Cellular dosimetry of 197Hg, 197mHg and 111In: comparison of dose deposition and identification of the cell and nuclear membrane as important targets.

PURPOSE: To examine the reliability to model cellular S-values for the Auger electron (AE) emitters, 111In, 197Hg and 197mHg with MCNP6 and their relative dose deposition in subcellular targets. METHODS: A model cell was defined as four concentric spheres consisting of the nucleus (N), cytoplasm (Cy), cell and nuclear membranes (CM, NM) in which radionuclides distributed homogeneously. The transport of AE, conversion electrons and photons were simulated by MCNP6 to calculate cellular S values (SN←CM, SN←Cy, SN←NM, SN←N, SCM←CM, SNM←NM). SN←CM, SN←Cy and SN←N were also calculated with MIRDcell. RESULTS: MIRDcell and MCNP6-calculated SN←N were in excellent agreement, but a slight discrepancy on SN←Cy and SN←CM was observed. The ratios of SCM←CM or SNM←NM vs. SN←N were 9.7-51.0 or 10.5-37.4, 7.9-41.8 or 8.4-31.8 and 7.2-36.9 or 8.0-28.1 for 111In, 197Hg, 197mHg, respectively. The mean S(197Hg)/S(111In) and S(197mHg)/S(111In) were 2.5 ± 0.5 and 2.5 ± 0.6, respectively. CONCLUSIONS: Cellular S-values were reliably calculated with MCNP6. 197Hg and 197mHg deposit two-fold more doses than 111In at the subcellular scale. All AE emitters deposit a higher self-dose in the CM and NM than in the N, which warrants studies on the effects of targeting the CM and NM by AE emitters.

Imaging of HER2-Positive Tumors in NOD/SCID Mice with Pertuzumab Fab-Hexahistidine Peptide Immunoconjugates Labeled with [99mTc]-(I)-Tricarbonyl Complex.

PURPOSE: Molecular imaging of tumor HER2 expression may allow patient selection for HER2-targeted therapies. Our aim was to introduce hexahistidine (His6) peptides into pertuzumab Fab to enable labeling with the [99mTc(CO)3(H2O)3]+ complex and study these radioimmunoconjugates for microSPECT/CT imaging of HER2-positive tumor xenografts in mice. PROCEDURES: Fab were produced by papain digestion of pertuzumab and reacted with sulfo-SMCC for conjugation to His6-containing peptides (CGYGGHHHHHH). His6-peptide conjugation was measured by a radiometric assay. His6-pertuzumab Fab were labeled at 0.4-1.0 MBq/µg with [99mTc(CO)3(H2O)3]+ for 1 h at 37 °C. HER2 immunoreactivity was assessed in a direct (saturation) binding assay using HER2-overexpressing SK-BR-3 human breast cancer (BC) cells. MicroSPECT/CT and biodistribution studies were performed in NOD/SCID mice with HER2-positive s.c. SK-OV-3 human ovarian cancer, or MDA-MB-361 or MDA-MB-231 human BC xenografts at 4 or 24 h post i.v. injection of [99mTc]His6-pertuzumab Fab (29-49 MBq, 70 µg). The specificity of tumor uptake was assessed by comparison to irrelevant [99mTc]Fab 3913 in SK-OV-3 tumor-bearing mice. RESULTS: SDS-PAGE analysis demonstrated cleavage of pertuzumab to produce Fab, which eluted as a single peak with a retention time of 13.8 min on SE-HPLC. Fab were conjugated to 2.1 ± 0.5 His6 peptides and labeled with [99mTc(CO)3(H2O)3]+ to a radiochemical purity of 92-97 % at 0.4-0.8 MBq/µg. [99mTc]His6-pertuzumab Fab exhibited saturable and specific binding to SK-BR-3 cells with a KD = 51.3 ± 5.2 × 10-9 M and Bmax = 3.5 ± 0.1 × 106 receptors/cell. SK-OV-3 tumors were imaged at 4 and 24 h p.i [99mTc]His6-pertuzumab Fab. Tumor uptake at 24 h p.i. was 4.1 ± 0.6 %ID/g, which was 13-fold significantly greater than [99mTc]Fab 3913 (0.3 ± 0.0 %ID/g; P < 0.01). MicroSPECT/CT imaged HER2-overexpressing MDA-MB-361 tumors but not MDA-MB-231 tumors with low HER2 expression. Tumor uptake was 5.2-fold significantly greater at 24 h p.i. in MDA-MB-361 than MDA-MB-231 tumors (3.2 ± 0.1 %ID/g vs. 0.8 ± 0.1 %ID/g; P < 0.05). CONCLUSIONS: MicroSPECT/CT with [99mTc]His6-pertuzumab Fab imaged tumors in NOD/SCID mice that exhibited intermediate or high HER2 expression, but not tumors with low HER2. [99mTc]His6-pertuzumab Fab is promising for SPECT imaging of tumor HER2 expression.

