Crown Ether Nanovesicles (Crownsomes) Repositioned Phenytoin for Healing of Corneal Ulcers.

Drug repositioning is an important drug development strategy as it saves the time and efforts exerted in drug discovery. Since reepithelization of the cornea is a critical problem, we envisioned that the anticonvulsant phenytoin sodium can promote reepithelization of corneal ulcers as it was repurposed for skin wound healing. Herein, our aim is to develop novel crown ether-based nanovesicles "Crownsomes" of phenytoin sodium for ocular delivery with minimal drug-induced irritation and enhanced efficacy owing to "host-guest" properties of crown ethers. Crownsomes were successfully fabricated using span-60 and 18-crown-6 and their size, morphology, polydispersity index, ζ potential, drug loading efficiency, conductivity, and drug release were characterized. Crownsomes exhibited favorable properties such as formation of spherical nanovesicles of 280 ± 18 nm and -26.10 ± 1.21 mV surface charges. Crownsomes depicted a high entrapment efficiency (77 ± 5%) with enhanced and controlled-release pattern of phenytoin sodium. The optimum crownsomes formulation ameliorated ex vivo corneal drug permeability (1.78-fold than drug suspension) through the corneal calcium extraction ability of 18-crown-6. In vivo study was conducted utilizing an alkali-induced corneal injury rabbit model. Clinical and histopathological examination confirmed that crownsomes exhibited better biocompatibility and minimal irritation due to complex formation and drug shielding. Further, they enhanced corneal healing, indicating their effectiveness as a novel drug delivery system for ocular diseases.

Neurotoxicity Comparison of Two Types of Local Anaesthetics: Amide-Bupivacaine versus Ester-Procaine.

Local anaesthetics (LAs) may lead to neurological complications, but the underlying mechanism is still unclear. Many neurotoxicity research studies have examined different LAs, but none have comprehensively explored the distinct mechanisms of neurotoxicity caused by amide- (bupivacaine) and ester- (procaine) type LAs. Here, based on a CCK8 assay, LDH assay, Rhod-2-AM and JC-1 staining, 2',7'-dichlorohy-drofluorescein diacetate and dihydroethidium probes, an alkaline comet assay, and apoptosis assay, we show that both bupivacaine and procaine significantly induce mitochondrial calcium overload and a decline in the mitochondrial membrane potential as well as overproduction of ROS, DNA damage and apoptosis (P < 0.05). There were no significant differences in mitochondrial injury and apoptosis between the bupivacaine and procaine subgroups (P > 0.05). However, to our surprise, the superoxide anionic level after treatment with bupivacaine, which leads to more severe DNA damage, was higher than the level after treatment with procaine, while procaine produced more peroxidation than bupivacaine. Some of these results were also affirmed in dorsal root ganglia neurons of C57 mice. The differences in the superoxidation and peroxidation induced by these agents suggest that different types of LAs may cause neurotoxicity via different pathways. We can target more accurate treatment based on their different mechanisms of neurotoxicity.

The cytotoxic effect of oxybuprocaine on human corneal epithelial cells by inducing cell cycle arrest and mitochondria-dependent apoptosis.

Oxybuprocaine (OBPC) is a widely used topical anesthetic in eye clinic, and prolonged and repeated usage of OBPC might be cytotoxic to the cornea, especially to the outmost corneal epithelium. In this study, we characterized the cytotoxic effect of OBPC on human corneal epithelial (HCEP) cells and investigated its possible cellular and molecular mechanisms using an in vitro model of non-transfected HCEP cells. Our results showed that OBPC at concentrations ranging from 0.025% to 0.4% had a dose- and time-dependent cytotoxicity to HCEP cells. Moreover, OBPC arrested the cells at S phase and induced apoptosis of these cells by inducing plasma membrane permeability, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation. Furthermore, OBPC could trigger the activation of caspase-2, -3, and -9, downregulate the expression of Bcl-xL, upregulate the expression of Bax along with the cytoplasmic amount of mitochondria-released apoptosis-inducing factor, and disrupt mitochondrial transmembrane potential. Our results suggest that OBPC has a dose- and time-dependent cytotoxicity to HCEP cells by inducing cell cycle arrest and cell apoptosis via a death receptor-mediated mitochondria-dependent proapoptotic pathway, and this novel finding provides new insights into the acute cytotoxicity and its toxic mechanisms of OBPC on HCEP cells.

