Semimechanistic Modeling of the Effects of Blast Overpressure Exposure on Cefazolin Pharmacokinetics in Mice.

Cefazolin is a first-line antibiotic to treat infection related to deployment-associated blast injuries. Prior literature demonstrated a 331% increase cefazolin liver area under the curve (AUC) in mice exposed to a survivable blast compared with controls. We repeated the experiment, validated the findings, and established a semimechanistic two-compartment pharmacokinetic (PK) model with effect compartments representing the liver and skin. We found that blast statistically significantly increased the pseudo-partition coefficient to the liver by 326% (95% confidence interval: 76-737%), which corresponds to the observed 331% increase in cefazolin liver AUC described previously. To a lesser extent, plasma AUC in blasted mice increased 14-45% compared with controls. Nevertheless, the effects of blast on cefazolin PK were transient, normalizing by 10 hours after the dose. It is unclear as to how this blast effect t emporally translates to humans; however, given the short-lived effect on PK, there is insufficient evidence to recommend cefazolin dosing changes based on blast overpressure injury alone. Clinicians should be aware that cefazolin may cause drug-induced liver injury with a single dose and the risk may be higher in patients with blast overpressure injury based on our findings. SIGNIFICANCE STATEMENT: Blast exposure significantly, but transiently, alters cefazolin pharmacokinetics in mice. The questions of whether other medications or potential long-term consequences in humans need further exploration.

Mesoporous silica pellets as bifunctional bone drug delivery system for cefazolin.

For decades, bone drug delivery systems dedicated for osteomyelitis treatment have been investigated as bifunctional materials that exhibit prolonged drug release and mineralization potential. Herein, composite-type pellets based on cefazolin-loaded amino-modified mesoporous silica SBA-15 and microwave-assisted hydroxyapatite were investigated as potential bone drug delivery system in vitro. Pellets were obtained by granulation, extrusion and spheronization methods in laboratory scale and studied in terms of physical properties, drug release, mineralization potential, antimicrobial activity and cytotoxicity towards human osteoblasts. The obtained pellets were characterized for hardness and friability which indicated the pellets durability during further investigations. Prolonged (5-day) release of cefazolin from pellets was observed. The pellets exhibited mineralization potential in simulated body fluid, i.e., a continuous layer of bone-like apatite was formed on the surface of pellets after 28 days of incubation. An antimicrobial assay of pellets revealed an antibacterial effect against Staphylococcus aureus strain during 6 days. No cytotoxic effects of pellets towards human osteoblasts were observed. The obtained results proved that proposed pellets appear to have potential applications as bone drug delivery systems.

Cefazolin Irreversibly Inhibits Proliferation and Migration of Human Mesenchymal Stromal Cells.

Drugs may have a significant effect on postoperative bone healing by reducing the function of human mesenchymal stromal cells (hMSC) or mature osteoblasts. Although cefazolin is one of the most commonly used antibiotic drugs in arthroplasty to prevent infection worldwide, there is a lack of information regarding how cefazolin affects hMSC and therefore may have an effect on early bone healing. We studied the proliferation and migration capacity of primary hMSC during cefazolin treatment at various doses for up to 3 days, as well as the reversibility of the effects during the subsequent 3 days of culture without the drug. We found a time- and dose-dependent reduction of the proliferation rate and the migratory potential. Tests of whether these effects were reversible revealed that doses ≥ 250 µg/mL or treatments longer than 24 h irreversibly affected the cells. We are the first to show that application of cefazolin irreversibly inhibits the potential of hMSC for migration to the trauma site and local proliferation. Cefazolin should be administered only at the required dosage and time to prevent periprosthetic infection. If long-term administration is required and delayed bone healing is present, cefazolin application must be considered as a cause of delayed bone healing.

Transformation of cefazolin during chlorination process: products, mechanism and genotoxicity assessment.

Large quantities of cephalosporins have entered into aquatic environment in recent years, posing potential adverse effect to human health and ecological safety. In this study, cefazolin, one of widely used cephalosporins, was targeted to explore its transformation behaviors in chlorination disinfection process. With the help of ultra high performance liquid chromatography and high resolution mass spectroscopy, one chlorinated product and four oxidation products were detected in cefazolin chlorination system. The corresponding transformation pathways of cefazolin were proposed. Two kinds of reactions occurred in chlorination system, one was oxidation of thioether-sulfur to sulfoxide and di-sulfoxide, and the other was base-catalyzed electrophilic substitution of alpha-H of amide by chlorine atom. The pH value determined the occurrence of reaction types, and increasing chlorine dose promoted transformation of cefazolin. More importantly, genotoxicity in SOS/umu assay had an elevation after chlorination, which might be attributed to the formation of chlorinated product and sulfoxide during chlorination process.

