A Combination of Tri-Leucine and Angiopep-2 Drives a Polyanionic Polymalic Acid Nanodrug Platform Across the Blood-Brain Barrier.

One of the major problems facing the treatment of neurological disorders is the poor delivery of therapeutic agents into the brain. Our goal is to develop a multifunctional and biodegradable nanodrug delivery system that crosses the blood-brain barrier (BBB) to access brain tissues affected by neurological disease. In this study, we synthesized a biodegradable nontoxic ß-poly(l-malic acid) (PMLA or P) as a scaffold to chemically bind the BBB crossing peptides Angiopep-2 (AP2), MiniAp-4 (M4), and the transferrin receptor ligands cTfRL and B6. In addition, a trileucine endosome escape unit (LLL) and a fluorescent marker (rhodamine or rh) were attached to the PMLA backbone. The pharmacokinetics, BBB penetration, and biodistribution of nanoconjugates were studied in different brain regions and at multiple time points via optical imaging. The optimal nanoconjugate, P/LLL/AP2/rh, produced significant fluorescence in the parenchyma of cortical layers II/III, the midbrain colliculi, and the hippocampal CA1-3 cellular layers 30 min after a single intravenous injection; clearance was observed after 4 h. The nanoconjugate variant P/LLL/rh lacking AP2, or the variant P/AP2/rh lacking LLL, showed significantly less BBB penetration. The LLL moiety appeared to stabilize the nanoconjugate, while AP2 enhanced BBB penetration. Finally, nanoconjugates containing the peptides M4, cTfRL, and B6 displayed comparably little and/or inconsistent infiltration of brain parenchyma, likely due to reduced trans-BBB movement. P/LLL/AP2/rh can now be functionalized with intra-brain targeting and drug treatment moieties that are aimed at molecular pathways implicated in neurological disorders.

Pharmacokinetics and bioavailability of chromium malate and its influence on trace metals absorption after oral or intravenous administration.

OBJECTIVES: In our preliminary study, chromium malate could decrease the blood glucose level in mice with diabetes and exhibits good benefits in treating glycometabolism and adipose metabolization obstacle in rats with type 2 diabetes. This study was aimed at assessing the pharmacokinetics and bioavailability of chromium malate and influence on trace metals absorption in rats. METHODS: BAPP 2.3 pharmacokinetic calculating program (China Pharmaceutical University Medicine Center) was used to calculate the pharmacokinetic parameters. Models of type 2 diabetic mellitus rats were applied to analyzed Ca, Mg, Fe, Cu, and Zn contents. RESULTS: The results showed that mean retention time (MRT) in chromium malate group was significantly prolonged and the area under the curve (AUC) and relative bioavailability of chromium malate (male) group were significant increase compared to chromium picolinate group. The serum Ca, Mg, Fe, Cu, and Zn contents in chromium malate (at doses of 15 and 20 µg Cr/kg bw) groups were significantly increased compared to control group, chromium trichloride group, and chromium picolinate group in type 2 diabetes mellitus rats. CONCLUSIONS: Those results indicated that chromium malate can significantly prolong MRT and increase AUC (male). Moreover, chromium malate is more effective at treating increased serum Ca, Mg, Fe, Cu, and Zn contents compared to chromium trichloride and chromium picolinate.

Dual-pH Sensitive Charge-reversal Nanocomplex for Tumor-targeted Drug Delivery with Enhanced Anticancer Activity.

