Cell-penetrating peptides meditated encapsulation of protein therapeutics into intact red blood cells and its application.

Red blood cells (RBCs) based drug carrier appears to be the most appealing for protein drugs due to their unmatched biocompatability, biodegradability, and long lifespan in the circulation. Numerous methods for encapsulating protein drugs into RBCs were developed, however, most of them induce partial disruption of the cell membrane, resulting in irreversible alterations in both physical and chemical properties of RBCs. Herein, we introduce a novel method for encapsulating proteins into intact RBCs, which was meditated by a cell penetrating peptide (CPP) developed in our lab-low molecular weight protamine (LMWP). l-asparaginase, one of the primary drugs used in treatment of acute lymphoblastic leukemia (ALL), was chosen as a model protein to illustrate the encapsulation into erythrocytes mediated by CPPs. In addition current treatment of ALL using different l-asparaginase delivery and encapsulation methods as well as their associated problems were also reviewed.

Co-administration of protein drugs with gold nanoparticles to enable percutaneous delivery.

An interesting nanoscale interfacial phenomenon mediated by gold nanoparticles (Au-NPs) was found, in that co-administration with Au-NPs enables percutaneous delivery of protein drugs. The Au-NPs with a mean size of 5 nm were revealed to be skin permeable, presumably due to the nano-bio interaction with skin lipids and the consequent induction of transient and reversible openings on the stratum corneum. Importantly, when simultaneously applied with Au-NPs, the protein drugs were also granted the ability to penetrate the skin barrier and migrate into the deep layers. This indicated that co-administration with the skin-permeable Au-NPs could mediate proteins across the skin barrier. Such co-delivery effect highlights a simple yet effective method for overcoming the skin barrier for percutaneous protein drug delivery. Employing this method, a non-invasive vaccine delivery strategy was developed, and by topically co-administrating antigens with Au-NPs, robust immune responses were elicited in the tested animals. The results provide the promise for achieving a needleless and self-administrable transcutaneous vaccination.

ATTEMPTS system: a macromolecular prodrug strategy for cancer drug delivery.

In order to reduce systemic toxicity and effectively deliver macromolecular drug into tumor cells, a system termed "ATTEMPTS" (antibody targeted, [protamine] triggered, electrically modified prodrug-type strategy) was developed in our laboratory. This approach was adapted from our previously reported heparin/protamine-based system for controlled delivery of protease drugs such as tissue- specific plasminogen activator (tPA). In this "ATTEMPTS" system, the cell-permeable protein drugs are synthesized by conjugating proteins to cell-penetrating peptides (CPPs). Cell penetration ability of such CPP-protein conjugates would initially be disabled, acting as a "prodrug", by forming polyelectrolyte complexes with a functionalized heparin-antibody moiety. The complexes would accumulate in tumor sites by the antibody targeting function, and then the local release of CPP-protein conjugates would be triggered by protamine. We applied this system to the macromolecular anticancer agents, such as the protein drugs (gelonin and asparaginase) as well as the polymerdrugs (polyrotaxane-doxorubicin and polyrotaxane-camptothecin). Both in vitro and preliminary in vivo studies demonstrated the regulable cell penetration behavior based on the competitive ionic interactions between CPP/heparin and heparin/protamine. Thus, this ATTEMPTS approach provides a multi-functionalized system incorporating the features of targeting, prodrug-like, triggerable release, and cell penetration ability for the delivery of macromolecular anticancer agents. A summary of our work on "ATTEMPTS" is presented in this review.

Specific down regulation of 3T3-L1 adipocyte differentiation by cell-permeable antisense HIF1alpha-oligonucleotide.