A comparison of DFO and DFO* conjugated to trastuzumab-DM1 for complexing 89Zr - In vitro stability and in vivo microPET/CT imaging studies in NOD/SCID mice with HER2-positive SK-OV-3 human ovarian cancer xenografts.

INTRODUCTION: Desferrioxamine (DFO) is conjugated to antibodies to chelate 89Zr for PET, but DFO forms a hexadentate complex with Zr4+ that exhibits instability contributing to bone uptake of 89Zr, while the cationic charge of the Zr4+-DFO complex may promote normal tissue uptake of the radioimmunoconjugates (RICs). DFO* is a novel chelator that forms a more stable octadentate and neutral complex with 89Zr. Our aim was to compare the in vitro stability of [89Zr]Zr-DFO*-human IgG (hIgG) and [89Zr]Zr-DFO-hIgG RICs, and the in vivo PET imaging properties of the antibody-drug conjugate (ADC), trastuzumab-DM1 (T-DM1), labeled with 89Zr by conjugation to DFO or DFO*. METHODS: SCN-pPhe-DFO and SCN-pPhe-DFO* were reacted with hIgG at a 14.6-fold excess or with T-DM1 at a 4.1-fold or 10-fold excess, respectively, purified and labeled with 89Zr. The number of DFO* introduced was determined by measuring the absorbance at 245/252 nm and the protein concentration was measured at 280 nm. The stability of [89Zr]Zr-DFO*-hIgG was studied in vitro in human plasma, and by challenge with a 385-fold excess (0.1 mM) of DFO or EDTA. An inverse stability study was performed with [89Zr]Zr-DFO-hIgG challenged with 0.1 mM DFO*. The HER2 binding affinity of [89Zr]Zr-DFO*-T-DM1 was measured in a direct (saturation) binding assay using SK-BR-3 human breast cancer cells or SK-OV-3 human ovarian cancer cells. The biodistribution of [89Zr]Zr-DFO*-T-DM1 and [89Zr]Zr-DFO-T-DM1 were compared in non-tumor bearing Balb/c mice and in NOD/SCID mice with s.c. SK-OV-3 xenografts at 96 h post-intravenous injection (p.i.). MicroPET/CT images were obtained at 96 h p.i. of the RICs. RESULTS: hIgG and T-DM1 were conjugated to 4.5-5.3 and 3.1 chelators (DFO or DFO*), respectively, and labeled with 89Zr to a final radiochemical purity of 91-99%. [89Zr]Zr-DFO*-hIgG was stable in vitro in human plasma or to challenge with 0.1 mM EDTA, but incubation with 0.1 mM DFO caused 26.0 ± 2.1% loss of 89Zr after 5 days. In contrast, incubation of [89Zr]Zr-DFO-hIgG with 0.1 mM DFO* resulted in 77.0 ± 3.9% loss of 89Zr after 5 days. [89Zr]Zr-DFO*-T-DM1 retained high affinity binding to HER2 on SK-BR-3 and SK-OV-3 cells with a Kd = 2.2 ± 0.3 nM and 1.9 ± 0.3 nM, respectively, and Bmax = 3.4 ± 0.1 × 105 and 1.1 ± 0.04 × 105 receptors/cell, respectively. Biodistribution studies of [89Zr]Zr-DFO-T-DM1 and [89Zr]Zr-DFO*-T-DM1 in Balb/c and NOD/SCID mice revealed significantly lower uptake in bone, liver, kidneys, and spleen for [89Zr]Zr-DFO*-T-DM1 than [89Zr]Zr-DFO-T-DM1. Uptake of [89Zr]Zr-DFO*-T-DM1 and [89Zr]Zr-DFO-T-DM1 in SK-OV-3 tumors was moderate [5.0 ± 1.8% injected dose/g (%ID/g) and 6.3 ± 0.6%ID/g, respectively; P = 0.18]. Tumors were imaged with both RICs. CONCLUSION: We conclude that DFO* conjugated to T-DM1 provides more stable complexation of 89Zr and therefore, [89Zr]Zr-DFO*-T-DM1 would be more useful than [89Zr]Zr-DFO-T-DM1 to probe the delivery of T-DM1 to tumors by PET, which we previously found is correlated with response to treatment with T-DM1 in mouse tumor xenograft models. ADVANCES IN KNOWLEDGE AND IMPLICATION FOR PATIENT CARE: This study is the first to directly compare the PET imaging properties of [89Zr]Zr-DFO*-T-DM1 and [89Zr]Zr-DFO-T-DM1 in a HER2-overexpressing tumor xenograft mouse model. Our results indicate that [89Zr]Zr-DFO*-T-DM1 provides superior imaging properties due to the greater stability of the [89Zr]Zr-DFO* than [89Zr]Zr-DFO complex.

