Recessive dystrophic epidermolysis bullosa (RDEB) and junctional epidermolysis bullosa (JEB) are severe blistering skin disorders caused by mutations in genes encoding type VII collagen (COL7A1) and laminin 332 (LAMA3, LAMB3, or LAMC2), respectively. In RDEB, 25% of patients carry nonsense mutations that result in premature termination codons (PTCs), while in JEB, the majority of mutations in LAMB3 are nonsense mutations (80%). CC-90009, an eRF3a degrader, is effective in inducing PTC readthrough in various in vitro models of diseases caused by nonsense mutations. This study evaluated CC-90009's ability, in combination with gentamicin, to suppress PTCs and promote the expression of type VII collagen (C7) in primary RDEB keratinocytes and fibroblasts, as well as laminin 332 in primary JEB keratinocytes with nonsense mutations. While CC-90009 alone demonstrated limited efficacy, its combination with low-dose gentamicin led to a dose-dependent increase in C7 and laminin β3 production, surpassing the effects of high-dose gentamicin alone. Furthermore, CC-90009/gentamicin reversed the hypermotility and poor substratum attachment characteristic of EB cells. Finally, C7 and laminin 332 induced by CC-90009/gentamicin localized to the dermal-epidermal junction in RDEB and JEB skin equivalents. Therefore, CC-90009/gentamicin may present a novel and safe treatment option for RDEB, JEB, and other inherited skin diseases arising from nonsense mutations.

Recessive dystrophic epidermolysis bullosa (RDEB) and junctional epidermolysis bullosa (JEB) are lethal blistering skin disorders resulting from mutations in genes coding for type VII collagen ( COL7A1 ) and laminin 332 ( LAMA3 , LAMB3, or LAMC2), respectively. In RDEB, 25% of patients harbor nonsense mutations causing premature termination codons (PTCs). In JEB, a majority of mutations in LAMB3 are nonsense mutations (80%). ELX-02, an aminoglycoside analog, has demonstrated superior PTC readthrough activity and lower toxicity compared to gentamicin in various genetic disorders. This study investigated the ability of ELX-02 to suppress PTCs and promote the expression of C7 and laminin 332 in primary RDEB keratinocytes/fibroblasts and primary JEB keratinocytes harboring nonsense mutations. ELX-02 induced a dose-dependent production of C7 or laminin b 3 that surpassed the results achieved with gentamicin. ELX-02 reversed RDEB and JEB cellular hypermotility and improved poor cell-substratum adhesion in JEB cells. Importantly, ELX-02-induced C7 and laminin 332 localized to the dermal-epidermal junction. This is the fi rst study demonstrating that ELX-02 can induce PTC readthrough and restore functional C7 and laminin 332 in RDEB and JEB caused by nonsense mutations. Therefore, ELX-02 may offer a novel and safe therapy for RDEB, JEB, and other inherited skin diseases caused by nonsense mutations.

Background Recessive dystrophic epidermolysis bullosa (RDEB) is an incurable widespread blistering skin disorder caused by mutations in the gene encoding for type VII collagen (C7), the major component of anchoring fibrils. Objectives To evaluate the efficacy and safety of intravenous (IV) gentamicin readthrough therapy in patients with RDEB harbouring nonsense mutations. The primary outcomes were increased expression of C7 in patients' skin and safety assessments (ototoxicity, nephrotoxicity, autoimmune response); secondary outcomes included measuring wound healing in target wounds and assessment by a validated Epidermolysis Bullosa Disease Activity and Scarring Index (EBDASI) scoring system. Methods An open-label pilot trial to assess two different IV gentamicin regimens between August 2018 and March 2020 with follow-up through to 180 days post-treatment was carried out. Three patients with RDEB with confirmed nonsense mutations in COL7A1 in either one or two alleles and decreased baseline expression of C7 at the dermal-epidermal junction (DEJ) of their skin participated in the study. Three patients received gentamicin 7.5 mg kg-1 daily for 14 days and two of the three patients further received 7.5 mg kg-1 IV gentamicin twice weekly for 12 weeks. Patients who had pre-existing auditory or renal impairment, were currently using ototoxic or nephrotoxic medications, or had allergies to aminoglycosides or sulfate compounds were excluded. Results After gentamicin treatment, skin biopsies from all three patients (age range 18-28 years) exhibited increased C7 in their DEJ. With both regimens, the new C7 persisted for at least 6 months post-treatment. At 1 and 3 months post-treatment, 100% of the monitored wounds exhibited > 85% closure. Both IV gentamicin infusion regimens decreased EBDASI total activity scores. Of the patients assessed with the EBDASI, all exhibited decreased total activity scores 3 months post-treatment. All three patients completed the study; no adverse effects or anti-C7 antibodies were detected. Conclusions IV gentamicin induced the readthrough of nonsense mutations in patients with RDEB and restored functional C7 in their skin, enhanced wound healing and improved clinical parameters. IV gentamicin may be a safe, efficacious, low-cost and readily available treatment for this population of patients with RDEB.