Poly(ß-L-malic acid) (PMLA), a natural aliphatic polyester, has been proven to be a promising carrier for anti-cancer drugs. In spite of excellent bio-compatibility, the application of PMLA as the drug carrier for cancer therapy is limited by its low cellular uptake efficiency. The strong negative charge of PMLA impedes its uptake by cancer cells because of the electrostatic repulsion. In this study, a dual pH-sensitive charge-reversal PMLA-based nanocomplex (PMLA-PEI-DOX-TAT@PEG-DMMA) was developed for effective tumor-targeted drug delivery, enhanced cellular uptake, and intracellular drug release. The prepared nanocomplex showed a negative surface charge at the physiological pH, which could protect the nanocomplex from the attack of plasma proteins and recognition by the reticuloendothelial system, so as to prolong its circulation time. While at the tumor extracellular pH 6.8, the DMMA was hydrolyzed, leading to the charge reversal and exposure of the TAT on the polymeric micelles, thus enhancing the cellular internalization. Then, the polymeric micelles underwent dissociation and drug release in response to the acidic pH in the lyso/endosomal compartments of the tumor cell. Both in vitro and in vivo efficacy studies indicated that the nanocomplex significantly inhibited the tumor growth while the treatment showed negligible systemic toxicity, suggesting that the developed dual pH-sensitive PMLA-based nanocomplex would be a promising drug delivery system for tumor-targeted drug delivery with enhanced anticancer activity.

L-Malate's Plasma and Excretion Profile in the Treatment of Moderate and Severe Hemorrhagic Shock in Rats.

Introduction. Malate is a standard component in fluid therapy within a wide range of medical applications. To date, there are insufficient data regarding its plasma distribution, renal excretion, and metabolism after infusion. This study aimed to investigate these three aspects in a rat model of moderate and severe hemorrhagic shock (HS). Methods. Male Wistar rats were subjected to HS by dropping the mean arterial blood pressure to 25-30 mmHg (severe) and 40-45 mmHg (moderate), respectively, for 60 minutes. Subsequently, reperfusion with Ringer-saline or a malate containing crystalloid solution (7 mM, 13.6 mM, and 21 mM, resp.) was performed within 30 minutes, followed by an observation period of 150 minutes. Results. In the present experiments, malate rapidly disappeared from the blood, while only 5% of the infused malate was renally excreted. In the resuscitation interval the urinary citrate and succinate amounts significantly increased compared to control. Conclusion. Malate's half-life is between 30 and 60 minutes in both, moderate and severe HS. Thus, even under traumatic conditions malate seems to be subjected to rapid metabolism with participation of the kidneys.

Safety Assessment of Dialkyl Malates as Used in Cosmetics.

The Cosmetic Ingredient Review Expert Panel (Panel) reviewed the safety of 6 dialkyl malate compounds used in cosmetics. These ingredients function mostly as skin-conditioning agents-emollients. The Panel reviewed relevant animal and human data related to the ingredients along with a previous safety assessment of malic acid. The similar structure, properties, functions, and uses of these ingredients enabled grouping them and using the available toxicological data to assess the safety of the entire group. The Panel concluded that these dialkyl maleate compounds are safe in the present practices of use and concentration as given in this safety assessment.

Preparation of poly(ß-L-malic acid)-based charge-conversional nanoconjugates for tumor-specific uptake and cellular delivery.

In this study, a multifunctional poly(ß-L-malic acid)-based nanoconjugate with a pH-dependent charge conversional characteristic was developed for tumor-specific drug delivery. The short branched polyethylenimine-modified poly(ß-L-malic acid) (PEPM) was first synthesized. Then, the fragment HAb18 F(ab')2 and 2,3-dimethylmaleic anhydride were covalently attached to the PEPM to form the nanoconjugate, HDPEPM. In this nanoconjugate, the 2,3-dimethylmaleic anhydride, the shielding group, could shield the positive charge of the conjugate at pH 7.4, while it was selectively hydrolyzed in the tumor extracellular space (pH 6.8) to expose the previously-shielded positive charge. To study the anticancer activity, the anticancer drug, doxorubicin, was covalently attached to the nanoconjugate. The doxorubicin-loaded HDPEPM nanoconjugate was able to efficiently undergo a quick charge conversion from -11.62 mV to 9.04 mV in response to the tumor extracellular pH. The electrostatic interaction between the positively charged HDPEPM nanoconjugates and the negatively charged cell membrane significantly enhanced their cellular uptake, resulting in the enhanced anticancer activity. Also, the tumor targetability of the nanoconjugates could be further improved via the fragment HAb18 F(ab')2 ligand-receptor-mediated tumor cell-specific endocytosis.

