Kidney-Protector Lipidic Cilastatin Derivatives as Structure-Directing Agents for the Synthesis of Mesoporous Silica Nanoparticles for Drug Delivery.

Mesoporous silica nanomaterials have emerged as promising vehicles in controlled drug delivery systems due to their ability to selectively transport, protect, and release pharmaceuticals in a controlled and sustained manner. One drawback of these drug delivery systems is their preparation procedure that usually requires several steps including the removal of the structure-directing agent (surfactant) and the later loading of the drug into the porous structure. Herein, we describe the preparation of mesoporous silica nanoparticles, as drug delivery systems from structure-directing agents based on the kidney-protector drug cilastatin in a simple, fast, and one-step process. The concept of drug-structure-directing agent (DSDA) allows the use of lipidic derivatives of cilastatin to direct the successful formation of mesoporous silica nanoparticles (MSNs). The inherent pharmacological activity of the surfactant DSDA cilastatin-based template permits that the MSNs can be directly employed as drug delivery nanocarriers, without the need of extra steps. MSNs thus synthesized have shown good sphericity and remarkable textural properties. The size of the nanoparticles can be adjusted by simply selecting the stirring speed, time, and aging temperature during the synthesis procedure. Moreover, the release experiments performed on these materials afforded a slow and sustained drug release over several days, which illustrates the MSNs potential utility as drug delivery system for the cilastatin cargo kidney protector. While most nanotechnology strategies focused on combating the different illnesses this methodology emphasizes on reducing the kidney toxicity associated to cancer chemotherapy.

Simultaneous administration of imipenem/cilastatin/relebactam with selected intravenous antimicrobials, a stewardship approach.

Imipenem/cilastatin/relebactam is a ß-lactam/ß-lactamase inhibitor that has been recently FDA approved for complicated intra-abdominal and urinary tract infections under the brand name Recarbrio®. It has activity against imipenem non-susceptible Pseudomonas species as well as KPC-producing Enterobacteriaceae. Optimization of PK/PD of antimicrobials particularly in critically-ill patients is essential, but unfortunately, is hindered by separate administration that requires significant resources. The objective of the study is to investigate the compatibility of Y-site administration of imipenem/cilastatin/relebactam with a wide range of antimicrobials. After admixture, physical characteristics, pH changes and turbidity were measured for each 2-drug combination at a time. With the exception of amphotericin B deoxycholate, and posaconazole, imipenem/cilastatin/relebactam was compatible with a variety of antimicrobial agents. The compatibility profile described, will facilitate incorporation into hospital protocols, contribute to therapy optimization and guide clinicians to avoid successive administration, consequently resulting in reduction of total infusion time, optimization of PK/PD, economizing nursing time and cost containment.

Y-site Administration of Imipenem/Cilastatin/Relebactam With Common Intravenous Medications.

PURPOSE: Imipenem/cilastatin/relebactam has shown efficacy in complicated intra-abdominal and urinary tract infections in the RESTORE IMI-1 study, and it was recently approved by the US Food and Drug Administration. A press release announced that another Phase III study (RESTORE IMI-2) in patients with hospital-acquired and ventilator-associated pneumonia has met the primary end point. Critically ill patients with multidrug-resistant infections are expected to receive several pharmaceutical intravenous drugs while admitted in hospitals, warranting the need for Y-site compatibility studies. This study was conducted to evaluate the physical compatibility of imipenem/cilastatin/relebactam for injection during Y-site administration with common injectable intravenous medications. METHODS: Imipenem/cilastatin/relebactam was prepared to the concentration of 5 mg/mL, and other intravenous tested drugs were reconstituted as per the package inserts. Y-site was simulated as a 2-drug combination by mixing 5 mL of each in a glass tube, with reversing of the order of mixing; physical characteristics were recorded, and pH changes and turbidity were measured at time intervals. FINDINGS: Imipenem/cilastatin/relebactam was found to be compatible with a wide range of intravenous medications, facilitating co-administration with various IV medications. IMPLICATIONS: The compatibility reported is limited to a 2-h observation period in this study to adequately cover imipenem/cilastatin/relebactam infusion time. In addition, it is based on the measured turbidity with no chemical assay of the components of the admixture.