Generalized severe junctional epidermolysis bullosa (GS-JEB) is an incurable and fatal autosomal recessively inherited blistering skin disease caused by mutations in the LAMA3, LAMB3, or LAMC2 genes. Most of these mutations are nonsense mutations that create premature termination codons that lead to impaired production of functional laminin 332, a protein needed for epidermal-dermal adherence. Gentamicin induces readthrough of nonsense mutations and restores the full-length protein in various genetic diseases. Using primary keratinocytes from three GS-JEB patients, we showed that gentamicin induced functional laminin 332 that reversed a JEB-associated, abnormal cell phenotype. In a subsequent open-label trial involving the same patients, we examined whether 0.5% gentamicin ointment applied topically to open skin wounds could promote nonsense mutation readthrough and create new laminin 332 in the patients' skin. Gentamicin-treated wounds exhibited increased expression of laminin 332 at the dermal-epidermal junction for at least 3 months and were associated with improved wound closure. There were no untoward side effects from topical gentamicin. The newly induced laminin 332 did not generate anti-laminin 332 autoantibodies in either the patients' blood or skin. Gentamicin readthrough therapy may be a treatment for GS-JEB patients with nonsense mutations.

Herlitz junctional epidermolysis bullosa (H-JEB) is an incurable, devastating, and mostly fatal inherited skin disease for which there is only supportive care. H-JEB is caused by loss-of-function mutations in LAMA3, LAMB3, or LAMC2, leading to complete loss of laminin 332, the major component of anchoring filaments, which mediate epidermal-dermal adherence. LAMB3 (laminin β3) mutations account for 80% of patients with H-JEB, and ∼95% of H-JEB-associated LAMB3 mutations are nonsense mutations leading to premature termination codons (PTCs). In this study, we evaluated the ability of gentamicin to induce PTC readthrough in H-JEB laminin β3-null keratinocytes transfected with expression vectors encoding eight different LAMB3 nonsense mutations. We found that gentamicin induced PTC readthrough in all eight nonsense mutations tested. We next used lentiviral vectors to generate stably transduced H-JEB cells with the R635X and C290X nonsense mutations. Incubation of these cell lines with various concentrations of gentamicin resulted in the synthesis and secretion of full-length laminin β3 in a dose-dependent and sustained manner. Importantly, the gentamicin-induced laminin β3 led to the restoration of laminin 332 assembly, secretion, and deposition within the dermal/epidermal junction, as well as proper polarization of α6β4 integrin in basal keratinocytes, as assessed by immunoblot analysis, immunofluorescent microscopy, and an in vitro 3D skin equivalent model. Finally, newly restored laminin 332 corrected the abnormal cellular phenotype of H-JEB cells by reversing abnormal cell morphology, poor growth potential, poor cell-substratum adhesion, and hypermotility. Therefore, gentamicin may offer a therapy for H-JEB and other inherited skin diseases caused by PTC mutations.