NKG2D blockade significantly attenuates ischemia-reperfusion injury in a cardiac transplantation model.

BACKGROUND: NKG2D (natural killer group 2 member D), are activating or coactivating receptor on NK cells, γδ T, and CD8(+) T cells, stimulates cytokine secretion by the former two and plays a costimulatory role for the last CD8(+) T cells. METHODS: Male Lewis rat hearts were flushed and stored in cold Bretschneider preservation solution for 8 hours. Anti-NKG2D monoclonal antibody (mAb) was administered before transplantation into syngeneic recipients. Expressions of Troponin-T, myeloperoxidase (MPO), tumor necrosis factor (INF), (ICAM) and interleukin (IL)-17 were examined on days 1, 3, and 7 after reperfusion. RESULTS: We observed that isografts from anti-NKG2D mAb-treated animals showed decreased cardiac troponin-T, low expression of MPO, TNF, and ICAM, and superior cardiac output. Furthermore, blockade of NKG2D significantly reduced the number of γδ T cells, which are the main source of IL-17 production. CONCLUSION: Blockade of NKG2D significantly attenuated ischemia-reperfusion injury in a cardiac transplantation model. The effect coincided with a low expression of TNFα, ICAM and a reduced number of infiltrating IL-17-producing γδ T cells.

Effect of topical anesthetic agents and ethanol on corneoepithelial wound healing in an ex vivo whole-globe porcine model.

PURPOSE: To assess the impact of topical anesthetic agents and ethanol on ocular surface wound healing using an ex vivo whole-globe porcine model. SETTING: Department of Ophthalmology, Inselspital, University of Bern, Bern, Switzerland. DESIGN: Experimental study. METHODS: Standardized corneoepithelial lesions (5.0 mm diameter, 40 µm depth) were created with excimer laser light in freshly enucleated porcine eyes. The globes (6 per group) were exposed to different concentrations of ethanol (2.0% to 99.0%), cocaine (2.0% to 10.0%), procaine hydrochloride (0.4%), tetracaine (0.5% to 1.0%), or lidocaine (2.0%), 3 drops/hour for 3 hours. Control solutions were physiologic saline, balanced salt solution, and tissue-culture medium. After 20 to 26 hours, wound-healing response was compared by measuring the diameter of each corneoepithelial lesion. RESULTS: The mean diameter of corneoepithelial lesions exposed to physiologic saline decreased from 4.78 mm ± 0.19 (SD) to 4.44 ± 0.17 mm between 20 and 26 hours. After 24 hours, the mean lesion size, compared with physiological saline, was larger after cocaine 5.0% (5.20 ± 0.26 mm) and 10.0% (5.39 ± 0.12 mm), tetracaine 0.5% (5.59 ± 0.35 mm) and 1.0% (5.55 ± 0.27 mm), and procaine hydrochloride 0.4% (5.76 ± 0.12 mm), but not after lidocaine 2.0% (5.01 ± 0.17 mm). Balanced salt solution, tissue-culture medium, ethanol 2.0% to 99.0%, and cocaine 2.0% did not inhibit the wound-healing response. CONCLUSIONS: In an ex vivo whole-globe porcine model, lidocaine 2.0% and cocaine 2.0% were the least toxic anesthetic agents. At all concentrations, ethanol had no impact on wound healing. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Procaína: Efeitos farmacológicos e toxicológicos / Procaine: Pharmacological and toxicological effects

Procaine is a local anesthetic used by dentists for decades. Nowadays it is being used to treat depression, increase the libido and act on inflammatory conditions and also to induce weight loss, among other uses. However, there has been criticism of such treatments with this substance, alone or in combination. The lack of a scientific basis makes its use subjective and unfounded and often potentially harmful to the individual. Therefore, the aim of this review is to find scientific evidence of systemic actions of procaine that demonstrate its efficacy for such purposes. From a review of the scientific literature, it was concluded that, except for a possible antidepressant effect, so far there are no data proving the alleged effects of procaine. In view of this, the current use of this substance in the treatment of chronic diseases or as an anti-aging drug would not be justified. Moreover, this review emphasizes the need for pharmacological and toxicological studies on procaine and the need to carry out in vivo and in vitro safety trials on pharmaceutical preparations containing this substance, in order to prove or disqualify the indications for its use.