Stability Studies of Antipyocyanic Beta-Lactam Antibiotics Used in Continuous Infusion.

In intensive care, beta-lactams can be reconstituted in 50 mL polypropylene syringes with NaCl 0.9 % and administered for 8 to 12 h at various concentrations with motor-operated syringe pumps. The feasibility and/or the stability of these antibiotic therapies are often poorly known by clinicians. The purpose of this study was to determine the stability of seven antipyocyanic beta-lactam antibiotics and cilastatin under real-life conditions. Stability indicating HPLC methods allowing quantification in pharmaceutical preparations and subsequent stability studies were performed. The stability studies showed that continuous infusion of piperacillin/tazobactam 80/10 mg/mL, of cefepime 20 and 40 mg/mL and of aztreonam 40 and 120 mg/mL can be used over 12 h. Moreover, continuous infusion of cefepime 120 mg/mL can be used over 10 h, whereas meropenem 10 and 20 mg/mL and ceftazidime 40 mg/mL remained stable only over 8 h, and meropenem 40 mg/mL was significantly degraded after 6 h. Finally, imipenem/cilastatin 5/5 mg/mL and piperacillin/tazobactam 320/40 mg/mL should not be used as continuous infusion. These data allow the establishment of protocols of administration of antipyocyanic beta-lactams by continuous infusion. Some of them are not appropriate to this mode of administration (imipenem/cilastatin, piperacillin/ tazobactam 320/40 mg/mL) or must be avoided if possible (ceftazidime 40 mg/mL).

Nontherapeutic equivalence of a generic product of imipenem-cilastatin is caused more by chemical instability of the active pharmaceutical ingredient (imipenem) than by its substandard amount of cilastatin.

BACKGROUND: We demonstrated therapeutic nonequivalence of "bioequivalent" generics for meropenem, but there is no data with generics of other carbapenems. METHODS: One generic product of imipenem-cilastatin was compared with the innovator in terms of in vitro susceptibility testing, pharmaceutical equivalence, pharmacokinetic (PK) and pharmacodynamic (PD) equivalence in the neutropenic mouse thigh, lung and brain infection models. Both pharmaceutical forms were then subjected to analytical chemistry assays (LC/MS). RESULTS AND CONCLUSION: The generic product had 30% lower concentration of cilastatin compared with the innovator of imipenem-cilastatin. Regarding the active pharmaceutical ingredient (imipenem), we found no differences in MIC, MBC, concentration or potency or AUC, confirming equivalence in terms of in vitro activity. However, the generic failed therapeutic equivalence in all three animal models. Its Emax against S. aureus in the thigh model was consistently lower, killing from 0.1 to 7.3 million less microorganisms per gram in 24 hours than the innovator (P = 0.003). Against K. pneumoniae in the lung model, the generic exhibited a conspicuous Eagle effect fitting a Gaussian equation instead of the expected sigmoid curve of the Hill model. In the brain infection model with P. aeruginosa, the generic failed when bacterial growth was >4 log10 CFU/g in 24 hours, but not if it was less than 2.5 log10 CFU/g. These large differences in the PD profile cannot be explained by the lower concentration of cilastatin, and rather suggested a failure attributable to the imipenem constituent of the generic product. Analytical chemistry assays confirmed that, besides having 30% less cilastatin, the generic imipenem was more acidic, less stable, and exhibited four different degradation masses that were absent in the innovator.

A novel chitosan-polyethylene oxide nanofibrous mat designed for controlled co-release of hydrocortisone and imipenem/cilastatin drugs.