BACKGROUND. Recessive dystrophic epidermolysis bullosa (RDEB) is an incurable disease caused by mutations in the gene encoding type VII collagen, the major component of anchoring fibrils (AF). We previously demonstrated that gentamicin produced functional type VII collagen in RDEB cells harboring nonsense mutations. Herein, we determined whether topical or intradermal gentamicin administration induces type VII collagen and AFs in RDEB patients.METHODS. A double-blind, placebo-controlled pilot trial assessed safety and efficacy of topical and intradermal gentamicin in 5 RDEB patients with nonsense mutations. The topical arm tested 0.1% gentamicin ointment or placebo application 3 times daily at 2 open erosion sites for 2 weeks. The intradermal arm tested daily intradermal injection of gentamicin solution (8 mg) or placebo into 2 intact skin sites for 2 days in 4 of 5 patients. Primary outcomes were induction of type VII collagen and AFs at the test sites and safety assessment. A secondary outcome assessed wound closure of topically treated erosions.RESULTS. Both topical and intradermal gentamicin administration induced type VII collagen and AFs at the dermal-epidermal junction of treatment sites. Newly created type VII collagen varied from 20% to 165% of that expressed in normal human skin and persisted for 3 months. Topical gentamicin corrected dermal-epidermal separation, improved wound closure, and reduced blister formation. There were no untoward side effects from gentamicin treatments. Type VII collagen induction did not generate anti-type VII collagen autoantibodies in patients' blood or skin.CONCLUSION. Topical and intradermal gentamicin suppresses nonsense mutations and induces type VII collagen and AFs in RDEB patients. Gentamicin therapy may provide a readily available treatment for RDEB patients with nonsense mutations.

Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited disorder characterized by skin fragility, blistering, and multiple skin wounds with no currently approved or consistently effective treatment. It is due to mutations in the gene encoding type VII collagen (C7). Using recombinant human C7 (rhC7) purified from human dermal fibroblasts (FB-rhC7), we showed previously that intravenously injected rhC7 distributed to engrafted RDEB skin, incorporated into its dermal-epidermal junction (DEJ), and reversed the RDEB disease phenotype. Human dermal fibroblasts, however, are not used for commercial production of therapeutic proteins. Therefore, we generated rhC7 from Chinese hamster ovary (CHO) cells. The CHO-derived recombinant type VII collagen (CHO-rhC7), similar to FB-rhC7, was secreted as a correctly folded, disulfide-bonded, helical trimer resistant to protease degradation. CHO-rhC7 bound to fibronectin and promoted human keratinocyte migration in vitro. A single dose of CHO-rhC7, administered intravenously into new-born C7-null RDEB mice, incorporated into the DEJ of multiple skin sites, tongue and esophagus, restored anchoring fibrils, improved dermal-epidermal adherence, and increased the animals' life span. Furthermore, no circulating or tissue-bound anti-C7 antibodies were observed in the mice. These data demonstrate the efficacy of CHO-rhC7 in a preclinical murine model of RDEB.

Patients with recessive dystrophic epidermolysis bullosa (RDEB) have severe, incurable skin fragility, blistering, and multiple skin wounds due to mutations in the gene encoding type VII collagen (C7), the major component of anchoring fibrils mediating epidermal-dermal adherence. Nearly 10-25% of RDEB patients carry nonsense mutations leading to premature stop codons (PTCs) that result in truncated C7. In this study, we evaluated the feasibility of using aminoglycosides to suppress PTCs and induce C7 expression in two RDEB keratinocyte cell lines (Q251X/Q251X and R578X/R906) and two primary RDEB fibroblasts (R578X/R578X and R163X/R1683X). Incubation of these cells with aminoglycosides (geneticin, gentamicin, and paromomycin) resulted in the synthesis and secretion of a full-length C7 in a dose-dependent and sustained manner. Importantly, aminoglycoside-induced C7 reversed the abnormal RDEB cell phenotype and incorporated into the dermal-epidermal junction of skin equivalents. We further demonstrated the general utility of aminoglycoside-mediated readthrough in 293 cells transiently transfected with expression vectors encoding 22 different RDEB nonsense mutations. This is the first study demonstrating that aminoglycosides can induce PTC readthrough and restore functional C7 in RDEB caused by nonsense mutations. Therefore, aminoglycosides may have therapeutic potential for RDEB patients and other inherited skin diseases caused by nonsense mutations.