Antibiotic prophylaxis in (sub)normothermic organ preservation: in vitro efficacy and toxicity of cephalosporins.

BACKGROUND: Bacterial contamination during cold organ preservation occurs without major complications. However, with organ preservation steering toward (sub)normothermic temperatures, bacterial contamination may be detrimental with limited evidence to support the choice of antibiotic. METHODS: This study aimed to determine the effective antibiotic prophylaxis for (sub)normothermic preservation by investigating whether Staphylococcus epidermidis was capable of growing in a subnormothermia-compatible preservation solution Polysol (PS) and in solutions designed for cold preservation (University of Wisconsin solution, histidine-tryptophan-ketoglutarate solution, and Belzer-machine perfusion solution). Various S. epidermidis and Staphylococcus aureus strains were exposed to ceftriaxone and cefazolin at concentrations from 0 to 1000 µg/mL under subnormothermic and normothermic conditions in PS. To mimic procedural conditions, the effect of cefazolin was determined after exposure of bacteria to 20-hr incubation at 28°C in the presence of cefazolin and subsequent incubation at 37°C in the absence of cefazolin. The toxicity of cefazolin was assessed by cell viability and caspase activation assays in porcine kidney endothelial cells. RESULTS: Without antibiotics, PS sustained bacterial growth under sub(normothermic) conditions, whereas growth was absent in cold preservation solutions. Cefazolin exhibited greater bactericidal effect on S. epidermidis than ceftriaxone. However, after inoculating PS with 10 colony-forming units/mL, only a cefazolin concentration of 1000 µg/mL was able to exert a complete bactericidal effect on S. epidermidis and S. aureus strains and maintain sterility after removal of cefazolin. Finally, 1000 µg/mL cefazolin showed no adverse effects on porcine kidney endothelial cells. CONCLUSIONS: Based on these findings, we recommend that high-dose cefazolin be used for prophylaxis in (sub)normothermic organ preservation with PS.

Investigation of the toxic functional group of cephalosporins by zebrafish embryo toxicity test.

2-mercapto-5-methyl-1,3,4-thiadiazole (MMTD) is the 3'-side chain of cephalosporin including cefazolin sodium (CFZL) and cefazedone (CFZD). It is not only present in finished products as the residual precursor, but also produced through drug degradation. Performing the zebrafish embryo toxicity test, we evaluated the toxicity effects of cefazolin sodium, cefazedone, their synthetic precursors and intermediates. Our results suggest that the teratogenic effect of cefazedone and cefazolin sodium on zebrafish embryonic development is associated with the structure of MMTD. They mainly interfere with the development of tissues and organs derived from embryonic ectoderm and mesoderm. We further consider the rationality of the quality control limit of MMTD (1.0%) in the specification. As the acceptable daily intakes (ADIs) of cefazolin is 10 µg/kg per day 16 and the minimum teratogenic concentration of MMTD is tenfold lower than that of cefazolin sodium, we recommend that the acceptable daily intakes of MMTD should be 1 µg/(kg day). In general, the therapeutic dose of cefazolin sodium is 2-4 g/day. Based upon the calculation of MMTD quality control limits (1.0%), MMTD intake can be 20-40 mg/day, which will be much more than the acceptable daily intake value of 1 µg/(kg day). Thus, MMTD should be recommended as a specified impurity and qualified as serious again.

Pro-convulsant effect of cefazolin sodium against pentylenetetrazol- or picrotoxin-induced convulsions in mice.