Antimicrobial chitosan-polyethylene oxide (CS-PEO) nanofibrous mats containing ZnO nanoparticles (NPs) and hydrocortisone-imipenem/cilastatin-loaded ZnO NPs were produced by electrospinning technique. The FE-SEM images displayed that the spherical ZnO NPs were ∼70-200nm in size and the CS-PEO nanofibers were very uniform and free of any beads which had average diameters within the range of ∼20-130nm. For all of the nanofibrous mats, the water uptakes were the highest in acidic medium but they were decreased in the buffer and the least swellings were obtained in the alkaline environment. The drug incorporated mat preserved its bactericidal activity even after it was utilized in the release experiment for 8days in the PBS buffer. The hydrocortisone release was increased to 82% within first 12h while the release rate of imipenem/cilastatin was very much slower so that 20% of the drug was released during this period of time suggesting this nanofibrous mat is very suitable to inhibit inflammation (by hydrocortisone) and infection (using imipenem/cilastatin antibiotic and ZnO NPs) principally for the wound dressing purposes.

Discovery of MK-7655, a ß-lactamase inhibitor for combination with Primaxin®.

ß-Lactamase inhibitors with a bicyclic urea core and a variety of heterocyclic side chains were prepared and evaluated as potential partners for combination with imipenem to overcome class A and C ß-lactamase mediated antibiotic resistance. The piperidine analog 3 (MK-7655) inhibited both class A and C ß-lactamases in vitro. It effectively restored imipenem's activity against imipenem-resistant Pseudomonas and Klebsiella strains at clinically achievable concentrations. A combination of MK-7655 and Primaxin® is currently in phase II clinical trials for the treatment of Gram-negative bacterial infections.

Nonvolatile salt-free stabilizer for the quantification of polar imipenem and cilastatin in human plasma using hydrophilic interaction chromatography/quadrupole mass spectrometry with contamination sensitive off-axis electrospray.

A hydrophilic interaction chromatography/mass spectrometry (HILIC-MS)-based assay for imipenem (IMP) and cilastatin (CIL) was recently reported. This orthogonal electrospray ion source-based (ORS) assay utilized nonvolatile salt (unremovable) to stabilize IMI in plasma. Unfortunately, this method was not applicable to conventional MS with off-axis spray (OAS-MS) because MS sensitivity was rapidly deteriorated by the nonvolatile salt. Therefore, we aimed to find a nonvolatile salt- and ion suppression-free approach to stabilize and measure the analytes in plasma using OAS-MS. Acetonitrile and methanol were tested to stabilize the analytes in the plasma samples. The recoveries, matrix effects and stabilities of the analytes in the stabilizer-treated samples were studied. The variations in MS signal intensities were used as the indicator of the assay ruggedness. The results show that a mixture of methanol and acetonitrile (1:1) is best for the storage and measurement of IMP and CIL in human plasma. Utilization of this precipitant not only blocked the hydrolysis of the analytes in plasma but also resulted in an ion suppression-free, fast (120 s per sample) and sensitive detection. The sensitivity obtained using the less sensitive OAS-MS (API3000, 4 pg on column) is much greater than that of the published ORS-MS-based assay (API4000, 77 pg on column). The ruggedness of the assay was demonstrated by its constant MS signal intensity. In conclusion, an improved HILIC/MS-based assay for IMP and CIL was established. The approach presented here provides a simple solution to the challenge of analyzing hydrolytically unstable ß-lactam antibiotics in biological samples.

Dipeptide hydrolysis by the dinuclear zinc enzyme human renal dipeptidase: mechanistic insights from DFT calculations.

The reaction mechanism of the dinuclear zinc enzyme human renal dipeptidase is investigated using hybrid density functional theory. This enzyme catalyzes the hydrolysis of dipeptides and beta-lactam antibiotics. Two different protonation states in which the important active site residue Asp288 is either neutral or ionized were considered. In both cases, the bridging hydroxide is shown to be capable of performing the nucleophilic attack on the substrate carbonyl carbon from its bridging position, resulting in the formation of a tetrahedral intermediate. This step is followed by protonation of the dipeptide nitrogen, coupled with C-N bond cleavage. The calculations establish that both cases have quite feasible energy barriers. When the Asp288 is neutral, the hydrolytic reaction occurs with a large exothermicity. However, the reaction becomes very close to thermoneutral with an ionized Asp288. The two zinc ions are shown to play different roles in the reaction. Zn1 binds the amino group of the substrate, and Zn2 interacts with the carboxylate group of the substrate, helping in orienting it for the nucleophilic attack. In addition, Zn2 stabilizes the oxyanion of the tetrahedral intermediate, thereby facilitating the nucleophilic attack.