Type VII collagen (C7) is a major component of anchoring fibrils, structures that mediate epidermal-dermal adherence. Mutations in gene COL7A1 encoding for C7 cause dystrophic epidermolysis bullosa (DEB), a genetic mechano-bullous disease. The biological consequences of specific COL7A1 mutations and the molecular mechanisms leading to DEB clinical phenotypes are unknown. In an attempt to establish genotype-phenotype relationships, we generated four individual substitution mutations that have been associated with recessive DEB, G2049E, R2063W, G2569R, and G2575R, and purified the recombinant mutant proteins. All mutant proteins were synthesized and secreted as a 290-kDa mutant C7 alpha chain at levels similar to wild type C7. The G2569R and G2575R glycine substitution mutations resulted in mutant C7 with increased sensitivity to protease degradation and decreased ability to form trimers. Limited proteolytic digestion of mutant G2049E and R2063W proteins yielded aberrant fragments and a triple helix with reduced stability. These two mutations next to the 39-amino acid helical interruption hinge region caused local destabilization of the triple-helix that exposed an additional highly sensitive proteolytic site within the region of the mutation. Our functional studies demonstrated that C7 is a potent pro-motility matrix for skin human keratinocyte migration and that this activity resides within the triple helical domain. Furthermore, G2049E and R2063W mutations reduced the ability of C7 to support fibroblast adhesion and keratinocyte migration. We conclude that known recessive DEB C7 mutations perturb critical functions of the C7 molecule and likely contribute to the clinical phenotypes of DEB patients.

Epidermolysis bullosa acquisita (EBA) is an acquired bullous disease of the skin characterized by IgG autoantibodies against type VII (anchoring fibril) collagen. We previously defined four immunodominant antigenic epitopes within the noncollagenous 1 (NC1) domain of type VII collagen. In this study, we produced an additional recombinant fusion protein from the NC1 domain corresponding to the N-terminal 227 amino acids (residues 1 to 227), which contains homology with cartilage matrix protein (CMP). Using enzyme-linked immunosorbent assay and immunoblot analysis, we tested sera from EBA patients (n = 32), bullous systemic lupus erythematosus patients (n = 3), bullous pemphigoid patients (n = 15), and normal humans (n = 12). Twenty-six of 32 EBA sera and two of three bullous systemic lupus erythematosus sera reacted with the CMP domain, whereas none of the control sera did. Affinity-purified anti-CMP EBA antibodies injected into hairless mice produced the clinical, histological, immunological, and ultrastructural features of EBA. F(ab')(2) fragments generated from anti-CMP EBA autoantibodies did not induce disease. Our studies provide the first evidence that EBA autoantibodies to the CMP subdomain of NC1 are pathogenic and induce blister formation. This is the first antigenic epitope on type VII collagen demonstrated to be a pathogenic target for EBA autoantibodies.

Epidermolysis bullosa acquisita (EBA) is an autoimmune sub-epidermal blistering disease characterized by autoantibodies to type VII (anchoring fibril) collagen. To date, however, direct evidence for a pathogenic role of human EBA autoantibodies has not been demonstrated. In this study, we affinity-purified anti-type VII collagen antibodies from EBA patients' sera and then injected them into adult hairless immunocompetent mice. Mice injected with EBA autoantibodies developed skin fragility, blisters, erosions, and nail loss on their paws-all features of EBA patients. By clinical, histological, immunological, and ultrastructural parameters, the induced lesions were reminiscent of human EBA. Histology showed bullous lesions with an epidermal-dermal separation. IgG and C3 deposits were observed at the epidermal-dermal junction. All mice had serum antibodies that labeled the dermal side of salt-split human skin like EBA sera. Direct immunogold electron microscopy specifically localized deposits of human IgG to anchoring fibrils. (Fab')(2) fragments generated from EBA autoantibodies did not induce disease. We conclude that EBA human patient autoantibodies cause subepidermal blisters and induce EBA skin lesions in mice. These passive transfer studies demonstrate that human EBA autoantibodies are pathogenic. This novel EBA mouse model can be used to further investigate EBA autoimmunity and to develop possible therapies.