A procaína é um anestésico local utilizado há décadas por dentistas. Atualmente, tem sido utilizada para tratar a depressão, aumentar a libido e agir em processos inflamatórios e no emagrecimento, entre outras utilidades. Porém, existem críticas acerca do tratamento com essa substância isolada ou associada. A falta de embasamento científico para sua utilização torna seu uso infundado e subjetivo, podendo ser muitas vezes nocivo ao indivíduo. Portanto, este artigo tem como objetivo buscar evidências científicas das ações sistêmicas da procaína que comprovem seus efeitos para tais finalidades. Foi realizado um levantamento na literatura científica e concluiu-se que, exceto por um possível efeito antidepressivo, até o momento não existem dados que comprovem os efeitos alegados para a procaína. Devido a isso, os usos atuais não se justificariam no tratamento de doenças crônicas ou no combate ao envelhecimento. Além disso, esta revisão enfatiza a necessidade da realização de estudos para avaliação farmacológica e toxicológica da procaína, bem como a necessidade de aplicar-se ensaios in vivo e in vitro na avaliação da segurança de preparações farmacêuticas que contenham essa substância, a fim de comprovar as inúmeras indicações de uso.

Evaluación experimental en roedores, de la toxicidad de la procaína en algunos esquemas de utilización como neural terapéutico / Experimental evaluation in rodents of the toxicity of procaine in some schemes of use as therapeutic neural

La procaína es el fármaco para la realización de la terapia neural. Su perfil de seguridad ha sido descrito para su uso como anestésico local, sin embargo, la literatura revisada no aporta información suficiente para su uso como neural terapéutico. El objetivo del presente trabajo es contribuir con el conocimiento sobre las bases teóricas de la Terapia Neural, mediante la evaluación de la toxicidad de la Procaína en roedores, según el esquema de uso como neural terapéutico. Se realizó un estudio de experimentación animal para evaluar la neurotoxicidad de la Procaína en administración repetida por vía intradérmica. El comportamiento animal se observó una hora después de cada aplicación y durante el mes siguiente, mediante una batería de pruebas observacionales para evaluar signos clínicos de neurotoxicidad. En ninguna de las pruebas aplicadas se observaron cambios sugestivos de neurotoxicidad, con lo que se puede concluir que la Procaína es segura al ser administrada en dosis bajas por vía intradérmica, teniendo en cuenta que hacen falta estudios para establecer el perfil completo de la Procaína como neural terapéutico.

Inherent toxicity of organ preservation solutions to cultured hepatocytes.

Organ preservation solutions have been designed to protect grafts against the injury inflicted by cold ischemia. However, toxicity of University of Wisconsin (UW) solution during rewarming has been reported. Therefore, we here assessed the toxicity of UW, histidine-tryptophan-ketoglutarate (HTK), Euro-Collins, histidine-lactobionate (HL), sodium-lactobionate-sucrose and Celsior solutions in cultured hepatocytes under hypothermic (4 degrees C), intermediate (21 degrees C) and physiological (37 degrees C) conditions. Marked toxicity of UW, HTK, HL and Euro-Collins solutions was observed at both 37 and 21 degrees C. With the exception of UW solution, these solutions also increased cell injury during cold incubation (LDH release after 18 h at 4 degrees C: HTK 76+/-2%, Euro-Collins 78+/-17%, HL 81+/-15%; control: Krebs-Henseleit buffer 20+/-6%). Testing of individual components using modified Krebs-Henseleit buffers suggested that histidine and phosphate are responsible for (part of) this toxicity. These potential toxicities should be taken into account in the development of future preservation solutions.