Hypoxia is a strong modulator of angiogenesis, accelerating adipose tissue expansion, suggesting that hypoxia inducible factor 1alpha (HIF1alpha) can be a novel target for anti-obesity. We conjugated antisense-HIF1alpha-oligonucleotide (ASO) with low molecular weight protamine (LMWP), a cell-penetrating peptide, to enhance its ability to block hypoxic-angiogenesis, thereby eliciting an anti-obesity effect. Nano-sized ASO-LMWP (AS-L) conjugates enhanced cellular uptake of ASO without yielding a cytotoxic effect and protected the ASO against enzymatic attack and chemical reduction. AS-L showed enhanced intra-cellular localization compared to naked ASO and the complex of ASO with lipofectamine during hypoxic-differentiation. Consequently AS-L induced significant down-regulation of leptin and VEGF gene expressions, thereby reducing fat accumulation in the cell. This proof-of-concept study shows that AS-L produces an inhibitory effect on adipogenesis and angiogenesis during differentiation, indicating LMWP mediated ASO delivery can potentially be a safe and promising treatment for obesity.

L-Asparaginase encapsulated intact erythrocytes for treatment of acute lymphoblastic leukemia (ALL).

As a primary drug for the treatment of acute lymphoblastic leukemia (ALL), encapsulation of L-asparaginase (ASNase) into red blood cells (RBC) has been popular to circumvent immunogenicity from the exogenous protein. Unlike existing methods that perturbs RBC membranes, we introduce a novel method of RBC-incorporation of proteins using the membrane-translocating low molecular weight protamine (LMWP). Confocal study of fluorescence-labeled LMWP-ovalbumin, as a model protein conjugate, has shown significant fluorescence inside RBCs. Surface morphology by scanning electron microscopy of the RBCs loaded with LMWP-ASNase was indistinguishable with normal RBCs. These drug loaded RBCs also closely resembled the profile of the native erythrocytes in terms of osmotic fragility, oxygen dissociation and hematological parameters. The in vivo half-life of enzyme activity after administering 8 units of RBC/LMWP-ASNase in DBA/2 mice was prolonged to 4.5+/-0.5 days whereas that of RBCs loaded with ASNase via a hypotonic method was 2.4+/-0.7 days. Furthermore, the mean survival time of DBA/2 mice bearing mouse lymphoma cell L5178Y was improved by approximately 44% compared to the saline control group after treatment with the RBC loaded enzymes. From these data, an innovative, novel method for encapsulating proteins into intact and fully functional erythrocytes was established for potential treatment of ALL.

Postmortem blood concentrations of organophosphorus pesticides.

Cases involving acute fatalities due to ingestion of organophosphorus pesticides (OPs), such as chlorpyrifos, diazinon, malathion and parathion, are presented. Solid-phase extraction (SPE) and gas chromatography/mass spectrometry (GC/MS) were used for the analysis of OPs in postmortem blood. After extraction with an Oasis HLB cartridge, the eluent was evaporated to dryness under a nitrogen stream at 35 degrees C, reconstituted with ethanol, and then analyzed by GC/MS. Terbufos was used as an internal standard. Verification procedures, such as the limit of detection, limit of quantification, linearity of the calibration, precision and recovery were performed. Validation data were adequate for analyzing OPs in blood. Chlorpyrifos, diazinon, malathion and parathion were detected in 31 postmortem blood samples. Parathion was the most frequently detected compound among the four pesticides. The mean concentrations of chlorpyrifos, diazinon, malathion and parathion were 0.72, 1.03, 0.82 and 2.90 mg/L, respectively.

Preliminary in vivo evaluation of the protein transduction domain-modified ATTEMPTS approach in enhancing asparaginase therapy.