Cefazolin injection (3000 mg/kg, i.v.) in mice showed several behavioral excitations such as wild running, jumping, rolling, and finally undergoing severe convulsions followed by death. It's lower doses (500-2000 mg/kg, i.v.) were unable to produce any convulsions or behavioral excitations in mice. However, cefazolin (500 or 1000 mg/kg, i.v.) when administered before different doses of pentylenetetrazol (PTZ; 40 or 60 mg/kg, i.p.) or picrotoxin (PTX; 4 or 8 mg/kg, i.p.), it produced severe tonic-clonic convulsions in mice. The convulsions or behavioral excitations produced by 3000 mg/kg, i.v. cefazolin was also reversed by different doses of diazepam (0.5-2 mg/kg, i.p.) further proving the GABAergic modulatory effect of cefazolin. The results conclude the pro-convulsant action of cefazolin on PTZ- or PTX-induced convulsions, and further confirm the clinical reports.

Toward refinement of the colony-forming unit-granulocyte/macrophage clonogenic assay: inclusion of a metabolic system.

This work represents a first attempt to refine the colony-forming unit-granulocyte/macrophage (CFU-GM) clonogenic assay by incorporating liver microsomes and co-factors as a metabolic system into the in vitro test system in response to an ECVAM recommendation. From the comparison of results obtained with the CFU-GM clonogenic assay currently used and with the new experimental protocol, different toxicity on granulocyte/macrophage precursors was demonstrated, when drugs with a known metabolism in vivo were tested.

Evaluation of antimicrobial agents for veterinary use in the ecotoxicity test using microalgae.

The influence of antimicrobial agents approved as veterinary drugs in Japan on the growth of green algae, Selenastrum capricornutum and Chlorella vulgaris, was studied in accordance with the OECD guidelines for testing chemicals. Among the agents tested, growth inhibitory activity was very varied, i.e. erythromycin showed the strongest activity (EC50, 50% effective concentration, = 0.037 mg/l), sulfa drugs had activity to some extent (EC50s of sulfamethoxazole, sulfadiazine, and sulfadimethoxine were 1.5, 2.2, and 2.3 mg/l, respectively), but ampicillin and cefazolin did not inhibit growth (EC50s>1000 mg/l). We also investigated synergistic effect of combining sulfa drugs with trimethoprim or pyrimethamine, which are commonly used as a combined drug. By adding trimethoprim, the growth inhibitory activity of sulfamethoxazole and sulfadiazine was significantly enhanced. Growth inhibition by sulfa drugs was reduced by the addition of folic acid, indicating that they inhibit folate synthesis in green algae.

Antibiotics induce apoptosis of human peritoneal mesothelial cells.

The peritoneal mesothelial cell is a critical component of the peritoneal membrane. The intraperitoneal use of several antibiotics to treat bacterial peritonitis is current clinical practice. Our previous study showed that cephalothin (CPL) and cefotaxime (CFT) have cytotoxic effects on human peritoneal mesothelial cells (HPMC), however, the exact mechanism of cytotoxicity has not been elucidated. In the present study, flow cytometry, TdT-mediated dUTP nick-end labelling (TUNEL) staining and electron microscopy were used to detect the apoptosis of HPMCs. Immunofluorescent staining was used to evaluate the cytochrome c distribution pattern. Western blotting was used to assess apoptotic signalling proteins. We found that CPL (0.5 mg/mL) and CFT (1 mg/mL) induced apoptosis of HPMCs, whereas cefazolin (0.5 mg/mL) and ceftriaxone (0.5 mg/mL) failed to induce apoptosis of HPMCs. While the DNA content of CFT- or CPL-treated cells was reduced, as determined by flow cytometry, cefazolin and ceftriaxone had no such effect. The CFT- or CPL-treated cells displayed the features of apoptosis both under the electron microscope and by using TUNEL staining. However, cefazolin and ceftriaxone produced the same result as the medium controls. Furthermore, CFT and CPL increased the expression of Bax and p53, and caused the translocation of cytochrome c from the mitochondria to the cytoplasm. The HPMC treated by CFT but not by CPL induced the cleavage of procaspase-3 to form active caspase-3. In conclusion, cefotaxime and cephalothin induce apoptosis of HPMCs in vitro. Signal transduction may be through the mitochondrial pathway.

[Drug interactions between nonsteroidal anti-inflammatory drug and pazufloxacin mesilate, a new quinolone antibacterial agent for intravenous use: convulsions in mice after intravenous or intracerebroventricular administration].

