intestinal absorption of many drugs, but its contribution to oral absorption of drugs is still controversial. The objective of this study is to quantitatively evaluate the contribution of PEPT1 to oral absorption of cephalexin, a typical substrate for PEPT1, in rats. MATERIALS AND METHODS: The absorbability of cephalexin via PEPT1 or passive diffusion was assessed in five intestinal segments by utilizing glycyl-proline as a competitive inhibitor by in-situ closed loop method. Absorption kinetics of cephalexin after oral administration was predicted by GI-Transit-Absorption model. RESULTS: Absorbability of cephalexin was segment-dependent, and concentration-dependent in all the segments except for the lower ileum. Intrinsic absorption rate constant via PEPT1 ranged from 0.64 to 4.07 h(-1). The absorption rate constants via passive diffusion ranged from 0.78 to 1.24 h(-1). Plasma concentration-time profile of cephalexin was successfully predicted and the substantial contribution of PEPT1 to the oral absorption was calculated to be from 46% to 60% of total absorption. Simulation study indicated that 83% bioavailability would be expected for cephalexin even though PEPT1 does not function. CONCLUSIONS: PEPT1 substantially contributes to oral absorption of cephalexin, around a half of total absorption. However, the function of PEPT1 can be compensated by passive diffusion for cephalexin.