Transplacental passage of Pt after treatment with the new triamine complex cis-diaminechloro-[2-(diethylamino) ethyl 4-amino-benzoate, N4]-chloride platinum (II) monohydrochloride monohydrate.

Cis-diaminechloro-[2-(diethylamino) ethyl 4-amino-benzoate, N4]-chloride platinum (II) monohydrochloride monohydrate (DPR) is a monofunctional Pt triamine complex synthesized starting from cisplatin and procaine hydrochloride, characterized by a good antitumor activity coupled with low toxic effects and able to impair prenatal development of mice but at doses outside or just in the upper range of therapeutic doses. In the present paper the transplacental passage of DPR-derived Pt was investigated in CD1 mice on days 9, 13, 16 and 18 of pregnancy, 24 h after ip administration of 21 mg/kg DPR. For comparison, groups of mice were treated with an equivalent Pt-containing dose of cisplatin (10.7 mg/kg). Similarly to cisplatin, small amounts of Pt were detected in fetuses on day 9. From day 13 of gestation the concentration of DPR- and cisplatin-derived Pt increased up to the highest fetal concentrations detected on day 16. On day 18 the concentration of Pt decreased. Most importantly, on days 13-18 of pregnancy cisplatin-derived Pt was always significantly higher than that assayed after DPR administration. In addition, on day 13 of pregnancy Pt exposure of fetuses was significantly higher when dams were treated with cisplatin (AUC(0.5-24)= 3.40 vs. 4.95 microg.h/g). Finally, it is worth noting that serum decay of Pt after DPR or cisplatin administration in adult female mice was similar with AUC0.13-2h s of 7.5 and 6.6 microg.h/ml, respectively. When we determined the concentration of Pt into the main organs of fetuses from dams treated with either DPR or cisplatin on day 18 of gestation, we observed a different organ distribution. In fact, while the concentration of DPR-derived Pt was greater in the heart (1.08+/-0.30 vs. 0.78 +/- 0.35 microg/g, p <0.10), an opposite situation was found in the kidney (0.51+/-0.20 vs. 0.69 +/- 0.22 microg/g, p <0.05). In conclusion, our data show that DPR may pass through the placenta with an efficiency significantly lower than that of cisplatin. This finding may represent one of the possible causes of the lower embryotoxic/teratogenic effect of DPR as compared to cisplatin.

Epithelial activity of hexokinase and glucose-6-phosphate dehydrogenase in cultured bovine lenses recovering from pharmaceutical-induced optical damage.

PURPOSE: In a previous toxicological study, cultured bovine lenses exposed to three topical anesthetics displayed distinct patterns of optical damage and recovery. This work investigated the epithelial activity of the metabolic enzymes hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PD) in lenses recovering from anesthetic-induced damage. METHODS: Cultured bovine lenses were exposed to the anesthetics Alcaine, Fluress and Fluoracaine for 2 h. An automated laser scanner was used to determine the focal length variability (FLV) of the lenses at time-points up to 24 h following their return to fresh culture medium. The epithelial enzyme activities for HK and G6PD were then assayed at the 24 h time-point. RESULTS: Lenses exposed to Alcaine displayed an abrupt increase in FLV, while Fluoracaine treated lenses exhibited optical damage at a slower rate. The FLV in these two groups recovered to near-control levels after 24 h. Fluress treated lenses did not differ in FLV from controls at any time. The activities of both HK and G6PD were significantly reduced in epithelial samples from each of the three anesthetic treatment groups, relative to controls. CONCLUSIONS: These results show that lens optical quality can recover despite a severe reduction in epithelial HK and G6PD activity, indicating that the optical function of the lens may not be directly related to epithelial metabolic activity. The ScanTox In Vitro Assay System provides an objective measure of lens optical quality, enabling a direct comparison of optical damage and recovery to lens biochemical changes.

Embryo-lethal and teratogenic effect of the new platinum compound DPR in pregnant mice.

