The induction of prolonged myelopoietic effects in monkeys by GW003, a recombinant human granulocyte colony-stimulating factor genetically fused to recombinant human albumin.

GW003, a genetic fusion protein of human serum albumin and granulocyte colony-stimulating factor (G-CSF), was developed based on a novel strategy for producing long-acting proteins. The purpose of this study was to evaluate the hematologic, pharmacokinetic, and toxicokinetic effects of GW003 on cynomolgus monkeys. We show that following a single subcutaneous administration of GW003, the absolute neutrophil count increased significantly compared with monkeys that received only the vehicle, and the magnitude of the neutrophilic response to GW003 was dose dependent. After an injection at equal molar dose, the clearance of GW003 in the monkeys was approximately fourfold slower, and the terminal half-life (T1/2 ) was fivefold longer than the corresponding values for recombinant methionyl human G-CSF. Interestingly, both the clearance and T1/2 decreased with increasing doses of GW003, and much faster elimination was observed after multidose exposure. In toxicokinetic studies, the serum concentration of GW003 after the eighth injection was much lower than it was after the first injection, and a neutralizing antibody against G-CSF was found to have a dose-dependent effect upon the treatment groups. Overall, the favorable pharmacokinetic and pharmacodynamic properties supported the selection and development of GW003 as a promising candidate for neutropenia therapy.

An LC-MS/MS assay to determine plasma pharmacokinetics of cyclic thymic hexapeptide (cTP6) in rhesus monkeys.

A robust and simple method for absolute quantification of a novel bidirectional immunomodulatory drug candidate, cyclic thymic hexapeptide (cTP6), in rhesus monkey plasma was developed and validated by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Plasma proteins were precipitated by adding four volumes of acetonitrile. Peptides in the supernatant were separated by liquid chromatography on an Agilent Zorbax Eclipse Plus-C18 chromatographic column with gradient elution using 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in methanol (mobile phase B) at 0.2 mL/min. The analytes were identified by triple quadrupole mass spectrometry in positive ion-mode. The assay was linear over a concentration range of 10-5000 ng/mL for cTP6, with a lower limit of quantification (LLOQ) of 10 ng/mL. Intra- and inter-day precision of the assay at three concentrations were 1.51-7.70% with accuracy of 95.1-104.2%. The average recovery of cTP6 for three concentration levels was 59.6-64.0%. No significant matrix effect was observed. Peptide cTP6 was detected in plasma of live rhesus monkeys up to 6-8h after intra-muscular injection. The half-life was 2.24-2.95 h. The result revealed a nonlinear pharmacokinetic response to increasing doses of cTP6 (100, 200, 500 µg/kg). For the multiple dose study of cTP6, the drug did not accumulate during daily administration at 100 µg/kg for 7 consecutive days in rhesus monkeys.

Qualitative and quantitative studies on human B7.1-Fc fusion protein and the application in pharmacokinetic study in rhesus monkeys.

A sensitive, accurate, and precise enzyme immunoassay (EIA) for the quantification of intact human B7.1-Fc in rhesus monkey serum was validated, and the characteristics of B7.1 and Fc moiety of fusion protein were identified by surface plasmon resonance (SPR) and flow-cytometric method, respectively. B7.1-Fc bound to CD28 and CTLA-4 with K(d) values of 45.1 and 9.58 nM, respectively, which were very closed to the previous reports and the function of Fc moiety of fusion protein was also confirmed by Fc receptor binding assay and IL-8 releasing assay. To monitor the intact protein, the EIA method employed a sandwich scheme in which a multiclonal anti-human IgG (Fc specific) antibody and a monoclonal anti-human B7.1 antibody were served as capture and detection antibody, respectively. This EIA has a range of reliable response of 0.5-32 ng/ml. The LLOQ was established at 0.5 ng/ml. The intra-assay precision and accuracy were 6.1-8.8% and (3.0-9.0)%, respectively with the inter-assay precision and accuracy were 5.7-11.5% and (10.7-9.1)%, respectively. Stability was established under certain conditions and no significant differences were found. This validated EIA assay was then successfully employed in the assessment of pharmacokinetic behavior of B7.1-Fc in rhesus monkeys after intravenous infusion, and a non-linear characteristics was established across the investigated dosage range (32-320 µg/kg).

Quantitative analysis of a novel HIV fusion inhibitor (sifuvirtide) in HIV infected human plasma using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry.