Tumor uptake and tumor/blood ratios for [89Zr]Zr-DFO-trastuzumab-DM1 on microPET/CT images in NOD/SCID mice with human breast cancer xenografts are directly correlated with HER2 expression and response to trastuzumab-DM1.

INTRODUCTION: Our objective was to determine correlations between the tumor uptake and T/B ratios for 89Zr-labeled T-DM1 (89Zr-DFO-T-DM1) in mice with human BC xenografts by microPET/CT and biodistribution studies with HER2 expression and response to treatment with trastuzumab-DM1 (T-DM1). METHODS: The tumor and normal tissue uptake and T/B ratios for 89Zr-DFO-T-DM1 (10 µg; 7.0 MBq) incorporated into a therapeutic dose (60 µg) were determined by microPET/CT and biodistribution studies at 96 h p.i. in NOD/SCID mice with s.c. MDA-MB-231 (5 × 104 HER2/cell), MDA-MB-361 (5 × 105 HER2/cell) and BT-474 (2 × 106 HER2/cell) human BC xenografts. Mice bearing these tumors were treated with T-DM1 (3.6 mg/kg every 3 weeks) and the tumor doubling time estimated by fitting of tumor volume vs. time curves. A tumor doubling time ratio (TDR) was calculated by dividing the doubling time for T-DM1 and normal saline treated control mice. The clonogenic survival (CS) of BC cells with increasing HER2 expression treated for 72 h in vitro with T-DM1 or trastuzumab (0-100 µg/mL) was compared. Correlations were determined between the T/B ratios for 89Zr-DFO-T-DM1 and HER2 expression, TDR and CS, and between CS and TDR. RESULTS: Uptake of 89Zr-DFO-T-DM1 in MDA-MB-231, MDA-MB-361 and BT-474 tumors was 2.4 ±â€¯0.4%ID/g, 6.9 ±â€¯2.2%ID/g and 9.8 ±â€¯1.1%ID/g, respectively. There was a non-linear but direct correlation between the T/B ratios for 89Zr-DFO-T-DM1 and HER2 expression with the T/B ratio ranging from 4.5 ±â€¯0.7 for MDA-MB-231 to 18.2 ±â€¯1.8 for MDA-MB-361 and 35.9 ±â€¯5.1 for BT-474 xenografts. Tumor intensity on microPET/CT images was proportional to HER2 expression. The standard uptake value (SUV) for the tumors on the images was strongly correlated with the T/B ratio in biodistribution studies. There was a direct linear correlation between the T/B ratio for 89Zr-DFO-T-DM1 and TDR, with TDR ranging from 0.9 for MDA-MB-231 to 1.6 for MDA-MB-361 and 2.1 for BT-474 tumors. The cytotoxicity of T-DM1 in vitro on BC cells was dependent on HER2 expression but T-DM1 was more potent than trastuzumab. There was an inverse correlation between the TDR for mice treated with T-DM1 and CS of BC cells exposed in vitro to T-DM1. CONCLUSIONS: Based on the direct correlations between the T/B ratio for 89Zr-DFO-T-DM1 by PET and HER2 expression and response to T-DM1, our results suggest that PET with 89Zr-DFO-T-DM1 may predict response of HER2-positive BC to treatment with T-DM1. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: Our results suggest that PET with 89Zr-DFO-T-DM1 may predict response to treatment with T-DM1 in HER-positive BC.