Natural and synthetic poly(malic acid)-based derivates: a family of versatile biopolymers for the design of drug nanocarriers.

The field of specific drug delivery is an expanding research domain. Besides the use of liposomes formed from various lipids, natural and synthetic polymers have been developed to prepare more efficient drug delivery systems either under macromolecular prodrugs or under particulate nanovectors. To ameliorate the biocompatibility of such nanocarriers, degradable natural or synthetic polymers have attracted the interest of many researchers. In this context, poly(malic acid) (PMLA) extracted from microorganisms or synthesized from malic or aspartic acid was used to prepare water-soluble drug carriers or nanoparticles. Within this review, both the preparation and the applications of PMLA derivatives are described emphasizing the in vitro and in vivo assays. The results obtained by several groups highlight the interest of such polyesters in the field of drug delivery.

Nanoparticles of esterified polymalic acid for controlled anticancer drug release.

Esterification of microbial poly(malic acid) is performed with either ethanol or 1-butanol to obtain polymalate conjugates capable to form nanoparticles (100-350 nm). Degradation under physiological conditions takes place with release of malic acid and the corresponding alcohol as unique degradation products. The anticancer drugs Temozolomide and Doxorubicin are encapsulated in nanoparticles with efficiency of 17 and 37%, respectively. In vitro drug release assays show that Temozolomide is almost completely discharged in a few hours whereas Doxorubicin is steadily released along several days. Drug-loaded nano-particles show remarkable effectiveness against cancer cells. Partially ethylated poly(malic acid) nano-particles are those showing the highest cellular uptake.

Polymalic acid nanobioconjugate for simultaneous immunostimulation and inhibition of tumor growth in HER2/neu-positive breast cancer.

Breast cancer remains the second leading cause of cancer death among women in the United States. Breast cancer prognosis is particularly poor in case of tumors overexpressing the oncoprotein HER2/neu. A new nanobioconjugate of the Polycefin(TM) family of anti-cancer drugs based on biodegradable and non-toxic polymalic acid (PMLA) was engineered for a multi-pronged attack on HER2/neu-positive breast cancer cells. An antibody-cytokine fusion protein consisting of the immunostimulatory cytokine interleukin-2 (IL-2) genetically fused to an antibody specific for human HER2/neu [anti-HER2/neu IgG3-(IL-2)] was covalently attached to the PMLA backbone to target HER2/neu expressing tumors and ensure the delivery of IL-2 to the tumor microenvironment. Antisense oligonucleotides (AON) were conjugated to the nanodrug to inhibit the expression of vascular tumor protein laminin-411 in order to block tumor angiogenesis. It is shown that the nanobioconjugate was capable of specifically binding human HER2/neu and retained the biological activity of IL-2. We also showed the uptake of the nanobioconjugate into HER2/neu-positive breast cancer cells and enhanced tumor targeting in vivo. The nanobioconjugate exhibited marked anti-tumor activity manifested by significantly longer animal survival and significantly increased anti-HER2/neu immune response in immunocompetent mice bearing D2F2/E2 murine mammary tumors that express human HER2/neu. The combination of laminin-411 AON and antibody-cytokine fusion protein on a single polymeric platform results in a new nanobioconjugate that can act against cancer cells through inhibition of tumor growth and angiogenesis and the orchestration of an immune response against the tumor. The present Polycefin(TM) variant may be a promising agent for treating HER2/neu expressing tumors and demonstrates the versatility of the Polycefin(TM) nanobioconjugate platform.

Assessment the levels of tartrate-resistant acid phosphatase (TRAP) on mice fed with eggshell calcium citrate malate.

