Distribution of drugs over whole blood: I. The transport function of whole blood for valproate

A method is presented for the determination of valproate (VPA) in approximately 40 microliters erythrocyte concentrations. Valproate partition between plasma proteins, ultrafiltrate, and erythrocytes at various concentrations was studied in vitro and in vivo. Partition ratios depended on VPA concentration in whole blood and the ratio of erythrocytes/ultrafiltrate, which increased with rising concentrations in the ultrafiltrate fraction. Shifts in ratios were studied by expanding the ultrafiltrate fraction of VPA-spiked blood. It appeared that VPA delivery to the expanded ultrafiltrate compartment originated in a disproportionately large part from erythrocytes. In vivo the half-life of VPA in the erythrocyte fraction was 0.7 h, whereas in the ultrafiltrate fraction and protein-bound fraction half-lives of 2 and 5 h were observed. It is concluded that for VPA in relation to erythrocytes a "last-come-first-go" system is plausible.     

Distribution of drugs over whole blood: III. The transport function of whole blood for hydrocortisone

Hydrocortisone (HC) distribution over the three main components of blood, i.e., plasma water, plasma proteins, and erythrocytes, was studied in vitro at various HC concentrations in plasma, in a suspension of washed erythrocytes in plasma water, and in whole blood. The distribution ratio of HC in the system of erythrocytes in plasma water was 2.1 when HC, 0.18-10.8 micrograms/ml, was added. In whole blood, however, this ratio was 2.4 for the same concentration range. In the HC range of 0.18-0.68 micrograms/ml of whole blood, the uptake percentage of HC by erythrocytes increased from about 16 to 28% of the amount of HC added. By adding blank ultrafiltrate to HC-enriched blood, it appeared that the erythrocyte fraction released HC in overproportional quantities compared with the release by plasma proteins. Similar findings have previously been reported regarding the drugs valproic acid and phenytoin. Migration of HC from HC-spiked plasma to blank erythrocytes reached an equilibrium within 5 min.     

Therapeutic monitoring of chlorpromazine II: Pitfalls in whole blood analysis

Pooled whole blood from healthy volunteers was spiked with pure, synthetic chlorpromazine, chlorpromazine sulfoxide, or chlorpromazine N-oxide and then made alkaline with either sodium hydroxide or sodium carbonate. The addition of alkali causes lysis of red cells, the contents of which spill into the plasma. The lysed samples were allowed to stand at room temperature for various timed intervals before extraction with organic solvents and analysis by high performance liquid chromatography. It was found that a portion (10-14%) of the chlorpromazine spike was oxidised to chlorpromazine sulfoxide, whether the blood was made alkaline with sodium hydroxide or sodium carbonate. Chlorpromazine N-oxide added to whole blood was entirely destroyed in the presence of alkali. The chlorpromazine N-oxide was rapidly reduced to chlorpromazine, a portion of which subsequently underwent oxidation to chlorpromazine sulfoxide. We have found that chlorpromazine N-oxide resides almost entirely in the plasma with only a small portion (less than 4%) distributed into the red cells. Hence, it is essential that red cells and plasma be separated before analysis. Chlorpromazine and chlorpromazine N-oxide can then be extracted from plasma by a method that does not lead to reduction of chlorpromazine N-oxide. Alkaline extraction methods must be avoided in the analysis of chlorpromazine in the red cell fraction.     

Toxicological screening of basic drugs in whole blood using UPLC-TOF-MS

Ultra performance liquid chromatography (UPLC) coupled with time-of-flight (TOF) mass spectrometry (MS) was established for toxicological screening of basic drugs in whole blood and tested on authentic samples. Whole blood samples (0.2 ml) were extracted using a Gilson apparatus equipped with Bond Elut Certify columns. Screening was performed for 175 compounds (psychotropic, cardiovascular, designer, and abused drugs). The drugs were separated in 15 min using a UPLC system (Waters ACQUITY BEH C18, 1.7 μm, 2.1 mm × 100 mm column) coupled to an LCT Premier XE (Waters) instrument. Data were processed by ChromaLynx XS using identification criteria of ± 0.2 min retention time, and ± 5 mDa mass tolerance. Whole blood was spiked with the 175 compounds in concentrations from 5-100 μg/kg to assess approximately the lowest concentrations that could be identified. This method was further applied to 119 samples from forensic investigations, leading to 302 hits, of which 291 (96%) were subsequently verified, in concentrations exceeding the lower limit of quantification (LLOQ), by a liquid chromatography (LC)-MS/MS confirmation method. In conclusion, this UPLC-TOF-MS method is a useful and effective screening method for basic drugs in whole blood.     