Asparaginase (ASNase) is an enzyme drug presently approved for the induction of remission in the treatment of patients with acute lymphoblastic leukemia (ALL). The cytotoxic effect of ASNase is derived from its ability to deplete asparagine, an essential amino acid required by certain types of leukemia cells for protein synthesis and survival. Despite its efficacy in enhancing disease remission rate and prolonging complete remission duration in ALL patients, ASNase therapy is nevertheless confounded by a number of serious toxic effects, particularly to organs associated with high protein production (e.g., liver, pancreas), due to the systemic depletion of asparagine. Presented herein is a modified version of our previously established ATTEMPTS protein delivery system that carries the potential to permit a tumor specific, intracellular delivery of ASNase, thereby allowing for a significant reduction of ASNase-induced systemic toxicity. In a previous paper, we already demonstrated the in vitro feasibility of this heparin/protamine-regulated, TAT-mediated system in delivering ASNase directly into ASNase-sensitive murine lymphoma cells. In this article, we further validated the in vivo applicability of this system in animals harboring ASNase-encapsulated L5178Y lymphoma cells. Preliminary results showed that animals inoculated with L5178Y cells containing TAT-ASNase exhibited an extended survival rate of approximately 13% over those harboring L5178Y cells without the encapsulation of ASNase. Furthermore, the TAT-ASNase-treated mice also displayed a significantly improved hematological and liver histological status than the control groups. These findings bring promise to the use of the modified ATTEMPTS delivery system in achieving enhanced ASNase therapy.

Determination of impurities in illicit methamphetamine seized in Korea and Japan.

Impurities produced during synthesis of methamphetamine (MA) show different patterns under various synthetic conditions. Valuable information on the origins and smuggling routes can be obtained by using impurities as chemical fingerprints. We have detected more than 100 compounds from 436 MA samples seized in Korea by gas chromatography-flame ionization detector and gas chromatography-mass spectrometer, among which 31 impurities and three additives were identified. Twenty-six impurity peaks including unknowns were selected as the indicators of similarity, and were used as variables for cluster analysis. Cluster analysis result showed that part of the MA samples seized in Japan might have the same origin as those seized in Korea. It means that broad-based cooperation is necessary for efficient regulation of MA. Synthetic trends of the MA seizures of Korea were monitored by cluster analysis with 16 MA samples synthesized by three different methods in the previous work. We could find comparable changes of synthetic trends, which might have been influenced by domestic regulations and international situations.

Analysis of ketamine and norketamine in urine by automatic solid-phase extraction (SPE) and positive ion chemical ionization-gas chromatography-mass spectrometry (PCI-GC-MS).

Ketamine (KT) is widely abused for hallucination and also misused as a "date-rape" drug in recent years. An analytical method using positive ion chemical ionization-gas chromatography-mass spectrometry (PCI-GC-MS) with an automatic solid-phase extraction (SPE) apparatus was studied for the determination of KT and its major metabolite, norketamine (NK), in urine. Six ketamine suspected urine samples were provided by the police. For the research of KT metabolism, KT was administered to SD rats by i.p. at a single dose of 5, 10 and 20mg/kg, respectively, and urine samples were collected 24, 48 and 72 h after administration. For the detection of KT and NK, urine samples were extracted on an automatic SPE apparatus (RapidTrace, Zymark) with mixed mode type cartridge, Drug-Clean (200 mg, Alltech). The identification of KT and NK was by PCI-GC-MS. m/z238 (M+1), 220 for KT, m/z 224 (M+1), 207 for NK and m/z307 (M+1) for Cocaine-D(3) as internal standard were extracted from the full-scan mass spectrum and the underlined ions were used for quantitation. Extracted calibration curves were linear from 50 to 1000 ng/mL for KT and NK with correlation coefficients exceeding 0.99. The limit of detection (LOD) was 25 ng/mL for KT and NK. The limit of quantitation (LOQ) was 50 ng/mL for KT and NK. The recoveries of KT and NK at three different concentrations (86, 430 and 860 ng/mL) were 53.1 to 79.7% and 45.7 to 83.0%, respectively. The intra- and inter-day run precisions (CV) for KT and NK were less than 15.0%, and the accuracies (bias) for KT and NK were also less than 15% at the three different concentration levels (86, 430 and 860 ng/mL). The analytical method was also applied to real six KT suspected urine specimens and KT administered rat urines, and the concentrations of KT and NK were determined. Dehydronorketamine (DHNK) was also confirmed in these urine samples, however the concentration of DHNK was not calculated. SPE is simple, and needs less organic solvent than liquid-liquid extraction (LLE), and PCI-GC-MS can offer both qualitative and quantitative information for urinalysis of KT in forensic analysis.

