A

FIGURE 9.3 (See color insert following page 522.) Scintigraphic images of human prostate tumor-bearing SCID mice 24 h post-intravenous injection of 99mTc labeled HPMA copolymer conjugates. HPMA copolymer-RGD4C conjugate shows higher localization in tumor as compared to the control, HPMA copolymer-RGE4C conjugate (solid arrow). Additional higher activity was found in the kidney (broken arrow). The mouse radiograph (center) shows anatomic correlation of tumor and other organs. Figure legends: (a) HPMA copo-lymer-RGD4C conjugates; (b) HPMA copolymer-RGE4C conjugates. (From Mitra, A., et al., J. Control. Release, 102, 191, 2005. With permission.)

16.000 14.000 12.000 10.000 8.000 6.000 4.000 2.000 0.000

16.000 14.000 12.000 10.000 8.000 6.000 4.000 2.000 0.000

Blood Heart Lung Liver Spleen Kidney Muscle Tumor

■ HPMA-RGD4C (n=6) □ RGD4C-DPK (n=6) a HPMA-RGE4C (n=6)

-TLn

Blood Heart Lung Liver Spleen Kidney Muscle Tumor

FIGURE 9.4 Residual radioactivity in % injected dose per gram of organ tissue (%ID/g) 24 h post-intravenous injection of 99mTc labeled HPMA copolymer conjugates and free peptides in (a) DU145 and (b) PC-3 prostate tumor xenograft-bearing SCID mice. The HPMA copolymers showed significantly higher tumor accumulation and reduced background localization than the peptides. The organ data are expressed as mean + SD (number of animals/group is shown). (From Line, B. R., et al., J. Nucl. Med., 46, 1152, 2005. With permission.)

xenograft bearing SCID mice, the tumor accumulation of the conjugate peaked at 72 h post-injection, whereas the accumulation in other major organs significantly decreased during that period. A single injection of the 90Y labeled conjugate at dose levels of 100 and 250 Ci caused significant reduction of tumor volume as compared to the untreated control that was evident from day 7 post-injection (Figure 9.5, top panel). Tumor histopathology showed cellular apoptosis in the treated groups, whereas no signs of acute toxicity were observed in the kidney, liver, and spleen (Figure 9.5, bottom panel).

The RGD4C peptide has a doubly cyclized structure containing two disulphide bonds that

178,179

affords high binding affinity and specificity for the aVp3 integrin. , 9 However, the solution instability of the RGD4C disulphide bonds is a potential disadvantage that can lead to significant reduction in aVb3 binding affinity. To overcome this problem, highly stable RGD peptides having monocyclic structure with head-to-tail cyclization containing a D-amino acid, e.g., RGDfK or RGDyK, have been synthesized.117 These have high affinity for aVb3 and have been used for delivery of imaging agents and therapeutics to tumor angiogenesis.19,21 The long term (up to 192 h) biodistribution and tumor targeting properties of multivalent HPMA copolymer

8 10 12 14 Days post-treatment

8 10 12 14 Days post-treatment

FIGURE 9.5 (See color insert following page 522.) TOP PANEL: Effect of 90Y labeled HPMA copolymer-RGD4C conjugate treatment on human prostate tumor (DU145) growth in SCID mice. Animal groups treated with single dose of 100 mCi (p<0.03) and 250 mCi (p<0.01) 90Y-HPMA-RGD4C conjugate showed significant tumor growth reduction as compared to the untreated controls by day seven post-treatment. Data are presented as mean of tumor volume (cm3) + SD (n = 6 mice per group). Figure legends: untreated control (closed square), 250 mCi of 90Y-HPMA copolymer-RGD4C conjugate (closed circle), and 100 mCi of 90Y-HPMA copolymer-RGD4C conjugate (open triangle). BOTTOM PANEL: Histological analysis of kidney and tumor samples at 21 days post-treatment following injection of 250 mCi 90Y labeled HPMA copolymer-RGD4C conjugate or untreated control. Kidney samples taken from (a) control group and (b) 250 mCi treatment group were similar and showed no radiation induced toxicity. There was a complete lack of evidence of any tubular epithelial injury. Normal glomerular and proximal tubular anatomy was evident in both control and 90Y treated specimens. (c) The tumor sections from the control animals showed high grade epithelial malignancy typical of DU145 xenografts. (d) Tumor samples from 250 mCi treatment animals showed large cellular drop out areas (black arrow), higher numbers of eosinophilic cytoplasmic hyaline globular bodies (thanato-somes, open arrow), and pronounced nuclear atypia (hatched arrow) indicative of treatment effect/induced cell damage (From Mitra, A., et al., Nucl. Med. Biol., 33, 43, 2006. With permission.)

conjugates of RGD4C and RGDfK peptides have been studied. Scintigraphic images and necropsy organ counts (Figure 9.6) showed that tumor accumulation of both HPMA-RGD4C and HPMA-RGDfK conjugates increased over time with peak accumulations at 4.9 + 0.9% (96 h p.i.) and 5.0+ 1.2% (48 h p.i.) ID/g, respectively. In contrast, the background organ distribution

01 t

24h 48 h 96 h 192h

GGG GGG GGG GGG GGG GGG GGG GGG GGG GGG GGG

Blood Heart Lung Liver Spleen Kidney Small Large Stomach

Intestine lntestine

1G.GGG 9.GGG 8.GGG 7.GGG , 6.GGG-S.GGG -4.GGG -3.GGG 2.GGG 1.GGG G.GGG

Lung

Liver Spleen Kidney

Small Large Stomach Intestine Intestine

FIGURE 9.6 (See color insert following page 522.) (a) Scintigraphic images of Lewis lung carcinoma-bearing mice up to 192 h post-intravenous injection of 111In labeled copolymers. (1) HPMA copolymer-RGD4C conjugate and (2) HPMA copolymer-RGDfK conjugate showed marked localization in tumor at 24 h p.i. and thereafter (solid arrow). The mouse radiograph shows anatomic correlation of tumor and other organs. Biodistribution of (b) 111In-HPMA copolymer-RGD4C conjugate and (c) 111In-HPMA copolymer-RGDfK conjugate. The organ activities, expressed as % injected dose per gram of organ tissue (%ID/g), showed persistent tumor localization and clearance of activity from the background organs (From Mitra, A., et al., J. Control. Release, 114, 175, 2006. With permission.)

Tail

Muscle

Tumor

Blood

Heart

Tail

Muscle

Tumor rapidly cleared over time, resulting in significant increase in T/B ratios. The radioactive dose to organs as indicated by the area under curve was highest for the tumor. The polymer conjugates of RGD4C or RGDfK provides a means to enhance tumor uptake, decrease background accumulation, and enable selective delivery of therapeutic or diagnostic agents to tumor sites. Because both HPMA-RGD4C and HPMA-RGDfK conjugates have similar tumor targeting abilities and pharmacokinetics, RGDfK can be a suitable ligand because of higher solution stability and easier synthetic manipulation than the 12 amino-acid RGD4C.

These studies with HPMA copolymer-RGD conjugates demonstrate the feasibility and advantages of using multivalent polymer-peptide conjugates. These conjugates show prolonged retention at the tumor site and enhanced T/B ratios. This increased contrast at the tumor site will be necessary for development of a clinically relevant diagnostic agent for therapy planning. Further, the improved therapeutic index will be ideal for angiogenesis directed chemo- or radiotherapy.

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