Hydrophilic interaction chromatography/tandem mass spectrometry for the simultaneous determination of three polar non-structurally related compounds, imipenem, cilastatin and an investigational beta-lactamase inhibitor, MK-4698, in biological matrices.

A method coupling hydrophilic interaction chromatography (HILIC) with tandem mass spectrometry (MS/MS) has been developed for the simultaneous determination of three polar non-structurally related compounds--a carbapenem antibiotic, imipenem (IMP), a renal dehydropeptidase inhibitor, cilastatin (CIL), and an investigational beta-lactamase inhibitor, MK-4698 (BLI), in rat plasma, monkey plasma and mouse blood. The analytes were extracted through protein precipitation, chromatographed on a Waters Atlantis HILIC column, and detected on a Sciex API4000 mass spectrometer using a Turbo-Ion Spray ion source in positive ionization mode following multiple-reaction monitoring (MRM). The assay dynamic range was 0.1-100 microg/mL for IMP, CIL and BLI, respectively, using a total of 20-25 microL biologic samples, and the total HPLC/MS/MS run time was 4 min/injection. The assay was found to be sensitive, selective and reproducible. The challenges, namely, sample stability, blood sample processing, matrix effect in monkey study samples, and dilution re-assays for the limited mouse blood samples, are resolved and discussed. This technique allowed rapid analysis of polar compounds in biologic matrixes with satisfactory chromatographic retention and increased throughput.

Comparison of in vivo nephrotoxicity in the rabbit by a pyrrolidinyl-thio Carbapenem CW-270031.

CW-270031 is a novel synthesized carbapenem antibiotic with a broad antimicrobial activity. Carbapenem antibiotics are well known for their nephrotoxicity. In this study, we evaluated the nephrotoxicity potential of this compound in rabbits, which are known for being more sensitive than other animals to renal insult. CW-270031 was administered to NZW male rabbits via an ear vein (200 mg/kg, single injection). Blood samples were collected on 2, 3, and 4 days after treatment. Urea nitrogen and creatinine in plasma were quantified. Four days after the treatment, all animals were autopsied and histopathological examinations were performed on their kidneys, revealing that cephaloridine and imipenem were highly nephrotoxic, and cefazolin had mild renal toxicity, whereas CW-270031 as well as meropenem and tienam had no toxicity to the kidney. The present findings suggest that CW-270031 is a potential carbapenem antibiotic with no nephrotoxicity.

CW-270033, a novel pyrrolidinyl-thio carbapenem, has potent antimicrobial activity in vitro and in vivo.

CW-270033, an injectable carbapenem, is a novel, synthesized pyrrolidinyl-thio carbapenem. In the present study, the in-vitro and in-vivo antibacterial activities of CW-270033 against wild-type strains and clinical isolates were compared with those of imipenem and meropenem. CW-270033 was more active than imipenem against Gram-negative (Escherichia coli and Pseudomonas aeruginosa) clinical isolates, but was slightly less active than meropenem. Against the Gram-positive clinical isolates methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE), CW-270033 was slightly more active than meropenem, but was less active than imipenem. CW-270033 displayed potent in-vivo activity against E. coli ATCC 25922, P. aeruginosa ATCC 27853, and S. aureus SMITH in the mouse systemic infection model; the efficacy of CW-270033 in this model was 2--7 fold higher than that of meropenem. This activity was comparable to the in-vitro activity of CW-270033. An intravenous injection of CW-270033 showed that the half-life of CW-270033 in serum in mice was about 20 min, which was about two times that of meropenem. CW-270033 was also found to be resistant to hydrolysis by the mouse renal dehydropeptidase I (DHP-I) enzyme.

A substrate variant as a high-affinity, reversible inhibitor: insight from the X-ray structure of cilastatin bound to membrane dipeptidase.