Targeting drugs to receptors involved in tumor angiogenesis is a novel and promising approach to improve cancer treatment. In this study, we evaluated the antitumor activity of paclitaxel (PTX) conjugated with a bicyclic peptide E[c(RGDyK)](2) (RGD) in a metastatic breast cancer cell line (MDA-MB-435). The cyclic RGD peptide selectively binds to alpha(v) integrin receptors that are highly expressed in metastatic cancer cells. PTX, an antimicrotubule agent, is a potent antitumor agent commonly used in the treatment of advanced metastatic breast cancer. The in vitro results showed that RGD peptide inhibited cell cycle proliferation by arresting cells in G(0)/G(1)-phase. The PTX-RGD conjugate inhibited cell proliferation with activity comparable to that observed for paclitaxel, both of which were mediated by an arrest of G(2)/M-phase of the cell cycle followed by apoptosis. Although the PTX-RGD conjugate showed slightly decreased integrin binding affinity than the unconjugated peptide, it indicated integrin specific accumulation in vivo. (125)I-Labeled PTX-RGD showed highest tumor uptake at 2 h postinjection (2.72 +/-0.16%ID/g) and best tumor/background contrast after 4 h postinjection. Our results demonstrate the potential of tumor-targeted delivery of paclitaxel based on the specific recognition of cell adhesion molecule alpha(v)beta(3) integrin to reduce toxicity and enhance selective killing of cancer cells.

A series of radiolabeled cyclic arginine-glycineaspartic acid (RGD) peptide ligands for cell adhesion molecule integrin αβ3-targeted tumor angiogenesis targeting are being developed in our laboratory. In this study, this effort continues by applying a positron emitter 64Cu-labeled PEGylated dimeric RGD peptide radiotracer 64Cu-DOTA-PEG-E[c(RGDyK)]2 for lung cancer imaging. The PEGylated RGD peptide indicated integrin αβ3 avidity, but the PEGylation reduced the receptor binding affinity of this ligand compared to the unmodified RGD dimer. The radiotracer revealed rapid blood clearance and predominant renal clearance route. The minimum nonspecific activity accumulation in normal lung tissue and heart rendered high-quality orthotopic lung cancer tumor images, enabling clear demarcation of both the primary tumor at the upper lobe of the left lung, as well as metastases in the mediastinum, contralateral lung, diaphragm. As a comparison, fluorodeoxyglucose (FDG) scans on the same mice were only able to identify the primary tumor, with the metastatic lesions masked by intense cardiac uptake and high lung background. 64Cu-DOTA-PEGE[c(RGDyK)]2 is an excellent positron emission tomography (PET) tracer for integrin-positive tumor imaging. Further studies to improve the receptor binding affinity of the tracer and subsequently to increase the magnitude of tumor uptake without comprising the favorable in vivo kinetics are currently in progress.