IL-18BPa:Fc cooperates with immunosuppressive drugs in human whole blood

The proinflammatory cytokine interleukin (IL)-18 appears to be involved in the pathogenesis of diseases associated with immunoactivation and inflammation. Consequently, blockage of IL-18 bioactivity by use of IL-18 binding protein (IL-18 BP) is likely a promising therapeutic concept. In the present study, we investigated immunomodulatory activities of IL-18 BPa:Fc in human whole blood cultures. We report that IL-18 BPa:Fc (200 ng/mL) significantly inhibited lipopolysaccharide (LPS, 10 ng/mL)/IL-12 (5 ng/mL)-induced release of interferon-gamma (IFNgamma) and matrix metalloproteinase-9 (MMP-9) from whole blood cultures of healthy donors. Notably, IL-18 BPa:Fc (200 ng/mL) further reinforced dexamethasone (5 nM)- or mycophenolic acid (2 microM)-mediated reduction of LPS/IL-12-induced IFNgamma production by an additional 50.5 or 49.9%, respectively. To investigate effects of IL-18 BP:Fc in the context of autoimmune diseases, experiments were performed with whole blood obtained from patients with systemic lupus erythematosus or Wegener's granulomatosis undergoing immunosuppressive therapy. After ex vivo stimulation with LPS (10 ng/mL), production of IFNgamma and MMP-9 was determined. Both mediators likely contribute to renal inflammation frequently seen in these diseases. In accord with the aforementioned data, LPS (10 ng/mL)-induced IFNgamma was significantly reduced by coincubation with IL-18 BPa:Fc at 200 ng/mL. IL-18 BPa:Fc also inhibited production of MMP-9. The present data demonstrate that IL-18 BPa:Fc has the potential to amplify anti-inflammatory actions of immunosuppressive drugs, and thus may prove to be a valuable novel pharmacological component in the treatment of human autoimmune diseases.     

Binding of chlorpromazine, phenytoin and aspirin to the erythrocytes and lipoproteins in whole human blood

The quantitative binding of 14C-labelled chlorpromazine, phenytoin or aspirin (at 10 microM) to blood cells and plasma lipoproteins in whole human blood or to the washed erythrocytes in an isotonic protein-free medium has been studied. The fractions of chlorpromazine, phenytoin and aspirin bound to the blood cells in whole blood amounted to about 40, 14 and 2% of the total amount added, and those to the lipoproteins amounted to 7, 2 and 1%, respectively. Their binding to the washed erythrocytes in protein-free medium was 95, 76 and 40%, respectively. Their octanol:water partition coefficients were 214, 170 and less than 0.1, respectively. These results suggest that the amphiphilic drugs with relatively high hydrophobicity may be bound to the blood cells, mainly to erythrocytes, to considerable extents when administered clinically, and also that their binding to plasma lipoproteins may not be negligible.     

Inhibition of phenytoin metabolism by other drugs used in epilepsy.

Phenytoin metabolism is saturable within its normal therapeutic range and, therefore, small changes in the activity of the enzyme can lead to marked changes in serum phenytoin concentrations. The anticonvulsant drugs sulthiame and pheneturide both inhibit the metabolism of phenytoin. The mechanism of this interaction appears to be different for these two drugs. Nortriptyline produces a small increase in serum phenytoin concentrations, but this is unlikely to be of clinical importance. Case reports suggest that both chlorpromazine and chloramphenicol inhibit phenytoin metabolism to a significant degree.     

Adenosine metabolism in human whole blood. Effects of nucleoside transport inhibitors and phosphate concentration

Adenosine (Ado, 10 microM) was metabolized in whole blood within 1 min, primarily to hypoxanthine and ATP. The concentration of Ado, the activities of adenosine deaminase (ADA) and Ado kinase, the Km values for Ado with ADA and Ado kinase, and the substrate inhibition of Ado kinase are factors that govern the Ado metabolism between deamination and phosphorylation. If ADA activity was blocked by 2'-deoxycoformycin (dCF, 5 microM), a tight-binding inhibitor of ADA, most of the Ado (96%) was incorporated into adenine nucleotides, whereas if Ado kinase activity was blocked with 5-iodotubercidin (10 microM), Ado was mainly (95%) metabolized into hypoxanthine. A high phosphate concentration (25 mM) caused marked increases in the formation of IMP. The nucleoside transport inhibitors dilazep (1 microM), dipyridamole (10 microM) and nitrobenzylthioinosine (NBMPR, 1 microM) strongly blocked cellular Ado metabolism. In the presence of nucleoside transport inhibitors, Ado which slowly enters the cell was metabolized principally by Ado kinase rather than ADA. Dilazep, NBMPR and dipyridamole were more effective in blocking Ado uptake and metabolism by erythrocytes suspended in a protein-free medium than by cells suspended in plasma.     