An analysis of the X-ray structure of cilastatin bound to membrane dipeptidase, together with docking studies, is presented here to reveal how a simple amide may act as a high-affinity, reversible, amidase inhibitor. Cilastatin binds as a normal substrate and is orientated in a perfect near-attack conformer for formation of a tetrahedral intermediate with the zinc-bound water/hydroxide. This intermediate is fated, however, only to revert to its starting components as scission of the amide bond is prevented by the precise fit of cilastatin within the active site. The cilastatin alkyl end groups that are tightly buttressed against amino acid residues on opposite sides of the active site, are aligned along the C-N reaction coordinate axis thereby preventing collapse of the intermediate via rupture of the C-N bond. Such a feature could have more general applicability in the explicit design of substrate variants as selective, tight-binding, and reversible inhibitors.

Identification by site-directed mutagenesis of three essential histidine residues in membrane dipeptidase, a novel mammalian zinc peptidase.

Membrane dipeptidase (EC 3.4.13.19) is a plasma membrane zinc peptidase that is involved in the renal metabolism of glutathione and its conjugates, such as leukotriene D4. The enzyme lacks the classical signatures of other zinc-dependent hydrolases and shows no homology with any other mammalian protein. We have used site-directed mutagenesis to explore the roles of five histidine residues in pig membrane dipeptidase that are conserved among mammalian species. When expressed in COS-1 cells, the mutants H49K and H128L exhibited a specific activity and Km for the substrate Gly-D-Phe comparable with those of the wild-type enzyme. However, the mutants H20L, H152L and H198K were inactive, but were expressed at the cell surface at equivalent levels to the wild-type, as assessed by immunoblotting and immunofluorescence. These three mutants were compared with regard to their ability to bind to the competitive inhibitor cilastatin, which binds with equal efficacy to native and EDTA-treated pig kidney membrane dipeptidase. Expressed wild-type enzyme and mutants H20L and H198K were efficiently bound by cilastatin-Sepharose, but H152L failed to bind. Thus His-152 appears to be involved in the binding of substrate or inhibitor, whereas His-20 and His-198 appear to be involved in catalysis. Membrane dipeptidase shares some similarity with a dipeptidase recently cloned from Acinetobacter calcoaceticus. In particular, His-20 and His-198 of membrane dipeptidase are conserved in the bacterial enzyme, as are Glu-125 and His-219, previously shown to be required for catalytic activity.

The pharmacology of meropenem, a new carbapenem antibiotic.

Meropenem, a new carbapenem antibiotic, is more active against gram-negative bacilli and less active against gram-positive cocci than is imipenem, and there are several important structural differences between meropenem and the older carbapenem. These differences may be responsible for the lower potential for the induction of epileptogenic activity observed with meropenem as well as for its increased stability to degradation by dehydropeptidase-I. The pharmacokinetics of meropenem are typical of those of a parenteral beta-lactam antibiotic with low protein binding and predominantly renal excretion. Dosage reduction is required in patients with reduced renal function; no dosage adjustment is required for patients with hepatic impairment. Meropenem has excellent penetration in abdominal tissues, bile, blister fluid, inflammatory exudate, cerebrospinal fluid (in the presence of inflammation), gynecologic tissues, respiratory tract tissues, and urinary tract tissues; tissue levels are generally equal to or above the levels needed for the treatment of patients with susceptible pathogens.

A new class of potent, slowly reversible dehydropeptidase inhibitors.

Dehydrodipeptide analogs whose scissile carboxamide has been replaced with a PO(OH)CH2 group have been found to be potent inhibitors of the zinc protease dehydrodipeptidase 1 (DHP-1, renal dipeptidase, EC 3.4.13.11). The best of these inhibitors, compound 25 (Ki = 0.52 nM), is two hundred times more potent than cilastatin 2 which is used clinically as a component of the broad-spectrum antibiotic combination Primaxin. Compound 25 is a tight binding inhibitor exhibiting slow binding kinetics with a remarkably slow off rate from DHP-1 (half life greater than 8 hours). The kinetics of its binding are consistent with a simple on-off mechanism whereas the less active D-enantiomer 26 appears to bind in an initial loose complex with the enzyme which slowly rearranges to a tighter complex (Ki = 83 nM).