Sprague Dawley rats and New Zealand White rabbits were purchased from Charles River Laboratories, Inc. (Willmington, MA). Tumor necrosis factor-alpha (TNF)-gold nanoparticle formulation (Aurimune™) and TNF ELISA (CytElisa™ kit) were provided by Cytimmune Sciences, Inc. (Rockville, MD). Heparin was purchased from Sigma-Aldrich (St. Louis, MO).
Animal rooms were kept at 50% relative humidity, 68–72 F with 12 h light/dark cycles. Rats were housed by treatment group, with two animals/cage (rat polycarbonate cage type), with 1/4” corncob bedding. Animals were allowed ad libitum
access to Purina 18% NIH Block and chlorinated tap water. NCI-Frederick is accredited by AAALAC International and follows the Public Health Service Policy for the Care and Use of Laboratory Animals
. Animal care was provided in accordance with the procedures outlined in [10
2.3. Rat pharmacokinetic studies
Double jugular catheterized 10-week-old female Sprague Dawley rats (approximate weights of 200 g) were purchased from Charles River Laboratories (Raleigh, N.C.). Rats, 4–5 animals per group, were treated intravenously by the right jugular catheter with 250 ug TNF / kg of either the Au–TNF (Aurimune™) nanoformulation or native TNF in PBS vehicle. Blood samples were collected from the left jugular catheter at 15, 30, 60, 120, 240, 480 and 1440 min. The blood samples were mixed 1:1 with heparin (1 mg/mL in PBS) and stored at 4 °C. Samples were analyzed for gold and TNF concentration by ICP-MS and ELISA, respectively.
2.4. Rabbit pharmacokinetic data
Rabbit pharmacokinetic data were obtained from a GLP compliant study conducted by BioCon, Inc. in New Zealand White rabbits (3 males and 3 females/group, 2.5–3 kg). The study was reviewed and approved by the BioCon, Inc IACUC. The rabbits were injected with 25 or 125 μg TNF /kg of the Au–TNF nanoformulation via the ear vein, followed by a 1 mL saline flush. The rabbit blood collection times were 5, 30, 60, 120, 240, 360, 480 and 1440 min. Blood was collected into heparinized tubes undiluted.
2.5. Human pharmacokinetic data
Human pharmacokinetic data were obtained from a GLP compliant clinical study conducted by NIH. Institutional Review Board (IRB) approval was granted by the NIH for the parent study titled “TNF-Bound Colloidal Gold in Treating Patients With Advanced Solid Tumors”, from which blinded clinical pharmacokinetic data was obtained for this retrospective analysis. Three oncology patients per dose level received 11 or 16 μg TNF /kg of the Au–TNF nanoformulation, administered as an intravenous bolus. Blood collection times were 5, 15, 30, 60, 120, 180, 240 and 480 min. Blood was collected into heparinized tubes undiluted.
2.6. Au ICP-MS analysis of blood and tissue
(Certain commercial instruments are identified in this paper to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by NIST, nor does it imply that the equipment identified is necessarily the best for the purpose.) The entire blood sample was transferred to a microwave cell. Four samples approximately 0.25 g each of RM 8012 (Gold Nanoparticles, Nominal 30 nm Diameter) were weighed into 4 microwave cells serving as controls. Six microwave cells were used for procedure blanks. To each blood sample, 4 mL of concentrated HNO3 and 1 mL concentrated HCl were added. To each tissue sample, 8 mL of concentrated HNO3 and 2 mL concentrated HCl were added. The microwave cells were capped and the samples were digested by microwave radiation. The resulting digest was quantitatively transferred to a pre-weighed 60-mL low density polyethylene (LDPE) bottle and diluted with water to approximately 50 g. The analytical portion was prepared by transferring 5 g of the sample digest into a 60-mL LDPE bottle and diluting the content to approximately 50 g with a diluent consisting of 1.5% HNO3 and 4% HCl by volume. The digest of a blood (or a tissue) sample from a control rat was used to prepare matrix matched calibration standards. Approximately 5 g of the digest was transferred to each of the six 60-mL LDPE bottles, into which 0 μg, 0.5 μg, 1.0 μg, 1.5 μg, 2.0 μg, 2.5 μg, 3.0 μg gold from SRM 3121 (Gold Standard Solution) were added, respectively. The content in each bottle was diluted to approximately 50 g with the acid diluent described above. Samples were quantified by using the quantitative analysis mode of the Agilent 7500cs ICP-MS. A solution containing 10 ng/g indium was used as the internal standard. Sample solution and the internal standard were picked up at two separate channels of the peristaltic pump. Solutions from the two channels were joined with a mixing T before entering the nebulizer. Ion counts at 115 amu and 197 amu were recorded for indium and gold, respectively.
2.7. TNF ELISA analysis of blood
An enzyme-linked immunosorbent assay (ELISA) method was used to determine free and total TNF in blood samples. This method is a sandwich ELISA, which measures TNF using a CytElisa™ kit (CytImmune Sciences, Inc.). Mouse anti-human TNF monoclonal antibodies coated on the plate were used to capture TNF in samples and rabbit antihuman TNF polyclonal antibodies were used as the detection antibodies. A secondary antibody, goat anti-rabbit antibody alkaline phosphatase conjugate, was used to develop a substrate to produce a visible color, which was detected at 490 nm. The amount of TNF in the sample was determined by comparing the sample response to that of a TNF standard curve, using a 4-parameter fit of the average uncorrected absorbance of the standard at each concentration. The standard curve used for the rat samples were 2500, 1875, 1250, 938, 625, 469 and 313, for the rabbit samples were 1000, 500, 250, 625, 312.5 156.75, 78.375 and 39, and for the human samples were 2500, 1875, 1250, 937.5 625, 468.75 and 312.5 pg TNF /mL.
2.8. Noncompartmental pharmacokinetic analysis
Noncompartmental pharmacokinetic parameters were determined by the following methods, using WinNonlin Version 4.1 software (Pharsight Corp., Mountain View, CA): the area under the time concentration curve (AUC) was calculated using the linear trapezoidal rule with extrapolation to time infinity; clearance (CL) was calculated from dose/AUC; apparent volume of distribution (Vd) was calculated from dose/Co (concentration at time zero calculated from extrapolation of the plasma time curve); plasma half-life (t1/2) was calculated from 0.693/slope of the terminal elimination phase (λ).
2.9. Compartmental pharmacokinetic analysis
The single compartment model expressed as the algebraic equation, C(t)=D/Vd*e(−K10*t), was fit to the pooled animal blood concentration–time data (C(t)) and dose (D). The volume of distribution (Vd) and first-order elimination rate constant (K10) parameters were estimated by nonlinear least-squares regression analysis, using 1/(Y*Y) weighting scheme (WinNonlin 4.1; Pharsight Corporation, Mountain View, CA).
For a description of the hyperbolic stability and Au–TNF release models, please refer to the results and discussion.
2.10. Allometric scaling
Allometric scaling of CL and V parameters were performed by fitting the power models, CL=a*BWb and V=c*BWd, where BW is the body weight and a–d are fitting parameters. Brain weight product scaling was performed by fitting the power models, CL=a*BWb/BrW and V=c*BWd/BrW, where BrW is the brain weight. Power model parameters were estimated by non-weighted, nonlinear least-squares regression analysis using WinNonlin Version 4.1 software (Pharsight Corp., Mountain View, CA). For allometric analysis, the standard body weights 75, 3 and 0.2 kg, and standard brain weights 1.5, 0.013 and 0.0018 kg were used for man, rabbit and rat, respectively.