A sensitive method for measuring sifuvirtide, a novel HIV fusion inhibitor peptide drug in HIV-1(+) human plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. The plasma samples were treated by solvent/detergent (S/D) method to inactivate viral activity before analysis. After protein precipitation sifuvirtide was determined by LC-MS/MS. A structure analog was used as internal standard (IS). The mass spectrometer was operated in positive ion and multiple reaction monitoring mode with transitions m/z 946.3-->159.0 for sifuvirtide and 951.7-->159.2 for IS. The intra-day precision ranged from 2.74% to 7.57% with accuracy from 91.63% to 102.53%. The inter-day precision ranged from 2.65% to 3.58% and the accuracy from 95.53% to 105.28%. Stability studies showed that sifuvirtide was stable both during the assay procedure and long-term storage. The lower limit of quantitation (LLOQ) was 9.75ngml(-1). The method was used for analyzing samples from phase IIa clinical study of sifuvirtide in China.

Development of a rapid and sensitive LC-MS/MS assay for the determination of combretastatin A4 phosphate, combretastatin A4 and combretastatin A4 glucuronide in beagle dog plasma and its application to a pharmacokinetic study.

This study details the development and validation of a simple and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the quantification of combretastatin A-4 3-O-phosphate (CA4P), combretastatin A4 (CA4) and its main metabolite, combretastatin A4 glucuronide (CA4G), in beagle dog plasma. Sample pretreatment includes simple protein precipitation by adding methanol to the plasma sample containing an internal standard (colchicine). LC separation was successfully accomplished on a Waters RP8 Symmetryshield column (3.0mmx150mm, i.d., 5microm) with a gradient mobile phase of methanol (0.1% formic acid, v/v) and water (20mM ammonium acetate) at a flow rate 0.8mLmin(-1). The three analytes were detected in the positive/negative ion alternate mode, negative ion mode for CA4G and positive ion mode for CA4P and CA4. Multiple reaction monitoring (MRM) was used for determination of three analytes. Calibration curves were linear in the concentration range of 5-5000ngmL(-1) for CA4P (r>or=0.999), 5-3000ngmL(-1) for CA4 (r>or=0.999) and 5-5000ngmL(-1) for CA4G (r>or=0.999). All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. The method was reliable and has been successfully applied to a pharmacokinetic study of CA4P in beagle dogs via intravenous drop infusion at dose rates of 1, 3 and 9mgkg(-1). After daily intravenous drop infusions at 1mgkg(-1) for 7 consecutive days, the accumulation ratio was approximately 1.0, indicating no accumulation from multiple doses in beagles.

Development and application of a real-time PCR method for pharmacokinetic and biodistribution studies of recombinant adenovirus.

A replication-deficient recombinant adenovirus (Ad5-LFA-3/IgG(1)) that encodes secreted LFA-3/IgG(1) was constructed for gene therapy treatment of psoriasis. The purpose of this study was to develop a real-time PCR method for pharmacokinetic and biodistribution studies of Ad5-LFA-3/IgG(1) within the circulation and organs. This method showed good specificity, sensitivity and reproducibility over a wide dynamic range of concentrations. Quantitative measurement of recombinant adenoviral DNA suggested that the level of Ad5-LFA-3/IgG(1) DNA in circulating blood peaked within 10 min following intravenous injection in rhesus macaques. Following this peak, the adenoviral DNA level dropped significantly to a very low level. Real-time PCR revealed that Ad5-LFA-3/IgG(1) DNA was enriched in the spleen, lung and liver after injection of the adenovirus into rats through the tail vein. The adenoviral DNA was barely detected in other tissues. These data provide important information for clinical trials of Ad5-LFA-3/IgG(1) and confirm the utility of the real-time PCR assay for monitoring gene therapy trials.

A new approach for pharmacokinetics of single-dose cetuximab in rhesus monkeys by surface plasmon resonance biosensor.

A novel assay method has been developed and validated, using surface plasmon resonance (SPR), for quantitation of cetuximab (C225) in monkey serum. By injecting non-labeled antibody samples onto a biosensor surface on which epidermal growth factor receptor (EGFR) was immobilized, the concentration of C225 can be accurately measured. This assay has a range of reliable response from 0.05 to 50 microg/ml C225 in monkey serum, which was well fitted with a sigmoidal model. The immobilized EGFR was found to be stable for at least 100 regeneration cycles at room temperature. Intra- and inter-assay CVs ranged from 3.20% to 8.89% and from 5.93% to 11.11%, accuracy from 92% to 107.52% and from 90% to 106.88%, respectively. Matrices such as 50% human serum, 50% Sprague Dawley rat serum, chimeric recombinant anti-CD20 monoclonal antibody, human gamma-globulin and chimeric recombinant her2 antibody did not interfere with C225 analysis on the sensor surface. This is the first report on the quantitation of C225 in monkey serum by an optical biosensor technology. This method was used to characterize the pharmacokinetics of C225 in rhesus monkeys. After a single-dose of intravenous infusion administration of 7.5, 24 and 75 microg/kg, average C(max) ranged from 168+/-28 to 1624+/-113 microg/ml, and AUC(0-infinity) ranged from 15,739+/-1059 to 295,017+/-44,533 microg h/ml. C225 elimination followed a bi-exponential profile with t(1/2) ranging from 2.7+/-0.7 to 6.7+/-0.1 h. It was non-linear serum pharmacokinetics of C225 across the investigated dosage range in monkeys (7.5-75 mg/kg).