The α v integrins, which act as cell adhesion molecules, are closely involved with tumor invasion and angiogenesis. In particular, α v β 3 integrin, which is specifically expressed on proliferating endothelial cells and tumor cells, is a logical target for development of a radiotracer method to assess angiogenesis and anti-angiogenic therapy. In this study, a dimeric cyclic RGD peptide E[c(RGDyK)] 2 was labeled with 18 F ( t 1 /2 = 109.7 min) by using a prosthetic 4-[ 18 F]fluorobenzoyl moiety to the amino group of the glutamate. The resulting [ 18 F]FB-E[c(RGDyK)] 2 , with high specific activity (200–250 GBq/μmol at the end of synthesis), was administered to subcutaneous U87MG glioblastoma xenograft models for micro-PET and autoradiographic imaging as well as direct tissue sampling to assess tumor targeting efficacy and in vivo kinetics of this PET tracer. The dimeric RGD peptide demonstrated significantly higher tumor uptake and prolonged tumor retention in comparison with a monomeric RGD peptide analog [ 18 F]FB-c(RGDyK). The dimeric RGD peptide had predominant renal excretion, whereas the monomeric analog was excreted primarily through the biliary route. Micro-PET imaging 1 hr after injection of the dimeric RGD peptide exhibited tumor to contralateral background ratio of 9.5 ± 0.8. The synergistic effect of polyvalency and improved pharmacokinetics may be responsible for the superior imaging characteristics of [ 18 F]FB-E[c(RGDyK)] 2 .

We have previously labeled cyclic RGD peptide c(RGDyK) with fluorine-18 through conjugation labeling via a prosthetic 4-[18F]fluorobenzoyl moiety and applied this [18F]FB-RGD radiotracer for αv-integrin expression imaging in different preclinical tumor models with good tumor-to-background contrast. However, the unfavorable hepatobiliary excretion and rapid tumor washout rate of this tracer limit its potential clinical applications. The aims of this study were to modify the [18F]FB-RGD tracer by inserting a heterobifunctional poly(ethylene glycol) (PEG, M.W. =3,400) between the 18F radiolabel and the RGD moiety and to test this [18F]FB-PEG-RGD tracer for brain tumor targeting and in vivo kinetics. [18F]FB-PEG-RGD was prepared by coupling the RGD-PEG conjugate with N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) under slightly basic conditions (pH=8.5). The radiochemical yield was about 20–30% based on the active ester [18F]SFB, and specific activity was over 100 GBq/μmol. This tracer had fast blood clearance, rapid and high tumor uptake in the subcutaneous U87MG glioblastoma model (5.2±0.5%ID/g at 30 min p.i.). Moderately rapid tumor washout was observed, with the activity accumulation decreased to 2.2±0.4%ID/g at 4 h p.i. MicroPET and autoradiography imaging showed a very high tumor-to-background ratio and limited activity accumulation in the liver, kidneys and intestinal tracts. U87MG tumor implanted into the mouse forebrain was well visualized with [18F]FB-PEG-RGD. Although uptake in the orthotopic tumor was significantly lower (P<0.01) than in the subcutaneous tumor, the maximum tumor-to-brain ratio still reached 5.0±0.6 due to low normal brain background. The results of H&E staining post mortem agreed with the anatomical information obtained from non-invasive microPET imaging. In conclusion, PEGylation suitably modifies the physiological behavior of the RGD peptide. [18F]FB-PEG-RGD gave improved tumor retention and in vivo kinetics compared with [18F]FB-RGD.