Embryo-lethal and teratogenic effects caused by the cisplatin-procaine complex cis-diaminechloro-[2-(diethylamino) ethyl 4-amino-benzoate, N(4)]-chlorideplatinum(II) monohydrochloride monohydrate (DPR) were examined in CD-1 mice after a single administration of 7, 14, 21 or 28 mg/kg, injected on day 6, 9, 13 or 16 of pregnancy. At day 18 of pregnancy fetuses were removed and carefully examined for external, visceral and skeletal malformations under a dissecting microscope. A significant reduction of maternal weight gain was observed in pregnant mice after the administration of 21 (day 13) or 28 mg/kg (days 9 and 13) DPR. The exposure to DPR during the organogenesis and early histogenesis periods of prenatal development (administration on day 9 or 13) induced a significant reduction of the mean percentage of live fetuses and a significant increase of the mean percentage of dead and resorbed fetuses. A dose-dependent reduction of fetal body weight was observed in surviving specimens exposed to DPR on embryonic day 9, 13 or 16. The analysis of surviving fetuses killed on day 18 of gestation showed that a few, but statistically significant, external malformations and visceral anomalies were observed after administration of 21 or 28 mg/kg DPR on embryonic day 13. External malformations consisted of three hepato-omphalocele and six palatoschisis (one random palatoschisis was also observed at 21 mg/kg DPR given on day 9), while visceral anomalies included only renal pelvis dilatation. Skeletal anomalies affected fetuses independently of the day of treatment and were more frequent at the highest doses of DPR. They consisted of a delay in skull ossifications, vertebral and sternal anomalies, and formation of extra ribs. A low and non-significant incidence of skeletal malformations (asymmetric sternum) was noticed in fetuses. Our data demonstrated that DPR can cause embryotoxic effects if administered during the period of organogenesis and early histogenesis. Beside embryo-lethality, DPR induced growth retardation and malformations in surviving fetuses.

Embryotoxicity of cisplatin and a cisplatin-procaine complex (DPR) studied in chick embryo.

Cisplatin is widely used as an antitumor drug. To reduce its toxic side effects in patients, cisplatin has been bound with procaine in a cisplatin-procaine complex (DPR). The lethal and teratogenic effects of cisplatin alone and of complexed cisplatin were determined in the chick embryo in ovo in order to compare their influence on rapidly proliferating embryonic tissues. The embryotoxic (lethal + teratogenic) effect was examined after a single intra-amniotic injection of one of six different doses, ranging from 0.03 to 30.0 microg, on embryonic days (ED) 3, 4 or 5. The minimal embryotoxic dose was lower for cisplatin alone (0.03-0.3 microg) than for cisplatin in the DPR complex (0.3-3.0 microg), suggesting that cisplatin alone is more embryotoxic than complexed cisplatin. Both substances caused malformations in the surviving embryos evaluated on ED 9. These malformations included microphthalmia, microcephaly, hypoplasia of the upper and lower jaw, cleft beak, and haemocephaly. Moreover, heart septum defects and limb reduction deformities were found after exposure to the DPR complex. The embryotoxicity of complexed cisplatin exhibited a stage-response effect. It was highest on day 3 and gradually decreased until ED 5. Such an apparent stage-response effect was not observed for cisplatin alone. The embryotoxicity of procaine hydrochloride - a component of the complex - was also tested. Procaine hydrochloride alone did not produce any embryotoxic effect, not even after a single injection of the maximal tested dose (100.0 microg per embryo). We also examined the protective effect of procaine hydrochloride, whose separate administration at ED 4 was followed by the injection of 0.3 microg cisplatin. We did not observe any protective effect of procaine hydrochloride if injected separately.

Comparison of 4 topical anesthetic agents for effect and corneal toxicity in rabbits.

PURPOSE: To compare the onset time, duration of action, corneal toxicity, and corneal epithelial healing time of 4 topical anesthetic agents in rabbits. SETTING: Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada. METHOD: Fifty-six rabbits were treated with 4 topical anesthetics (bupivacaine, lidocaine, procaine, and benzocaine) at different concentrations and different pH of solutions. Corneal sensation, corneal toxicity, and corneal epithelial healing time were measured. RESULTS: The onset time of all 4 anesthetic agents was within 1 minute; however, bupivacaine and lidocaine produced significantly longer action than procaine or benzocaine (P < .05). Buffered bupivacaine and lidocaine had a significantly longer anesthetic effect than that of the nonbuffered solutions (P < .05). No significant effect on corneal epithelial healing time or corneal toxicity was observed. CONCLUSION: Topical bupivacaine and lidocaine had a longer anesthetic effect, particularly in buffered solutions. No significant corneal toxicity was observed.