Positron-Emission Tomography of HER2-Positive Breast Cancer Xenografts in Mice with 89Zr-Labeled Trastuzumab-DM1: A Comparison with 89Zr-Labeled Trastuzumab.

Our aim was to synthesize 89Zr-labeled trastuzumab-emtansine (89Zr-DFO-T-DM1) to probe the delivery of trastuzumab-emtansine (T-DM1) to HER2-positive breast cancer (BC) by positron emission tomography (PET). We further aimed to compare the tumor and normal tissue uptake of 89Zr-DFO-T-DM1 with 89Zr-DFO-trastuzumab. T-DM1 was modified with 3.0 ± 0.2 desferrioxamine (DFO) chelators for complexing 89Zr by reaction with a 14-fold molar excess of p-NCS-Bz-DFO. The number of DFO chelators per T-DM1 molecule was quantified spectrophotometrically at 430 nm after the reaction with FeCl3. SDS-PAGE and SE-HPLC demonstrated a pure and homogeneous immunoconjugate. DFO-T-DM1 and DFO-trastuzumab were labeled to high efficiency (>97%) with 89Zr at a specific activity of 0.55 MBq/µg in a 2 M Na2CO3/0.5 M HEPES buffer, pH 7.0, at RT for 60-90 min. The labeling efficiency was measured by instant thin layer-silica gel chromatography (ITLC-SG) and SE-HPLC. HER2 immunoreactivity was measured in a saturation binding assay using SK-BR-3 human BC cells. 89Zr-DFO-T-DM1 exhibited high affinity HER2 binding ( Kd = 3.7 ± 0.4 nM) that was not significantly different than 89Zr-DFO-trastuzumab (4.4 ± 0.5 nM; P = 0.06). The optimal time for tumor imaging with 89Zr-DFO-T-DM1 was 96 h post-injection in NOD-scid mice with s.c. HER2 overexpressing (HER2 3+) BT-474 human BC xenografts. Tumor uptake was dependent on the level of HER2 expression in mice with s.c. BT-474 (HER2 3+), MDA-MB-231/H2N (HER2 2+), MDA-MB-231 (HER2 0-1+), or MDA-MB-468 (HER2 0) human BC xenografts injected with 89Zr-DFO-T-DM1 (10 µg, 5.2 MBq). All tumors were visualized by microPET/CT, but the tumor intensity was greatest for BT-474 and MDA-MB-231/H2N xenografts. The tumor uptake of 89Zr-DFO-T-DM1 was 4.1-fold significantly higher than 89Zr-DFO-trastuzumab in mice with s.c. BT-474 (HER2 3+) xenografts (43.5 ± 4.3%ID/g vs 10.6 ± 5.4%ID/g, respectively; P < 0.001). Tumor uptake of 89Zr-DFO-T-DM1 in MDA-MB-231/H2N xenografts (HER2 2+) was 3.7-fold significantly higher than 89Zr-DFO-trastuzumab (10.1 ± 3.6%ID/g vs 2.7 ± 0.5%ID/g; P < 0.001). The higher tumor uptake of 89Zr-DFO-T-DM1 compared to 89Zr-DFO-trastuzumab was not due to a higher HER2 binding affinity or to differences in the residence time in the blood or tumor size. We conclude that 89Zr-DFO-T-DM1 is a useful probe to assess the delivery of T-DM1 to HER2-positive BC. PET with 89Zr-DFO-trastuzumab has been studied clinically to predict response to T-DM1, but our results suggest that 89Zr-DFO-T-DM1 may be more accurate due to the differences in the tumor uptake observed in the preclinical BC xenograft mouse models.