Overexpression of gastrin-releasing peptide (GRP) receptor (GRPR) in both androgen-dependent (AD) and androgen-independent (Al) human neoplastic prostate tissues provides an attractive target for prostate cancer imaging and therapy. The goal of this study was to develop Cu-64-radio labeled GRP analogs for PET imaging of GRPR expression in prostate cancer xenografted mice. Methods: [Lys(3)]bombesin ([Lys(3)]BBN) was conjugated with 1,4,7,10-tetraazadodecane-N,N',N",N"'-tetraacetic acid (DOTA) and labeled with the positron-emitting isotope Cu-64 (half-life = 12.8 h, 19% beta(+)). Receptor binding of DOTA-[Lys(3)]BBN and internalization of (CU)-C-64-DOTA[Lys(3)]BBN by PC-3 prostate cancer cells were measured. Tissue biodistribution, microPET, and whole-body autoradiographic imaging of the radiotracer were also investigated in PC-3 (Al)/CRW22 (AD) prostate cancer tumor models. Results: A competitive receptor-binding assay using I-125-[Tyr(4)]BBN against PC-3 cells yielded a 50% inhibitory concentration value of 2.2 +/- 0.5 nmol/L for DOTA-[Lys3]BBN. Incubation of cells with the Cu-64-labeled radiotracer showed temperature- and time-dependent internalization. At 37degreesC, >60% of the tracer was internalized within the first 15 min and uptake remained constant for 2 h. Radiotracer uptake was higher in Al PC-3 tumor (5.62 +/- 0.08 %ID/g at 30 min after injection, where %ID/g is the percentage of injected dose per gram) than in AD CWR22 tumor (1.75 +/- 0.05 %ID/g at 30 min after injection). Significant accumulation of the activity in GRPR-positive pancreas was also observed ((10.4 +/- 0.15 % ID/g at 30 min after injection). Coinjection of a blocking dose of [Lys(3)]BBN inhibited the activity accumulation in PC-3 tumor and pancreas but not in CWR22 tumor. microPET and autoradiographic imaging of Cu-64-DOTA[Lys(3)]BBN in athymic nude mice bearing subcutaneous PC-3 and CWR22 tumors showed strong tumor-to-background contrast. Conclusion: This study demonstrates that PET imaging of Cu-64-DOTA-[Lys(3)]BBN is able to detect GRPR-positive prostate cancer.

PURPOSE:Alphavbeta3 and alphavbeta5 integrins are cell adhesion molecules that play a vital role in tumor angiogenesis and metastasis. The ability to visualize and quantify integrin expression in vivo will foster our understanding of the role of integrins alphavbeta3 and alphavbeta5 in tumor angiogenesis and allow for direct assessment of anti-angiogenic treatment efficacy based on integrin antagonists. This study compared the tumor targeting characteristics of two dimeric 64Cu-labeled RGD peptide agonists of alphavbeta3 integrin. PROCEDURES:Dimeric RGD peptides E[c(RGDyK)]2 and E[c(RGDfK)]2 were conjugated with 1,4,7,10-tetraazadodecane-N,N',N",N"'-tetraacetic acid (DOTA) and labeled with positron emitter 64Cu(t(1/2)=12.8 h, beta+=19%). Both 64Cu-DOTA-E[c(RGDyK)]2 and 64Cu-DOTA-E[c(RGDfK)]2 were used in biodistribution, microPET imaging and whole-body autoradiography studies in athymic female nude mice with orthotopically growing MDA-MB-435 breast carcinoma xenografts. RESULTS:At all time points, activity accumulation of 64)Cu-DOTA-E[c(RGDyK)]2 in tumors was significantly higher compared to the D-Phe analog. Liver uptake of the D-Tyr derivative was lower than the D-Phe derivative at early time points but the difference became marginal with time. Overall, 64Cu-DOTA-E[c(RGDyK)]2 yielded better position emission tomography (PET) images in orthotopic MDA-MB-435 bearing mice than did 64Cu-DOTA-E[c(RGDfK)]2. Both radiotracers had alphav-integrin specific tumor activity accumulation, as demonstrated by significant reduction of uptake with a coinjected blocking dose of c(RGDyK). CONCLUSIONS:The radiolabeled dimeric RGD peptides 64Cu-DOTA-E[c(RGDyK)]2 and 64Cu-DOTA-E[c(RGDfK)]2 have high and specific tumor uptake in a human breast cancer tumor xenograft, with the D-Tyr derivative showing better in vivo kinetics than the D-Phe derivative, most likely due to the increased hydrophilicity of the D-Tyr. Both dimeric peptides showed better tumor retention than the previously tested monomeric RGD counterparts, presumably because of bivalency and increase in apparent molecular size.