Comparison study of the extraction methods of paraquat in post-mortem human blood samples.

The most important step for Paraquat analysis in post-mortem human blood (PMB) is its extraction from the specimens, as Paraquat is insoluble in organic solvents due to its ionic form. The most common extraction method, solid phase extraction (SPE), has been used for the extraction of Paraquat from PMB. However, SPE procedures are somewhat time-consuming, and resulted in unsatisfactory recovery in our laboratory. Therefore, SPE procedures, with five extraction solvents for the liquid-liquid extraction (LLE) of paraquat in PMB, were compared using HPLC, and the chloroform-ethanol (7:3, v/v) solvent mixture was found to be the most effective. The recoveries of Paraquat using the 7:3 solvent mixture in human whole blood samples, which were already spiked with paraquat standards (1.05, 2.10 and 4.21 microg/mL) averaged 98.20, 105.71 and 99.40%, but the recoveries from the SPE were about 74.29, 78.50 and 80.10%, respectively. Linearity was obtained for the range of Paraquat standards, with a correlation coefficient; r2 > 0.999. The limit of detection (LOD, with S/N > or =3) and limit of quantitation (LOQ, with S/N > or =10) were 0.01 and 0.05 microg/mL, respectively. The extraction method was successfully applied to seven real post-mortem cases involving paraquat poisoning.

Bronchial washing yield before and after forceps biopsy in patients with endoscopically visible lung cancers.

BACKGROUND AND OBJECTIVE: The need for routine use of bronchial washing in addition to forceps biopsy has been debated in the diagnosis of endoscopically visible lung tumours. Moreover, the optimal sequence for obtaining bronchial washing and forceps biopsy specimens from endoscopically visible tumours through a flexible bronchoscope has not been well established. METHODS: A multicentre 13-month prospective randomized study was performed. Two hundred and thirty consecutive patients with endoscopically visible tumours were randomly assigned into a bronchial washing before forceps biopsy (pre-biopsy) group and a bronchial washing after forceps biopsy (post-biopsy) group. Bronchial washing and forceps biopsy were performed according to the assigned sequence. RESULTS: Two hundred and seven patients with a definite cytological or histological diagnosis of lung cancer were included in the analyses. One hundred and three were in the pre-biopsy group and 104 were in the post-biopsy group. The diagnostic yield of bronchial washing was 57.3% (59/103) in the pre-biopsy group and 55.8% (58/104) in the post-biopsy group (P = 0.88). In addition, bronchial washing provided the diagnosis in six patients without definite diagnosis from forceps biopsy, and its addition to forceps biopsy significantly increased the overall diagnostic yield of bronchoscopy from 93.7% to 96.6% (P = 0.03). CONCLUSION: The sequence for performing bronchial washing before or after forceps biopsy did not affect the diagnostic yield of bronchial washing in patients with endoscopically visible lung cancers. However, bronchial washing led to a significant increase in the overall diagnostic yield of bronchoscopy in patients with lung cancers.

Monitoring precursor chemicals of methamphetamine through enantiomer profiling.

Smuggling of methamphetamine is affected by enforced regulation and international situation, resulting in changes of precursors and synthetic methods used. Enantiomer ratio of methamphetamine can provide information concerning its precursor and synthetic method. This information is useful for the prevention of smuggling methamphetamine and its precursor, and resultant reduction of methamphetamine abuse. In the present study, we investigated on the enantiomer ratios of 433 crystalline methamphetamine samples seized in Korea from 1994 to 2005. Excluding 17 samples of low purity, 416 samples were used for enantiomer profiling. The methamphetamine samples were derivatized with (S)-(+)-alpha-methoxy-alpha-(trifluoromethyl)phenylacetyl chloride ((S)-(+)-MTPACl), and the derivatives were analyzed by GCMS in selected ion monitoring (SIM) mode. The enantiomer ratios of the samples were calculated from the standard calibration curves of each enantiomer, both of which showed good linearity in the range of 0-1.2 microg. Most of the seizures were pure S(+)-enantiomer, but 21% (95 of 416 samples) contained R(-)-enantiomer above 1%. They began to appear from 1997, and increased continuously up to 50% in the year 2005 (55 of 111 samples). From this study, we could find out that alternative precursors have been used recently for the illicit manufacture of methamphetamine seized in Korea.