Convulsant activity of pazufloxacin mesilate (PZFX mesilate), a new quinolone antibacterial agent for intravenous use, in combination with nonsteroidal anti-inflammatory drug (NSAID) was investigated in mice after intravenous or intracerebroventricular administration. Following results were obtained. 1. In combination with 4-biphenylacetic acid (BPAA) at an oral dose of 100 mg/kg, PZFX mesilate did not induce any convulsions at intravenous doses up to 200 mg/kg. Reference quinolones induced convulsions at the following intravenous doses: Enoxacin (ENX), 3.13 mg/kg or more; norfloxacin (NFLX) and lomefloxacin (LFLX), 6.25 mg/kg or more; ciprofloxacin (CPFX), 50 mg/kg or more; sparfloxacin (SPFX) and temafloxacin (TMFX), 100 mg/kg or more; fleroxacin (FLRX), 200 mg/kg. 2. PZFX mesilate at an intravenous dose of 50 mg/kg did not induce convulsions in mice after oral administration of any of 14 kinds of NSAIDs. It induced convulsions at 200 mg/kg in combination with aspirin at an oral dose of 600 mg/kg, while it did not with the other 13 kinds of NSAIDs. 3. Convulsion-inducing dose of PZFX mesilate after intracerebroventricular administration was 100 mg/body, which was higher than those of reference quinolones (NFLX, CPFX, ENX, LFLX, TMFX, levofloxacin, ofloxacin, FLRX and SPFX) and beta-lactam antibiotics (penicillin G, cefazoline, imipenem/cilastatin and panipenem/betamipron). In addition, concurrent dosing of BPAA (1 microgram/body) did not reduce the convulsion-inducing dose of PZFX mesilate. These results suggest that PZFX mesilate has remarkably weak convulsant activity.

Ciprofloxacin inhibition of experimental fracture healing.

BACKGROUND: Fluoroquinolones, such as ciprofloxacin, have an adverse effect on growing cartilage and endochondral ossification in children. This study was carried out to determine whether ciprofloxacin also has an adverse effect on the healing of experimental fractures. METHODS: Sixty male 300-gram Wistar rats were divided equally into three groups, which received ciprofloxacin, cefazolin, or no treatment for three weeks, beginning seven days after production of a closed, nondisplaced, bilateral femoral fracture. The serum concentrations of the ciprofloxacin and the cefazolin were 2.4 and 146 micrograms per milliliter, respectively. Radiographic, histological, and biomechanical studies were used to evaluate fracture-healing. RESULTS: Radiographs revealed significantly more advanced healing of the control fractures compared with the fractures in the ciprofloxacin-treated group (average stage, 2.1 compared with 1.5, p = 0.01). The cefazolin-treated group was not different from the controls with respect to radiographic healing (average stage, 1.8 compared with 2.1, p = 0.18). Torsional strength-testing of fracture callus exposed to ciprofloxacin revealed a 16 percent decrease in strength compared with the controls (284 compared with 338 newton-millimeters, p = 0.04) and a 49 percent decrease in stiffness (twenty compared with thirty-nine newton-millimeters per degree, p = 0.001). The biomechanical strength in the cefazolin-treated group was not different from that of the controls. Fracture calluses in the animals treated with ciprofloxacin showed abnormalities in cartilage morphology and endochondral bone formation and a significant decrease in the number of chondrocytes compared with the controls (0.77 x 10(4) compared with 1.3 x 10(4) cells per square millimeter, p = 0.004). CONCLUSIONS: These data suggest that experimental fractures exposed to therapeutic concentrations of ciprofloxacin in serum demonstrate diminished healing during the early stages of fracture repair. The administration of ciprofloxacin during early fracture repair may compromise the clinical course of fracture-healing.

Low convulsive activity of a new carbapenem antibiotic, DK-35C, as compared with existing congeners.