Optimized conditions were obtained by one-factor-at-a-time test (OFAT) and ternary quadratic regression orthogonal composite design (TQROCD) respectively. By pulse electric fields (PEF) technology, the process of eggshell calcium citrate malate (ESCCM), eggshell calcium citrate (ESCC) and eggshells calcium malate (ESCM) were comprehensive compared. The levels of tartrate-resistant acid phosphatase (TRAP) and the bioavailability on mice fed with eggshell calcium citrate malate (ESCCM) treated by pulsed electric field (PEF) were evaluated. Results showed that the rates of calcium dissolution of the different acids studied can be arranged as ESCCM (7.90 mg/mL)>ESCC (7.12 mg/mL)>ESCM (7.08 mg/mL) from highest to lowest rate of dissolution. At the same dose 133.0 mg kg(-1) d(-1), the levels of TRAP in the ESCCM treatment groups were significantly lower than those in ESCM and ESCC (P<0.05). Bone calcium content in the mice fed with ESCCM was generally higher than fed with ESCM and ESCC.

Cellular delivery of doxorubicin via pH-controlled hydrazone linkage using multifunctional nano vehicle based on poly(ß-l-malic acid).

Doxorubicin (DOX) is currently used in cancer chemotherapy to treat many tumors and shows improved delivery, reduced toxicity and higher treatment efficacy when being part of nanoscale delivery systems. However, a major drawback remains its toxicity to healthy tissue and the development of multi-drug resistance during prolonged treatment. This is why in our work we aimed to improve DOX delivery and reduce the toxicity by chemical conjugation with a new nanoplatform based on polymalic acid. For delivery into recipient cancer cells, DOX was conjugated via pH-sensitive hydrazone linkage along with polyethylene glycol (PEG) to a biodegradable, non-toxic and non-immunogenic nanoconjugate platform: poly(ß-l-malic acid) (PMLA). DOX-nanoconjugates were found stable under physiological conditions and shown to successfully inhibit in vitro cancer cell growth of several invasive breast carcinoma cell lines such as MDA-MB-231 and MDA-MB- 468 and of primary glioma cell lines such as U87MG and U251.

Enhanced anti-diabetic activity of a combination of chromium(III) malate complex and propolis and its acute oral toxicity evaluation.

In order to obtain the additional benefit of anti-diabetic activity and protective effects of liver injury for diabetes, the anti-diabetic effect and acute oral toxicity of a combination of chromium(III) malate complex (Cr(2)(LMA)(3)) and propolis were assessed. The anti-diabetic activity of the combination of the Cr(2)LMA(3) and propolis was compared with Cr(2)(LMA)(3) and propolis alone in alloxan-induced diabetic mice by daily oral gavage for a period of 2 weeks. Acute oral toxicity of the combination of the Cr(2)LMA(3) and propolis was tested using ICR mice at the dose of 1.0-5.0 g/kg body mass by a single oral gavage and observed for a period of 2 weeks. The results of the anti-diabetic activity of the combination from the aspects of blood glucose level, liver glycogen level, and the activities of aspartate transaminase, alanine transaminase, and alkaline phosphatase indicated that the increased anti-diabetic activity and the protective efficacy of liver injury for diabetes were observed. In acute toxicity study, LD(50) (median lethal dose) value for the combination was greater than 5.0 g/kg body mass. The combination of Cr(2)LMA(3) and propolis might represent the nutritional supplement with potential therapeutic value to control blood glucose and exhibit protective efficacy of liver injury for diabetes and non-toxicity in acute toxicity.

Inhibition of brain tumor growth by intravenous poly (ß-L-malic acid) nanobioconjugate with pH-dependent drug release [corrected].

