In this review, we present a timeline that shows the origin of mixed chelate copper (II) complexes, registered as Mark Title Casiopeínas^®, as the first copper (II) compounds proposed as anticancer drugs in 1988 and 1992. In the late twentieth century, the use of essential metals as anticancer agents was not even considered, except for their antifungal or antibacterial effects; also, copper, as gold salts, was used for arthritis problems. The use of essential metals as anticancer drugs to diminish the secondary toxic effects of Cisplatin was our driving force: to find less toxic and even more economical compounds under the rational design of metal chelate complexes. Due to their chemical properties, copper compounds were the choice to continue anticancer drug development. In this order of ideas, the rational designs of mixed chelate–copper (II) complexes (Casiopeínas, (Cas) homoleptic or heteroleptic, depending on the nature of the secondary ligand) were synthesized and fully characterized. In the search for new, more effective, and less toxic drugs, Casiopeína^® (Cas) emerged as a family of approximately 100 compounds synthesized from coordinated Cu(II) complexes with proven antineoplastic potential through cytotoxic action. The Cas have the general formula [Cu(N–N)(N–O)]NO₃ and [Cu(N–N)(O–O)]NO₃, where N–N is an aromatic substituted diimine (1,10-phenanthroline or 2,2′-bipyridine), and the oxygen donor (O–O) is acetylacetonate or salicylaldehyde. Lately, some similar compounds have been developed by other research groups considering a similar hypothesis after Casiopeína’s discoveries had been published, as described herein. As an example of translational medicine criteria, we have covered each step of the established normative process for drug development, and consequently, one of the molecules (Casiopeína III ia (CasIIIia)) has reached the clinical phase I. For these copper compounds, other activities, such as antibacterial, antiparasitic and antiviral, have been discovered.

A rapid and simple high-performance liquid chromatography (HPLC) method using extraction with zinc sulfate was developed for determining Casiopeina III-ia (CasIII-ia) levels and then validated for the linear 10-120 µg/mL range in 200 µL of rabbit blood. An HPLC method for quantifying CasIII-ia in plasma has been previously reported; however, studies on its distribution found that CasIII-ia concentrations were higher in whole blood than in plasma. The analysis was performed using the following parameters: methanol-sodium phosphate buffer (pH 6.5; 0.01 M) (40:60 v/v) mobile phase, using a 250 x 4.6 mm I.D Symmetry® C18 column and a particle size of 5 µm (Waters Associates, Milford, MA, USA) with a C18 5 µm guard column (Phenomenex®) was kept at a isocratic flow-rate of 0.8 ml/min, room temperature at a wavelength of 262 nm. Acetaminophen was used as the internal standard. The results showed that the assay is sensitive at 10 µg/mL. A linear relationship of r2=0.9954 for Cass-III-ia was plotted against concentrations ranging from 10 to 120 µg/mL; the analytical method complies with linearity. The maximum intra-day relative standard deviation (RSD) was 5.10 %. An average 84.8 % intra-day (n=15) and 92.5 % inter-day (n=30) recovery % of CasIII-ia was recovered for the whole blood samples. The results demonstrated the applicability of this method for obtaining it’s in vitro distribution and also it´s for use in pharmacokinetic studies at the preclinical level on rabbits. The present study shows an assay rapid, simple, precise, and accurate for quantifying Cas III-ia in rabbit whole blood. The pharmacokinetic study, carried out in rabbits, obtained the following pharmacokinetic parameters: (kel) = 0.0150 min-1; half-life time (T1/2) = 53.9 min; with an apparent volume of distribution (Vd) = 202.8 mL; clearance (Cl) = 2.0 mL /mi; and area under the curve (AUC)=23163.8 µg/mL.min. The results contribute to the preclinical characterization of Cas III-ia. Resumen. Se desarrolló un método rápido y sencillo por cromatografía líquida de alta resolución (HPLC) utilizando la extracción por precipitación con sulfato de zinc para determinar los niveles de Casiopeína III-ia (CasIII-ia), se validó en un rango lineal de 10-120 mg/mL en 200 µL de sangre de conejo. Un método HPLC para cuantificar Cas III-ia en plasma ha sido reportado previamente; sin embargo, estudios realizados sobre su distribución encontraron que las concentraciones de Cas III-ia eran más altas en sangre total que en plasma. El análisis se realizó utilizando los siguientes parámetros: fase móvil fue metanol y solución amortiguadora de fosfato sódico (pH 6,5; 0,01 M) (40:60 v/v) a un flujo de 0,8 mL/min, temperatura ambiente a una longitud de onda de 262 nm. Se utilizó acetaminofén como estándar interno. Los resultados obtenidos mostraron que el ensayo es sensible a 10 µg/mL. Se encontró una relación lineal de r2=0.9954 para CasIII-ia contra concentraciones que van de 10 a 120 µg/mL; el método analítico cumple con la linealidad. La desviación relativa estándar (RSD) fue de 5.10 %. La media del recobro fue de 84.8 % intra-dia (n=15) y de 92.5 % inter-día (n=30) para Cas III-ia en muestras de sangre total. Los resultados demuestran la aplicabilidad de este método para la obtención de la distribución in vitro y para su uso en estudios farmacocinéticos a nivel preclínico en conejos. El presente estudio muestra un ensayo rápido, sencillo, preciso y exacto para cuantificar CasIII-ia en sangre total de conejo. El estudio farmacocinético, realizado en conejos se obtuvieron los siguientes parámetros farmacocinéticos: (kel) = 0,0150 min-1; tiempo de semivida (T1/2) = 53,92 min; volumen aparente de distribución (Vd) = 202,8 mL; aclaramiento (Cl) = 2,0 mL /mi; y área bajo la curva (AUC)=23163,8 µg/mL.min. Los resultados contribuyen a la caracterización preclínica de Cas III-ia.