Validation of a sensitive gas chromatographic-mass spectrometric method for the simultaneous determination of beta-elemene and beta-elemenal in human plasma.

A sensitive gas chromatographic-mass spectrometric assay was described for determination of beta-elemene and beta-elemenal in human plasma, which has been successfully applied in clinical trial. After liquid-liquid extraction and gas chromatographic separation, the analytes were identified and quantitated. Calibration curves were linear in range from 31.25 to 8000 ng mL(-1) and the limit of quantification for both was 31.25 ng mL(-1). Intra- and inter-day precision at three concentrations were 2.3-8.3% with accuracy of -8.5 to 6.1% for elemene and 3.0-9.9% with accuracy of -2.3 to 5.9% for elemenal. The method was validated to be suitable for further pharmacokinetic study.

Single- and multiple-dose pharmacokinetics of exendin-4 in rhesus monkeys.

A radioimmunoassay (RIA) for the measurement of exendin-4 concentration in rhesus monkeys serum was developed and validated. The radioimmunoassay described here was sensitive, linear, accurate, precise, and reproducible. Range of the assay was 25-2000 pg/ml. Using this method we characterized the pharmacokinetics and accumulation of exendin-4 in rhesus monkeys. Following s.c. administration at doses rate of 1, 3 and 10 microg/kg, average C(max) ranged from 2.26+/-0.15 to 22.72+/-1.54 ng/ml, and AUC(0-infinity) ranged from 3.43+/-0.05 to 47.1+/-0.10 ng h/ml. As compared to the i.v. administration at a single dose of 3 microg/kg, the absolute bioavailability after s.c. administration were estimated to be 67.3+/-5.3, 75.1+/-4.7 and 72.7+/-8.4% for 1, 3 and 10 microg/kg dose, respectively. After daily s.c. administration at 10 microg/kg for 7 consecutive days, the accumulation ratio was approximately to 1.0, indicating no accumulation upon multiple doses in the monkeys.

Naphthoquine phosphate and its combination with artemisinine.

Naphthoquine phosphate and artemisinine are two antimalarials developed in China. Both drugs have proven to be efficacious and well tolerated as monotherapy as well as in combination in patients suffering from malaria. The Co-naphthoquine, a novel antimalarial combination, is an oral fixed combination tablet of the naphthoquine phosphate and artemisinine. Artemisinin is characterised by a rapid onset of schizonticidal action and a short half-life. Parasite clearance is, however, often incomplete when it is employed as a single agent unless high dosages are used over several days, but such a regimen may reduce patient compliance and increase the danger of toxicity. Naphthoquine phosphate, by contrast, has a slower onset of action and a longer half-life, associated with a low recrudescence rate. The two components act synergistically in animal, and clinically provide more rapid relief of symptoms and a higher cure rate than either component alone. The combination tablet was initially developed by the Academy of Military Medical Sciences (AMMS), Beijing, China.

[Efficacy of naphthoquine, artemisinine and a combination of the two drugs in the treatment of falciparum malaria].

OBJECTIVE: To compare the efficacy and safety of naphthoquine, artemisinine and a combination of the two drugs in the treatment of faciparum malaria. METHODS: Of 230 patients, 100 patients were treated with combined regime (Co-NQ), 100 patients were treated with naphthoquine (NQ) and 30 patients were treated with artemisinine (QHS). All patients were hospitalized for 7 days and followed up for 28 days. RESULTS: The mean fever clearance time for Co-NQ, NQ, and QHS was (17.5 +/- 12.3)h, (32.7 +/- 17.7)h and (18.1 +/- 9.7)h respectively; the mean parasite clearance time was (30.0 +/- 8.8)h, (45.5 +/- 10.0)h and (29.1 +/- 6.0)h respectively; and the 28 days cure rate was 97.0%, 100.0% and 66.7% respectively. CONCLUSION: The Co-NQ possesses benefits of both naphthoquine and artemisinine, acting rapidly, with a short course of only one dose and a high cure rate. The regime is well tolerated by patients.