Anestesia general con procaína intravenosa en el adulto / General anesthesia with intravenous procaine in adults

La procaína es un aminoéster derivado del ácido paraaminobenzoico y del dietilaminoetanol. La concentración que alcanza en el sistema nervioso central depende de la concentración plasmática de su fracción libre no disociada. La demostración hecha por Burstein de que la procaína administrada por vía intravenosa era capaz de inhibir las arritmias cardíacas producidas por la estimulación mecánica del corazón, dió origen a su empleo como anestésico general. La popularidad que aún conserva el procedimiento en muchos países de nuestro continente y en otras partes del planetas se debe a las siguientes ventajas: es una técnica muy económica, muy adecuada para el paciente ambulatorio, se acompaña de una excelente tolerancia al tubo traqueal; la incidencia de naúseas o vómitos en el postoperatorio es despreciable; se acompaña de gran estabilidad cardíaca; no contamina el ambiente quirúrgico. Entre las desventajas podemos mencionar las siguientes: puede ser riesgosa para el paciente en niños menores de 5 años de edad; requiere una venoclisis independiente y segura; para la inducción deben emplearse forzosamente otros agentes; precipita en presencia del tiopental y de otros fármacos intravenosos; no es aconsejable utilizarla en pacientes que presentan graves trastornos de la conducción auriculoventricular; su utilización como anestésico general i.v. se acompaña de un sangrado en napa de los tejidos seccionados. Cuando se la administra por vía intravenosa durante 5 minutos a una velocidad de 1.5 mg/kg/min, considerada por nosotros como la dosis de inducción o de impregnación, la concentración plasmática pico varía entre los 5 y los 20 µg/ml. Una vez que cesa la administración de procaína, su concentración plasmática declina rápidamente. La vida media para la fase alfa (t ½ alfa) varía entre los 2.27 y los 2.73 minutos. Debido a la velocidad de su hidrólisis, sólo el 10 por ciento de la procaína administrada por vía intravenosa se elimina por orina, sin sufrir modificaciones, y ninguno de sus dos metabolitos principales (el ácido paraaminobenzoico y el dietilaminoetanol) tienen efecto anestésico ni producen manifestaciones tóxicas. La procaína administrada por vía i.v. tiene una potencia analgésica 35 veces menor que la ketamina. Esta débil potencia analgésica hace conveniente su asociación con analgésicos opioides (meperidina, fentanilo o alfentanilo). La dosis de mantenimiento utilizada por nosotros es de 0.5 a 0.8 mg/kg/min...

Combination of cisplatin-procaine complex DPR with anticancer drugs increases cytotoxicity against ovarian cancer cell lines.

DPR, cis-diamminechloro-[2-(diethylamino)ethyl 4-amino-benzoate, N4]-chlorideplatinum(II) monohydrochloride monohydrate, is a newly developed water-soluble platinum compound which possess minimal cross-resistance to cisplatin and shows relatively less side effects. In an attempt to establish whether the combination of DPR with other conventional anticancer drugs would be of any benefit we assessed in vitro the cytotoxic effects of combinations of DPR with the antimetabolites 5-fluorouracil (5-FU) and methotrexate (MTX), the alkylating agents mitomycin C (MMC) and cisplatin, the antimicrotubule agent taxol (TAX), and the intercalating agent of the anthracycline group doxorubicin (DOX) on murine M5076 ovarian reticulosarcoma and human A2780 ovarian carcinoma cells. These agents were selected because of their common use in the clinic and because they represent four distinct categories of antineoplastic mechanisms. Cells were incubated for 72 h in the presence of single or combined drugs, and the cytotoxic effect was determined by the MTT assay. The analysis of combination treatment was made by the isobole method. In human A2780 cells, an overall synergy was found for DPR combined with 5-FU, DOX and cisplatin. Synergistic effects were also observed for most combinations with MTX or MMC. A DPR concentration-dependent additivity and antagonism was seen at the highest MTX concentration (1 microM), while additive effects were observed for the combined treatments of DPR and low concentrations of MMC (0.008 and 0.0016 microM). Additive effects were also observed for the association of DPR and TAX over most combinations tested. In murine M5076 cells, synergism was the prevailing result observed when DPR was combined with 5-FU, DOX, MMC or cisplatin. When administered together with MTX we observed additivity over most combinations tested. These findings suggest that DPR, when simultaneously administered with standard anticancer agents, may be advantagious for cytokilling.