Analysis of the impurities in the methamphetamine synthesized by three different methods from ephedrine and pseudoephedrine.

Organic impurities of methamphetamine may show different patterns by synthesis. In the present study, we tried to find the impurities reflecting the conditions of synthesis by comparing impurity patterns of the methamphetamine samples synthesized by different methods. Sixteen methamphetamine samples were synthesized from ephedrine and pseudoephedrine by the three different manufacturing methods of Emde, Nagai and Moscow. The synthesized samples were extracted with ethyl acetate containing four internal standards, and the patterns of the organic impurities were investigated by GC-MS and GC-FID . Through the investigation, we found 10 peaks appearing in the latter part of GC chromatograms are characteristic to synthesis. The areas of the selected peaks were converted to the variables suitable for the statistical calculation, and the synthesized samples could be classified into four groups through the resultant cluster analysis.

Standardization of method for the analysis of dextromethorphan in urine.

Dextromethorphan (DMP), an antitussive, is one of the most popular drugs among the younger generation in Korea. It usually is taken for its hallucinogenic properties and overdoses have been responsible for the fatalities that have been reported frequently. To control the abuse of DMP, the authorities restricted its use through classifying it as a controlled drug on October 2003. The purpose of this study is to provide a standard method for the analysis of DMP and its main metabolite, dextrorphan (DTP) in biological specimens. At first we established a standard operating procedure (SOP) for DMP/DTP in urine, and a method validation was performed. We also quantified DMP from 16 drug abuser's urine samples all of which were positive in the screening test for DMP. For the detection of DMP/DTP, urine samples were adjusted with 6N NaOH (pH 11) and extracted with ethylacetate. Thin layer chromatography was used as the screening test, and the final identification for DMP/DTP was used by GC/MS. The ions (m/z 271 for DMP, m/z 257 for DTP and m/z 86 for lidocaine as internal standard) were extracted from the full scan mass spectrum and were used for quantification. The selectivity, linearity of calibration, accuracy, within- and between day precision, limit of detection and quantification, recovery and stability were examined as parts of the method validation. Extracted calibration curves were linear from 100 to 2000 ng/mL for DMP and DTP with correlation coefficients better than 0.999. Limit detection was 50 ng/mL for DMP and DTP. Within-run precision (%CV) for DMP and DTP at three different concentrations (100, 500 and 1000 ng/mL) was 6.10-18.85%, and between-run precision was 1.70-7.86% for DMP and DTP. Absolute recovery for DMP and DTP was 57-74%, and relative recovery (extraction efficiency) was 80-89%. For 16 drug abuser's urine samples, the concentrations of DMP and DTP were 0.16-52.63 and 0.41-23.75 microg/mL, respectively. Method validation is an important requirement in the practice of chemical analysis, and it will be particularly useful in verifying the reliability of analytical results in the field of forensic science.

Effect of alpha-tocopherol and deferoxamine on methamphetamine-induced neurotoxicity.