Diabetes mellitus is a common chronic degenerative disease around the world. With the Encuesta Nacional de Salud y Nutrición (ENSANUT or National Health and Nutrition Survey) ranking the disease among the highest in terms of morbidity in Mexico[1], allied to the increase in metabolic syndrome worldwide, the development of new medicines for the treatment of diabetes mellitus is essential. Among these medicines is 5-O-β-D-galactopyranosyl-7-methoxy-3’-4’-dihydroxy-4-phenylcoumarin, an organic compound isolated from the leaves of Hintonia latiflora, also known as copalchi [2,3]. An effective preclinical assessment of a drug candidate molecule requires various preclinical tests, including the measurement of apparent permeability in in vitro models. [4-7] Using HPLC-UV, an analytical method was developed and then validated by means of both the FDA validation guidelines for bioanalytical methods (2001) and NOM-177-SSA1-2013 [8,9], with the method found to be reproducible, sensitive, and selective for the determination of 5-O-β-D-galactopyranosyl-7-methoxy-3',4'-dihydroxy-4-phenylcoumarin, which is a phytodrug (4-FC) corresponding to the Madin-Darby canine kidney (MDCK) cell line. The cytotoxicity of the phytodrug was determined for concentrations ranging from 0.1 to 100 μg/mL, [10] using cis-platinum as a positive control (0.01 to 10 μg/mL). The results obtained showed a non-significant cytotoxic effect in a range of 0.10 to 100 µg/mL, while, at concentrations of 4, 8 and, 16 μg/mL, the phytodrug presented a permeation rate (dQ/dt) higher than ranitidine hydrochloride (Ran) and lower than propranolol hydrochloride (Pro), thus suggesting a moderate permeation rate. The present study developed and validated a method for quantifying the analyte of interest, with cytotoxicity (IC50) and permeability tests also carried out in MDCK cells. Resumen. La diabetes mellitus es una enfermedad crónico degenerativa común a nivel mundial. En México, la ENSANUT reporta que dicha enfermedad ocupa primeros lugares de morbilidad. [1] El aumento del síndrome metabólico a nivel mundial, hace indispensable el desarrollo de nuevos medicamentos para tratar la diabetes mellitus, tal es el caso de la 5-O-β-D-galactopiranosil-7-metoxi-3’-4’-dihidroxi-4-fenilcumarina; un compuesto orgánico aislado de las hojas de Hintonia latiflora conocida como “Copalchi” [2,3]. Para una adecuada evaluación a nivel preclínico de moléculas candidatas a fármaco es necesario realizar diversas pruebas a nivel preclínico, entre ellas la permeabilidad aparente en modelos in vitro. [4-7] Se desarrollo y se realizó la validación de un método analítico por HPLC-UV, considerando las guías de validación de Métodos Bioanalíticos por la FDA (2001) y la NOM-177-SSA1-2013. [8,9], el mmétodo resultóser reproducible, sensible y selectivo para la determinación de la 5-O- β-D-galactopiranosil-7-metoxi-3',4'-dihidroxi-4-fenilcumarina un fitofármaco (4-FC) en la línea celular epitelio de riñón canino Madin-Darby (MDCK). Se determinó la citotoxicidad del fitofármaco en un rango de concentraciones de 0.1 a 100 μg/mL, [10] utilizando cis-platino como control positivo (0.01 a 10 μg/mL). Los resultados mostraron un efecto citotóxico no significativo en un rango de 0.10 a 100 µg/mL del fitofármaco a las concentraciones 4, 8 y 16 μg/mL, presentó una velocidad de permeación (dQ/dt) mayor que el clorhidrato de ranitidina (Ran) y menor que el clorhidrato de propranolol (Pro); lo que sugiere una velocidad de permeación moderada para dicho fitofármaco. En este trabajo se desarrollo, se validó el método para la cuantificación del analito de interés, se hizo el estudio de citotoxicidad (IC50) y el estudio de permeabilidad en células MDCK.