Comparison of the toxicities of cisplatin and a new cisplatin-procaine complex to rat renal cortical slices.

1. Procaine has previously been shown to diminish the nephrotoxicity of cisplatin and the nephrotoxic effects of cisplatin and a new cisplatin complex (cis-diamminechloro-[2-(diethylamino) ethyl-4-aminobenzoate, N4]-chlorideplatinum (II) monohydrochloride monohydrate; DPR), that contains procaine hydrochloride were compared with rat renal cortical slices. 2. Cisplatin at 1 mM caused toxicity to the slices, as shown by an increase in the leakage of aspartate aminotransferase and lactate dehydrogenase from the slices into the incubation medium and a decrease in the reduction of a tetrazolium dye (MTT assay). Addition of procaine (1 mM) protected against cisplatin-induced toxicity. DPR either at 1 mM or at 4 mM had no effect either on the enzyme leakage or MTT reduction by the renal slices, but DPR at 10 mM produced a similar magnitude of enzyme leakage to cisplatin (1 mM). 3. DPR lowered the concentration of ATP and glutathione (GSH) in the slices but was less potent than cisplatin. Thiobarbituric acid reactive substances, indicators of lipid peroxidation, released into the medium were increased by the highest concentration of DPR (10 mM), which suggests that DPR has the potential to cause oxidative stress. 4. The results suggest that DPR was far less toxic than either cisplatin alone or a mixture of cisplatin and procaine.

Cytotoxicity and cellular accumulation of a new cis-diammineplatinum (II) complex containing procaine in murine L1210 cells sensitive and resistant to cis-diamminedichloroplatinum (II).

The emergence of drug resistance during tumor chemotherapy is one of the main problems associated with cancer treatment, particularly with cisplatin (cis-DDP). In the hope of overcoming this problem, various cis-DDP-derived compounds have been synthesized, and their pharmacological activity was compared with that of cis-DDP. In this paper we report on studies on the cytotoxic activity induced by cis-diamminechloro-[2-(diethylamino)ethyl-4-aminobenzoate, N4]- chlorideplatinum(II) monohydrochloride monohydrate (DPR), a new complex of platinum containing procaine. All experiments were carried out on murine leukemic cells, which were either sensitive (L1210) or resistant (L1210/DDP) to cis-DDP. A tetrazolium dye (MTT) assay conducted 5 days after a 2-h exposure of cells to both drugs was utilized to determine the resistance factor (RF) of L1210/DDP cells as compared with the sensitive wild-type cells. Drug accumulation and efflux, together with the amount of platinum bound to DNA, were also investigated. The activity of DPR on sensitive cells was not significantly different from that of cis-DDP. Conversely, DPR was 4.3 times more effective than cis-DDP on resistant cells. A decreased drug accumulation is one of the mechanisms of resistance to cis-DDP of L1210/DDP cells. However, DPR accumulation was not significantly different in sensitive and resistant L1210 cells. Under culture conditions that yielded similar intracellular platinum concentrations, treatment with DPR produced significantly greater DNA platination than did treatment with cis-DDP in both cell lines. No difference in efflux was observed between L1210 and L1210/DDP cells exposed to either cis-DDP or DPR. Our results show that in parental cells, DPR is as potent as cis-DDP on a molar basis, and it is also minimally cross-resistant with cis-DDP in L1210/DDP cells. A direct implication of our results is that DPR could be useful in those human tumors showing a mechanism of resistance similar to that of L1210/DDP cells.