In vivo distribution of hydrocortisone over whole blood: a novel method for the extraction of erythrocytes

Ten mg hydrocortisone (HC) was administered intravenously to a healthy volunteer after a dexamethasone suppression test and HC concentrations were determined from 1-270 min in plasma, plasma water and on erythrocytes. HC was extracted from erythrocyte concentrates with high efficiency by HC-poor plasma or by human or bovine albumin solutions. Determination of HC in the plasma of the volunteer mainly gave insight about the concentration time course of HC bound to plasma proteins. One minute after HC injection the amount associated with erythrocytes was about half the amount bound to plasma proteins. Decrease of HC in plasma, and hence on plasma proteins, was monophasic from 30-270 min with a half-life of 116 min. Decrease of HC associated with erythrocytes and HC free in plasma water was biphasic from 30-270 min and initially HC diminished about five times faster from these compartments than from plasma proteins. At the end of the observation period half-lives on plasma proteins, erythrocytes and in plasma water were similar, i.e., at 120 min. It is concluded from these as well as from previous in vitro experiments that erythrocytes gain importance as HC carriers at increasing HC blood concentrations. Once charged, erythrocytes yield HC much more readily than do plasma proteins. This "last come first go" phenomenon of association of HC with erythrocytes is known also to exist for certain drugs. It indicates erythrocytes as important transporters of non-freely in water soluble compounds.     

Optimizing the lymphocyte transformation test in whole blood. II. Kinetics of thymidine incorporation

From the 2nd day of incubation on, the thymidine incorporation per incubated lymphocyte increased exponentially with time. The duration of the exponential growth phase was inversely correlated with the number of cells. Under optimum conditions the average time for the doubling of thymidine incorporation (Ti) was 15.7 h. Ti after 1 day of incubation was taken as an equivalent for the number of proliferating cells. It was estimated that less than 20% of the incubated lymphocytes are stimulated by PHA under optimum conditions. In Ficoll-separated mononuclear cells, the percentage of cells stimulated by PHA was lower than in whole blood; the proliferation rate was not decreased.     

A single-step extraction for screening whole blood for basic drugs by capillary GC/NPD

Basic drugs were routinely extracted from whole blood under alkaline conditions into n-butyl acetate. An aliquot of the n-butyl acetate was injected into a gas chromatograph equipped with a nitrogen-phosphorous detector and a wide-bore cross-linked 50% phenylmethyl silicone capillary column. Absolute and relative retention times were recorded for more than 100 extracted drug standards. Recovery from whole blood was determined for some of the more frequently encountered drugs. This one-step extraction proved to be reliable for general screening and has been used routinely in forensic and clinical toxicological analyses.     

Self-nanoemulsifying drug delivery systems (SNEDDS) for oral delivery of protein drugs: II. In vitro transport study

To develop a self-nanoemulsifying drug delivery system (SNEDDS) for protein drugs, and particularly, to test the in vitro transport of beta-lactamase (BLM) by SNEDDS across the cell monolayer. Fluorescently labeled BLM (FITC-BLM), a model protein, formulated into 16 SNEDDS preparations through a solid dispersion technique were studied for transport across MDCK monolayer. All the SNEDDS nanoemulsions resulted in higher transport rate than the free solution. The transport rate by SNEDDS depends on the SNEDDS composition. SNEDDS NE-12-7 (oil: Lauroglycol FCC, surfactant: Cremophor EL and a cosurfactant: Transcutol HP) at the ratio of 5:4:3, rendered the highest transportation rate, 33% as compared to negligible transport by the free solution. FITC-BLM solution mixed with the surfactant and the cosurfactant of SNEDDS NE-12-7 or with blank SNEDDS NE-12-7 increased the transport only by 3.3 and 1.5 folds, respectively, compared to free solution alone. It was found that the monolayer integrity was not compromised in the presence of SNEDDS NE-12-7 or its surfactant/cosurfactant. The SNEDDS significantly increased the transport of FITC-BLM across MDCK monolayer in vitro. SNEDDS may be a potential effective delivery system for non-invasive protein drug delivery.     

Radioimmunoassay screening and GC/MS confirmation of whole blood samples for drugs of abuse

From 1981 to 1984, an average of 300 radioimmunoassay screens on whole blood were performed each week in the authors' laboratory. Most samples were screened for opiates, phencyclidine and its analogs, barbiturates, and cocaine or its metabolite benzoylecgonine. A commercially available radioimmunoassay was used with modifications to facilitate screening of whole blood. Increasing sample size increased the sensitivity of the assay. Changing reagent concentration (1:1 dilution), incubation time, sample matrix (water, urine, or blood), or fraction counted (precipitate or supernatant) did not affect the utility of the standard curve or the sensitivity of the assay. All positive results for phencyclidine, opiates, cocaine, and related compounds were confirmed by GC/MS. Barbiturate positives were confirmed by UV spectrophotometry. The rate of confirmation in postmortem bloods from coroner's cases for 1981-84 was: cocaine/benzoylecgonine, 57%; opiates, 79%; phencyclidine, 49%; and barbiturates, 58%.