Effect of drug-polymer interactions on the aqueous solubility of milled solid dispersions.

The role of molecular interactions in ball milled solid dispersions in determining the aqueous solubility of the poorly water-soluble drug, griseofulvin (GF) has been examined. Ball milled solid dispersions of GF and hydroxypropylmethylcellulose acetate succinate (HPMCAS) and GF and polyvinylpyrrolidone (PVP) were prepared and characterized by laser diffraction, scanning electron microscopy and X-ray powder diffraction and the aqueous saturation solubility measured and analyzed using one way ANOVA. The results showed that solid dispersions of GF and HPMCAS possessed an aqueous GF saturation solubility of about ten times higher than the GF solubility achieved from PVP-based solid dispersions. Furthermore, although the aqueous solubility of GF did not vary with the milling conditions used to prepare the solid dispersions with PVP, significant changes in solubility were observed upon changing the milling conditions for preparation of the GF/HPMCAS solid dispersions. Surprisingly, the GF/HPMCAS solid dispersion prepared using spray drying exhibited a significantly lower aqueous solubility than those prepared by bead milling despite their smaller particle size and GF being fully in its amorphous form. It is thought that the higher surface energy of the spray-dried solid dispersions negatively affected the aqueous solubility of GF. In conclusion, the results suggest that the molecular interactions occurring between GF and HPMCAS affect the aqueous solubility of GF and that the molecular interactions appear to remain in the liquid state. In contrast no molecular interactions were evident in the GF/PVP solid dispersions.

Investigation of griseofulvin and hydroxypropylmethyl cellulose acetate succinate miscibility in ball milled solid dispersions.

Solid dispersions of varying weight ratios compositions of the nonionic drug, griseofulvin and the hydrophilic, anionic polymer, hydroxylpropylmethyl cellulose acetate succinate, have been prepared by ball milling and the resulting samples characterized using a combination of Fourier transform infra-red spectroscopy, X-ray powder diffraction and differential scanning calorimetry. The results suggest that griseofulvin forms hydrogen bonds with the hydroxylpropylmethyl cellulose acetate succinate polymer when prepared in the form of a solid dispersion but not when prepared in a physical mixture of the same composition. As anticipated, the actual measured glass transition temperature of the solid dispersions displayed a linear relationship between that predicted using the Gordon-Taylor and Fox equations assuming ideal mixing, but interestingly only at griseofulvin contents less than 50 wt%. At griseofulvin concentrations greater than this, the measured glass transition temperature of the solid dispersions was almost constant. Furthermore, the crystalline content of the solid dispersions, as determined by differential scanning calorimetry and X-ray powder diffraction followed a similar trend in that the crystalline content significantly decreased at ratios less than 50 wt% of griseofulvin. When the physical mixtures of griseofulvin and the hydroxylpropylmethyl cellulose acetate succinate polymer were analyzed using the Flory-Huggins model, a negative free energy of mixing with an interaction parameter of -0.23 were obtained. Taken together these results suggest that anionic hydrophilic hydroxylpropylmethyl cellulose acetate succinate polymer is a good solvent for crystalline nonionic griseofulvin with the solubility of griseofulvin in the solid dispersion being was estimated to be within the range 40-50 wt%. Below this solubility limit, the amorphous drug exists as amorphous glassy solution while above these values the system is supersaturated and glassy suspension and solution may coexist.