Methamphetamine (MA)-induced dopaminergic neurotoxicity is believed to be associated with the increased formation of free radicals. This study examined the effect of alpha-tocopherol (alpha-TC), a scavenger of reactive oxygen species, and deferoxamine (DFO), an iron chelator, on the MA-induced neurotoxicity. Male rats were treated with MA (10 mg/kg, every 2 h for four injections). The rat received either alpha-TC (20 mg/kg) intraperitoneally for 3 days and 30 min prior to MA administration or DFO (50 mg/kg) subcutaneously 30 min before MA administration. The concentrations of dopamine (DA), serotonin and their metabolites decreased significantly after MA administration, which was inhibited by the alpha-TC and DFO pretreatment. alpha-TC and DFO attenuated the MA-induced hyperthermia as well as the alterations in the locomotor activity. The level of lipid peroxidation was higher and the reduced glutathione concentration was lower in the MA-treated rats. These changes were significantly attenuated by alpha-TC and DFO. This suggests that alpha-TC and DFO ameliorate the MA-induced neuronal damage by decreasing the level of oxidative stress.

Case of complete recovery of pancytopenia after treatment of hypopituitarism.

We describe a 55-year-old woman who presented with pancytopenia with a normocytic and normochromic anemia which was progressive despite conventional treatments such as folic acid, vitamin B6, and oxymetholone. Her physical findings and history of a previous massive postpartum hemorrhage suggested Sheehan's syndrome, and the pituitary hormonal studies revealed panhypopituitarism. After 4 months of thyroxine and glucocorticoid replacement therapy, her pancytopenia and bone marrow hypoplasia recovered completely. Pancytopenia is a rare manifestation of a hormonal abnormality, but hematologists need to be aware of panhypopituitarism as a differential diagnosis when women showing features of hypopituitarism present with pancytopenia because it can be reversed with adequate hormone replacement.

Lidocaine intoxication: two fatal cases.

We present two fatal cases, a 41-year-old male (case 1) and his 8-year-old daughter (case 2), resulting from acute lidocaine poisoning. Lidocaine was quantified by gas chromatography (GC) analysis using nitrogen-phosphorus detection. The lidocaine concentrations of cases 1 and 2 were: liver, 27.7 microg/g and 24.9 microg/g; spleen, 70.1 microg/g and 29.9 microg/g; and gastric contents, 23.6 microg/g and 42.8 microg/g, respectively.

Interpretations of the TDxFLx calibration data of the abused drugs.

The useful TDxFLx calibration data was obtained for the interpretation of the interactions of the abused drugs to sheep antiserum protein. The antibody of TDxFLx calibrators was prepared from sheep antiserum. Furthermore these data can be used to interpret the abused drug-protein binding phenomena in human body and the TDxFLx screening results of the abused drugs in urine samples. TDxFLx system uses fluorescence polarization immunoassay technique that is a competitive binding immunoassay methodology to allow tracer-labeled antigen (*Drug) and patient antigen (Drug) to compete for the same binding sites on the antibody molecules of sheep antiserum. To obtain the binding parameters, binding constant (K) and number of independent binding site (n), generally, Scatchard equation is used. This Scatchard equation is expressed in the concentration terms of free drug, bound drug, and protein (antibody). The binding parameters can not be obtained by applying the TDxFLx calibration data to the Scatchard equation directly because the TDxFLx calibration data are composed of the fluorescence polarization and the total drug concentrations. To obtain the binding parameters from the TDxFLx calibration data the new useful equation which was expressed in the total concentrations of drug and fluorescence polarization should be derived. Derivation of new equation was based on the Scatchard equation. The TDxFLx calibration data was curve fitted to the derived equation using KaleidaGraph program and Macintosh computer. The binding constant (K) and the number (n(P(t))) of binding site of 11-nor-delta(9)-tetrahydrocannabinol-9-carboxylic acid (COOH.THC) on the antibody were 1.14 x 10(8)l/mole and 4.04 x 10(-7)M, respectively. The binding constant and the number (n(P(t))) of binding site of amphetamine were 5.15 x 10(5)l/mole and 2.05 x 10(6)M, respectively. In case of COOH.THC the fluorescence polarization decreased linearly with the concentration. However, in case of amphetamine or the other three abused drugs the fluorescence polarizations decreased exponentially with their concentrations.