UNLABELLED:The alphav-integrins, cell adhesion molecules that are highly expressed on activated endothelial cells and tumor cells but not on dormant endothelial cells or normal cells, present an attractive target for tumor imaging and therapy. We previously coupled a cyclic Arg-Gly-Asp (RGD) peptide, c(RGDyK), with 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) and labeled the RGD-DOTA conjugate with 64Cu (half-life, 12.8 h; 19% beta+) for solid tumor targeting, with high tumor-to-background contrast. The rapid tumor washout rate and persistent liver and kidney retention of this tracer prompted us to optimize the tracer for improved pharmacokinetic behavior. In this study, we introduced a polyethylene glycol (PEG; molecular weight, 3,400) moiety between DOTA and RGD and evaluated the 64Cu-DOTA-PEG-RGD tracer for microPET imaging in brain tumor models.METHODS:DOTA was activated in situ and conjugated with RGD-PEG-NH2 under slightly basic conditions. alphavbeta3-Integrin-binding affinity was evaluated with a solid-phase receptor-binding assay in the presence of 125I-echistatin. Female nude mice bearing subcutaneous U87MG glioblastoma xenografts were administered 64Cu-DOTA-PEG-RGD, and the biodistributions of the radiotracer were evaluated from 30 min to 4 h after injection. microPET (20 min of static imaging at 1 h after injection) and then quantitative autoradiography were used for tumor visualization and quantification. The same tracer was also applied to an orthotopic U87MG model for tumor detection.RESULTS:The radiotracer was synthesized with a high specific activity (14,800-29,600 GBq/mmol [400-800 Ci/mmol]). The c(RGDyK)-PEG-DOTA ligand showed intermediate binding affinity for alphavbeta3-integrin (50% inhibitory concentration, 67.5 +/- 7.8 nmol/L [mean +/- SD]). The pegylated RGD peptide demonstrated rapid blood clearance (0.57 +/- 0.15 percentage injected dose [%ID]/g [mean +/- SD] at 30 min after injection and 0.03 +/- 0.02 %ID/g at 4 h after injection). Activity accumulation in the tumor was rapid and high at early time points (2.74 +/- 0.45 %ID/g at 30 min after injection), and some activity washout was seen over time (1.62 +/- 0.18 %ID/g at 4 h after injection). Compared with (64)Cu-DOTA-RGD, this tracer showed improved in vivo kinetics, with significantly reduced liver uptake (0.99 +/- 0.08 %ID/g vs. 1.73 +/- 0.39 %ID/g at 30 min after injection and 0.58 +/- 0.07 %ID/g vs. 2.57 +/- 0.49 %ID/g at 4 h after injection). The pegylated RGD peptide showed higher renal accumulation at early time points (3.51 +/- 0.24 %ID/g vs. 2.18 +/- 0.23 %ID/g at 30 min after infection) but more rapid clearance (1.82 +/- 0.29 %ID/g vs. 2.01 +/- 0.25 %ID/g at 1 h after injection) than 64Cu-DOTA-RGD. The integrin receptor specificity of this radiotracer was demonstrated by blocking of tumor uptake by coinjection with nonradiolabeled c(RGDyK). The high tumor-to-organ ratios for the pegylated RGD peptide tracer (at 1 h after injection: tumor-to-blood ratio, 20; tumor-to-muscle ratio, 12; tumor-to-liver ratio, 2.7; and tumor-to-kidney ratio, 1.2) were confirmed by microPET and autoradiographic imaging in a subcutaneous U87MG tumor model. This tracer was also able to detect an orthotopic brain tumor in a model in which U87MG cells were implanted into the mouse forebrain. Although the magnitude of tumor uptake in the orthotopic xenograft was lower than that in the subcutaneous xenograft, the orthotopic tumor was still visualized with clear contrast from normal brain tissue.CONCLUSION:This study demonstrated the suitability of a PEG moiety for improving the in vivo kinetics of a 64Cu-RGD peptide tracer without compromising the tumor-targeting ability and specificity of the peptide. Systematic investigations of the effects of the size and geometry of PEG on tumor targeting and in vivo kinetics will lead to the development of radiotracers suitable for clinical applications such as visualizing and quantifying alphav-integrin expression by PET. In addition, the same ligand labeled with therapeutic radionuclides may be applicable for integrin-targeted internal radiotherapy.