Effective treatment of brain neurological disorders such as Alzheimer's disease, multiple sclerosis, or tumors should be possible with drug delivery through blood-brain barrier (BBB) or blood-brain tumor barrier (BTB) and targeting specific types of brain cells with drug release into the cell cytoplasm. A polymeric nanobioconjugate drug based on biodegradable, nontoxic, and nonimmunogenic polymalic acid as a universal delivery nanoplatform was used for design and synthesis of nanomedicine drug for i.v. treatment of brain tumors. The polymeric drug passes through the BTB and tumor cell membrane using tandem monoclonal antibodies targeting the BTB and tumor cells. The next step for polymeric drug action was inhibition of tumor angiogenesis by specifically blocking the synthesis of a tumor neovascular trimer protein, laminin-411, by attached antisense oligonucleotides (AONs). The AONs were released into the target cell cytoplasm via pH-activated trileucine, an endosomal escape moiety. Drug delivery to the brain tumor and the release mechanism were both studied for this nanobiopolymer. Introduction of a trileucine endosome escape unit resulted in significantly increased AON delivery to tumor cells, inhibition of laminin-411 synthesis in vitro and in vivo, specific accumulation in brain tumors, and suppression of intracranial glioma growth compared with pH-independent leucine ester. The availability of a systemically active polymeric drug delivery system that passes through the BTB, targets tumor cells, and inhibits glioma growth gives hope for a successful strategy of glioma treatment. This delivery system with drug release into the brain-specific cell type could be useful for treatment of various brain pathologies.

Roles of organic acids and nitrate in the long-distance transport of cobalt in xylem saps of Alyssum murale and Trifolium subterraneum.

Roles of organic acids and nitrate in the long-distance transport of cobalt (Co) in xylem saps of hyperaccumulator Alyssum murale and non-hyperaccumulator Trifolium subterraneum were studied under hydroponic conditions. Organic acids (oxalic, malic, malonic, citric, and fumaric) and nitrate in xylem sap samples were separated and determined simultaneously by reversed-phase high performance liquid chromatography after solid-phase extraction with nanosized hydroxyapatite. Results indicated that Co treatment significantly increased the concentrations of xylem oxalic and malic acids for the hyperaccumulator A. murale compared to the control but significantly decreased the concentrations of xylem nitrate and malonic acid; concentrations of citric acid in xylem sap samples did not show significant difference between the control and Co treatments. By analyzing the relationship between the concentrations of organic acids, nitrate, and concentrations of Co in xylem saps, it could be concluded that oxalic and malic acids in xylem saps seemed to participate in the long-distance Co translocation process, and citric acid did not relate to the xylem Co transport of A. murale and T. subterraneum. Our work might be very useful for understanding the mechanism of long-distance transport of heavy metals in hyperaccumulator.

Evaluación del efecto irritante e hidratante de ésteres de alfa-hidroxiácidos / Evaluation of irritation and moisturizing effect of alfa-hydroxy acids

El objetivo de este trabajo fue evaluar la irritación y el efecto hidratante de los ésteres de AHA´s. Se evaluó el efecto irritante de los ésteres málico, láctico y salicílico en comparación a sus respectivos ácidos utilizando dos grupos de voluntarios. Las concentraciones de ésteres y AHA´s evaluadas en el primer grupo fueron 1 por ciento, 2,5 por ciento y 5 por ciento en aceite mineral como vehículo. En el segundo grupo se aplicaron ésteres y AHA´s a una concentración de 4 por ciento, 6 por ciento y 8 por ciento en carbomer al 1 por ciento como vehículo. Se determinó el efecto hidratante agudo de los ésteres málico, láctico y salicílico incluidos en una emulsión mediante utilización de un Corneómetro CM 820. Las mediciones se realizaron a los 0,60 y 120 minutos sobre los brazos de voluntarios sanos con piel normal. Los resultados obtenidos, a partir de la Prueba del Parche, fueron sometidos a un análisis estadístico con el que se concluyó que no existieron diferencias estadísticamente significativas entre el potencial de irritación de los ésteres de ácido láctico (EAL), málico (EAM) y salicílico (EAS) en relación a sus respectivos ácidos. Los valores obtenidos en la evaluación del efecto hidratante se analizaron por un método estadístico sin obtener diferencias significativas entre las mediciones (AU)

Absorption of calcium fumarate salts is equivalent to other calcium salts when measured in the rat model.