Since carbapenems and cephalosporins have been suggested to induce convulsive side effects through an inhibitory action on the central gamma-aminobutyric acid (GABA)-mediated inhibitory transmission, the present study evaluated the convulsive activity of a new carbapenem antibiotic (1R,5S,6S)-6[(R)-1-hydroxyethyl]-2-[(3S,5S)-5(S-methyl-4thiomorpholin ylcarbonyl)pyrrolidin-3-thio]-l-methylcarbapen-2-em-3- carboxylic acid (DK-35C) in in vitro and in vivo experiments, in comparison with cefazolin, imipenem and meropenem. In in vitro experiments, their abilities to inhibit [3H]muscimol (5 nM) binding to GABA(A) receptors were measured using crude synaptic membranes prepared from the rat cerebral cortex. The concentrations (mM) of the antibiotics which inhibit 50% of the specific binding, were 0.6 for imipenem, 1.8 for cefazolin, 15.4 for DK-35C and 27.6 for meropenem. In in vivo experiments, intracerebroventricular (i.c.v.) injections of cefazolin, imipenem and DK-35C induced convulsions in a dose-dependent manner in rats. The doses (nmol/rat) of the antibiotics which induce convulsions in 50% of rats, were 57 for imipenem, 96 for cefazolin, 377 for DK-35C and >3000 for meropenem. In the mouse pentylenetetrazole (PTZ) convulsive model, intravenous pretreatment with cefazolin (800 mg/kg) or imipenem (200 mg/kg) shifted the dose-response curve of PTZ (i.p.) to the left, indicating enhancement of the convulsive activity of PTZ. However, pretreatment with cefazolin, meropenem or DK-35C at a dose of 400 mg/kg did not produce any marked effects on the convulsive activity of PTZ compared with the saline vehicle-pretreated control. The results clearly demonstrate a good correlation between in vitro GABA(A) receptor binding assay and in vivo i.c.v. convulsive model using rats, and suggest that DK-35C may possess a relatively weak convulsive activity mediated through an interaction with GABA(A) receptors.

The in vivo and in vitro comparative nephrotoxicity of cefazolin and gentamicin.

Biochemical, histopathological and cell culture evaluations compared the nephrotoxicity of cefazolin with that of gentamicin. New Zealand rabbits were dosed with 250 mg cefazolin/animal i.m. twice daily, 3 mg gentamicin/kg i.m. twice daily, or 0.9% NaCl solution for 10 d. The rabbits in drug-treated groups had necrosis of proximal kidney tubules and elevated urinary-gamma glutamyl transferase (uGGT) levels. The results of the histopathological examinations, uGGT analyses and effects in cell culture indicated the nephrotoxicities of both antibiotics were similar.

Effects of interleukin-2 on various models of experimental epilepsy in DBA/2 mice.

The effects of interleukin-2 (IL-2) on various models of experimental epilepsy were studied after intracerebroventricular administration in DBA/2 mice, a strain genetically susceptible to sound-induced seizures. Convulsions were induced by physical stimulus (sound of 109 dB. 12-16 kHz) or by chemical compounds (bicuculline, cephazolin or kainate). The present study demonstrated that human recombinant IL-2 (hr-IL-2) and mouse recombinant IL-2 (mr-IL-2) not only did not antagonize audiogenic seizures in DBA/2 mice but increased the incidence of seizures after the highest doses studied. In addition, hr-IL-2 and mr-IL-2 dose dependently facilitated sound-induced seizures at subthreshold sound exposure (83 dB). Pretreatment with monoclonal rat-antimouse IL-2 antibodies significantly affected the changes of occurrence of audiogenic seizures in DBA/2 mice induced by mr-IL-2. In addition, pretreatment with anti-IL-2 receptor monoclonal antibodies (anti-Tac) was able to completely antagonize or reduce the effects of IL-2 on audiogenic seizures. The effects of mr-IL-2 were also studied in two different models of epilepsy: the bicuculline and cephazolin models, due to impairment of GABAergic transmission, and the kainate model, due to an increase in excitatory amino acid transmission. In all models, mr-IL-2 demonstrated to facilitate the seizures induced by these chemoconvulsants. Since the proconvulsant properties of IL-2 were antagonized by specific monoclonal antibodies, we suggest that some epileptic phenomena may be linked to stimulation of IL-2 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of zinc on microbial growth and bacterial killing by cefazolin in a Staphylococcus aureus abscess milieu.

Microbial growth and antimicrobial bacterial killing are both diminished in abscesses. It was postulated that zinc depletion in abscesses, perhaps secondary to a neutrophil protein resembling calprotectin, may be partly responsible for these effects. In a rabbit tissue-cage abscess model, pooled abscess supernatant concentration of zinc was < 1.53 microM. The addition of 41.7 microM zinc had no effect on Staphylococcus aureus growth or the bacterial killing effect of cefazolin in serum. In abscess fluid supernatants, bacterial growth without antibiotic and bacterial killing by cefazolin were both enhanced by the addition of zinc. Fractionation of the abscess fluid with ultrafiltration membranes showed that these effects could be reproduced with the fraction between 30 and 50 kDa. These findings suggest that a protein in abscess fluid supernatants that resembles the neutrophil protein calprotectin may, through its zinc binding effects, inhibit microbial growth within an abscess but also inhibit the activity of bactericidal antibiotics.