The effect of plain 0.5% 2-chloroprocaine on venous endothelium after intravenous regional anaesthesia in the rabbit.

The possible venous endothelial toxicity of 0.5% 2-chloroprocaine without additives in intravenous regional anaesthesia (IVRA) was evaluated in rabbits. After exsanguination of a hind limb with an Esmarch's bandage a neonatal blood pressure cuff around the thigh was inflated (250 mmHg). For IVRA 4 ml of either plain 0.5% 2-chloroprocaine (pH 3.7), 0.9% NaCl (pH 6.0) or acidified NaCl (pH 3.7) was injected i.v. to the exsanguinated limb in a randomized, double-blind fashion. Each group comprised 15 rabbits. Eleven rabbits received 4 ml of 0.5 M or 1.0 M KCl, for the production of positive controls. Two hours after injection of the test solution the tourniquet was deflated and venous biopsies were taken one and 24 hours later for histological and immunocytochemical examination. Five to eight 24-hour samples from each group were also processed for electron microscopy. A macroscopic thrombus formation was observed in four rabbits after KCl and in two after acidified NaCl administration. No inflammatory changes were observed at histologic and immunocytochemical examination of any of the vein samples. Electron microscopy revealed that KCl had caused severe damage to the venous endothelium of four out of five samples and acidified NaCl had caused moderate damage to the endothelium of two out of seven samples. 2-chloroprocaine had caused moderate damage in four and severe damage in two of the vein samples; two samples were normal. No thrombus formation occurred. It is concluded that additive-free 2-chloroprocaine caused damage to the venous endothelium in rabbits when used for IVRA.

Endothelial cell toxicity of preservation solutions: comparison of endothelial cells of different origin and dependence on growth state.

Previously, we have shown that cultured liver endothelial cells are affected by an energy-dependent injury when incubated in cold University of Wisconsin (UW) or histidine-tryptophan-ketoglutarate solution. Here, we studied the susceptibility of other endothelial cells to this type of injury. Aortic endothelial cells in early-confluent, i.e., still proliferating, monolayer cultures were damaged more quickly during cold incubation in UW solution than during cold incubation in Krebs-Henseleit buffer. At this stage the addition of KCN did not alter the loss of viability in UW solution, but when the culture period was prolonged, cells were protected by the addition of cyanide. A paradoxical, protective effect of KCN could also be observed in late-confluent, i.e., nonproliferating, cultures of coronary endothelial cells incubated in UW solution. Similarly, liver endothelial cells in subconfluent, growing cultures were damaged by the addition of cyanide (loss of viability after 48 h, 3 +/- 1% in UW, 65 +/- 19% in UW + KCN), whereas in late-confluent cultures the addition of cyanide to UW solution was protective (loss of viability after 48 h, 100 +/- 0% in UW, 31 +/- 15% in UW + KCN). Variations of culture period and seeding density and the use of inhibitors of cell proliferation demonstrated that liver endothelial cells acquire their susceptibility to energy-dependent injury along with confluence. Subcultured cells retained this susceptibility for some hours. These results suggest that the energy-dependent injury described previously is not confined to liver endothelial cells and that the occurrence of energy-dependent injury requires a capacity of the cells that develops only after cultures have grown to confluence.

The effect of local anaesthetics on the thermal sensitivity of HTC cells.

Local anaesthetics caused death of HTC cells, and the order of toxicity (dibucaine > tetracaine > procaine) correlated with their oil:water partition coefficients. Cytotoxic effects of hyperthermia were enhanced by subtoxic levels of anaesthetics and again, their potency was related to the oil:water partition coefficients. DPH fluidity in plasma membranes at 37 degrees C was increased by dibucaine and tetracaine but not by procaine up to 5mM. At 43 degrees C, plasma membrane fluidity was increased by dibucaine but not by tetracaine or procaine. The results suggest that fluidisation of the hydrophobic core of the membrane may contribute to anaesthetic potentiation of heat cell death.