Calcium absorption from fumarate salts (calcium fumarate and calcium malate fumarate), which have recently been considered for use as sources for food and beverage enrichment, was compared to that from calcium citrate malate, calcium citrate, and calcium carbonate. Salts were instrinsically labeled with 45Ca and orally administered to Sprague-Dawley rats. Fractional absorption of calcium from each salt was determined using the femur uptake model. Fractional absorption from the five salts (0.30-0.27) was not significantly different (p > 0.05). Thus, when measured in the rat model, calcium from calcium fumarate and calcium malate fumarate is absorbed equally well as compared to other salts, which are common calcium sources in many foods, beverages, and supplements.

Boronic acid receptors for alpha-hydroxycarboxylates: high affinity of Shinkai's glucose receptor for tartrate.

The glucose receptor 1 developed by Shinkai was synthesized by known methods and with modifications involving the final synthetic step, installation of the phenylboronic acid moieties. Binding of the bis(alpha-hydroxycarbolxylate), tartrate, was assessed and compared to the corresponding bis(diol), erythritol, as well as the corresponding mono(alpha-hydoxycarboxylate), malate. These results suggest that bisboronate/bis(alpha-hydoxycarboxylate) interactions are stronger than the corresponding bisboronate/bis(diol) interactions. Furthermore, we report that the receptor is an order of magnitude more selective for tartrate than malate.

Cerebral dicarboxylate transport and metabolism studied with isotopically labelled fumarate, malate and malonate.

Transport and metabolism of dicarboxylates may be important in the glial-neuronal metabolic interplay. Further, exogenous dicarboxylates have been suggested as cerebral energy substrates. After intrastriatal injection of [(14) C]fumarate or [(14) C]malate, glutamine attained a specific activity 4.1 and 2.6 times higher than that of glutamate, respectively, indicating predominantly glial uptake of these four-carbon dicarboxylates. In contrast, the three-carbon dicarboxylate [(14) C]malonate gave a specific activity in glutamate which was approximately five times higher than that of glutamine, indicating neuronal uptake of malonate. Therefore, neurones and glia take up different types of dicarboxylates, probably by different transport mechanisms. Labelling of alanine from [(14) C]fumarate and [(14) C]malate demonstrated extensive malate decarboxylation, presumably in glia. Intravenous injection of 75 micromol [U-(13) C]fumarate rapidly led to high concentrations of [U-(13) C]fumarate and [U-(13) C]malate in serum, but neither substrate labelled cerebral metabolites as determined by (13) C NMR spectroscopy. Only after conversion of [U-(13) C]fumarate into serum glucose was there (13) C-labelling of cerebral metabolites, and only at <10% of that obtained with 75 micromol [3-(13) C]lactate or [2-(13) C]acetate. These findings suggest a very low transport capacity for four-carbon dicarboxylates across the blood-brain barrier and rule out a role for exogenous fumarate as a cerebral energy substrate.

Synthesis and biological evaluation of analogues of the antibiotic pantocin B.

Strains of the bacteria Erwinia herbicola produce antibiotics that effectively control E. amylovora, the bacterial pathogen responsible for the plant disease fire blight. Pantocin B was the first of these antibiotics to be characterized, and a flexible synthesis of various analogues is reported. Embedded in the "pseudo-tripeptide" backbone of pantocin B are a methylenediamine and a methyl sulfone, both unusual structural features in natural products. The peptidic nature of pantocin B facilitated a series of structure-activity relationship studies that probed the roles of these functional groups in determining the biological activity of pantocin B. A clear demarcation of the roles between the N- and C-terminal portions of the antibiotic was determined as a result of the structure-activity relationship studies. The N-terminal L-alanyl group is needed for cellular import but not for interaction with the intracellular target, the arginine biosynthetic enzyme N-acetylornithine aminotransferase. The methylenediamine and methyl sulfone portions were found to be essential for antibiotic activity, presumably due to extensive interactions with N-acetylornithine aminotransferase.