Quinolones potentiate cefazolin-induced seizures in DBA/2 mice.

The behavioral and convulsant effects of cefazolin, a beta-lactam derivative, were studied after intraperitoneal administration to DBA/2 mice, a strain genetically susceptible to sound-induced seizures, and Swiss mice. DBA/2 mice were more susceptible to seizures induced by cefazolin than were Swiss mice. The proconvulsant effects of some quinolones on seizures evoked by intraperitoneal administration of cefazolin were also evaluated in DBA/2 mice. Our study also demonstrated that the order of proconvulsant activity in our epileptic model was pefloxacin > enoxacin > ofloxacin > rufloxacin > norfloxacin > cinoxacin > ciprofloxacin > nalidixic acid. The relationships between the chemical structures and proconvulsant activities of quinolone derivatives were studied. The relationship between lipophilicity and proconvulsant activity was also investigated.

[Nephrotoxicity of cefpirome sulfate in rabbits--single and multiple intravenous administration].

Nephrotoxic potential of cefpirome sulfate (CPR) was examined by single and multiple intravenous administrations to Japanese white male rabbits. In single administration studies, no nephrotoxic symptoms were observed at the dose level of 320 mg/kg of CPR or less. At the dose level of 500 mg/kg or more, nephrotoxic findings were noted in both CPR and cefazolin sodium(CEZ) groups, such as proteinuria, glucosuria, increase in serum level of urea nitrogen, creatinine and uric acid, and necrosis and calcification in the proximal tubular epithelium of kidney. Renal phenolsulfonphthalein(PSP) excretion was suppressed at the dose level of 500 mg/kg of CPR or more, and 2000 mg/kg of CEZ. In 14 and 21 days repeated administration studies, no nephrotoxic symptoms were observed at the dose level of 100 mg/kg of CPR and CEZ or less. At the dose level of 200 mg/kg of CPR or more, urinary and serum biochemical findings mentioned above were observed, and histopathological changes in the kidney described above were added in 400 mg/kg group. The similar nephrotoxic symptoms including histopathological changes of the kidney were observed in the groups of 100 mg/kg of cefaloridine(CER) and 200 mg/kg of CEZ. In addition, renal PSP excretion was suppressed in the group of 200 mg/kg of CEZ. The results would suggest that CPR is less nephrotoxic than CER and that the nephrotoxic potential of CPR is comparable to CEZ because CPR caused more severe renal failures in single administration study and less severe renal failures in multiple administration study than CEZ.

[Nephrotoxicity of cefodizime sodium in rats--single and 14-day repeated intravenous administration].

Nephrotoxicity of cefodizime sodium (THR-221), a new cephem antibiotic, was studied in rats by comparing its toxic effect with those of other cephem antibiotics including cephaloridine (CER), cefazolin (CEZ) and cefmetazol (CMZ). Each drug was administered single and consecutive 14-day dosage with its alone or in combination with either furosemide or gentamicin. The results are summarized as follows: 1. In the single dosage study, the rats treated with THR-221 at dose levels of 1200 mg/kg and more showed slight changes in urinary protein and glucose. In rats treated with CER at a dose of 1200 mg/kg, creatinine level in plasma and weights of kidneys were increased, and degeneration and/or necrosis of the renal proximal tubular epithelia were observed. Furthermore, by the consecutive dosage of CER at a dose of 1000 mg/kg, remarkable renal responses including increase in urinary protein and glucose, and weights of kidneys, were observed. In addition, histopathological examinations showed degeneration and/or regeneration of the renal proximal tubular epithelia. 2. No enhanced effect of nephrotoxicity by combination with furosemide or gentamicin was observed except in case of combination of CER with furosemide. 3. The above results indicate that the nephrotoxic potency among these four antibiotics on the single and consecutive dosage studies is CER much greater than THR-221 greater than or equal to CEZ = CMZ and CER much greater than THR-221 = CEZ = CMZ, in the decreasing order.