Pharmacokinetics and Tolerability of the Cancer-Targeting MRI Contrast Agent MT218 in Healthy Males.

OBJECTIVE: The aim of this study was to evaluate the pharmacokinetics and safety profile of MT218, a peptide-targeted gadolinium-based contrast agent, in healthy males. MATERIALS AND METHODS: This was a double-blind, randomized, placebo-controlled, single-ascending-dose study including 30 healthy male subjects. In each dose group (0.01, 0.02, 0.04, and 0.08 mmol/kg), 4 subjects received MT218 and 2 subjects received placebo (saline) in bolus injections. The highest dose group (0.08 mmol/kg) was assessed in 2 cohorts, 1 fasted and 1 nonfasted. Clinical laboratory tests, vital signs, and electrocardiograms were investigated. Gadolinium concentrations were measured in plasma samples collected before administration and over a 24-hour period postinjection, and in urine specimens collected until 22 days. A noncompartmental model was used for pharmacokinetic analysis. A clinical and biological safety follow-up was carried out for up to 6 months. RESULTS: No clinically significant modifications in biochemistry, hematology, urinalysis, electrocardiogram parameters, or vital signs were reported at any time point for any treatment group. No serious adverse events were observed in any dose group. Transient dizziness, hyperhidrosis, and injection site coldness were the main adverse events reported in both the MT218 and placebo groups. The mean total apparent clearance decreased slightly with increasing dose, and the median plasma t 1/2 ranged from 1.7 hours in the 0.01 mmol/kg group to 2.7 hours in the 0.08 mmol/kg nonfasted group. MT218 was rapidly excreted via renal filtration with 42.9% to 52.8% of the injected dose measured in urine within the first hour after administration, and 92.5% to 117.3% in urine within 24 hours. No Gd was detected by inductively coupled plasma mass spectrometry in urine after 21 days. CONCLUSION: Single intravenous administration of MT218 was safely tolerated in the healthy males. Its pharmacokinetic parameters and safety profile are well aligned with those of other gadolinium-based contrast agents.

Evaluating Dual-Targeted ECO/siRNA Nanoparticles against an Oncogenic lncRNA for Triple Negative Breast Cancer Therapy with Magnetic Resonance Molecular Imaging.

Differentiation antagonizing noncoding RNA (DANCR) is recognized as an oncogenic long noncoding RNA (lncRNA) overexpressed in triple negative breast cancer (TNBC). We showed in a previous study that RNAi with targeted multifunctional ionizable lipid ECO/siRNA nanoparticles was effective to regulate this undruggable target for effective treatment of TNBC. In this study, we developed dual-targeted ECO/siDANCR nanoparticles by targeting a tumor extracellular matrix oncoprotein, extradomain B fibronectin (EDB-FN), and integrins overexpressed on cancer cells for enhanced delivery of siDANCR. The treatment of Hs578T TNBC cells and MCF-7 estrogen receptor-positive cells in vitro resulted in significant down-regulation of DANCR and EDB-FN and suppressed invasion and 3D spheroid formation of the cells. Magnetic resonance molecular imaging (MRMI) with an EDB-FN-targeted contrast agent, MT218, was used to noninvasively evaluate tumor response to treatment with the targeted ECO/siDANCR nanoparticles in female nude mice bearing orthotopic Hs578T and MCF-7 xenografts. MRMI with MT218 was effective to differentiate between aggressive TNBC with high DANCR and EDB-FN expression and ER+ MCF-7 tumors with low expression of the targets. MRMI showed that the dual-targeted ECO/siDANCR nanoparticles resulted in more significant inhibition of tumor growth in both models than the controls and significantly reduced EDB-FN expression in the TNBC tumors. The combination of MRMI and dual-targeted ECO/siDANCR nanoparticles is a promising approach for image-guided treatment of TNBC by regulating the onco-lncRNA.

PET Imaging of Hepatocellular Carcinoma Using ZD2-(68Ga-NOTA).

Purpose: We tested a recently developed short peptide radioligand for PET imaging of hepatocellular carcinoma (HCC) by targeting an oncoprotein, extra-domain B fibronectin (EDB-FN) in the tumor microenvironment. Methods: The radioligand consists of a small linear peptide ZD2 with 68Ga-NOTA chelator, and specifically binds to EDB-FN. PET images were acquired dynamically for 1 hour after intravenously (i.v.) injecting 37 MBq (1.0 mCi) of the radioligand into the woodchuck model of naturally occurring HCC. Woodchuck HCC originated from chronic viral hepatitis infection, which recapitulates the corresponding human primary liver cancer. The animals were euthanized post-imaging for tissue collection and validation. Results: For ZD2 avid liver tumors, the radioligand accumulation plateaued a few minutes after injection, while the liver background uptake stabilized 20 min post-injection. The status of EDB-FN in woodchuck HCC was confirmed by histology and validated by PCR and western blocking. Conclusion: We have showed the viability of using the ZD2 short peptide radioligand targeting EDB-FN in liver tumor tissue for PET imaging of HCC, which can potentially impact the clinical care for HCC patients.

Effective gene therapy of Stargardt disease with PEG-ECO/pGRK1-ABCA4-S/MAR nanoparticles.

Stargardt disease (STGD) is the most common form of inherited retinal genetic disorders and is often caused by mutations in ABCA4. Gene therapy has the promise to effectively treat monogenic retinal disorders. However, clinically approved adeno-associated virus (AAV) vectors do not have a loading capacity for large genes, such as ABCA4. Self-assembly nanoparticles composed of (1-aminoethyl)iminobis[N-(oleoylcysteinyl-1-amino-ethyl)propionamide (ECO; a multifunctional pH-sensitive/ionizable amino lipid) and plasmid DNA produce gene transfection comparable with or better than the AAV2 capsid. Stable PEG-ECO/pGRK1-ABCA4-S/MAR nanoparticles produce specific and prolonged expression of ABCA4 in the photoreceptors of Abca4 -/- mice and significantly inhibit accumulation of toxic A2E in the eye. Multiple subretinal injections enhance gene expression and therapeutic efficacy with an approximately 69% reduction in A2E accumulation in Abca4 -/- mice after 3 doses. Very mild inflammation was observed after multiple injections of the nanoparticles. PEG-ECO/pGRK1-ABCA4-S/MAR nanoparticles are a promising non-viral mediated gene therapy modality for STGD type 1 (STGD1).

Evaluation of Physicochemical Properties, Pharmacokinetics, Biodistribution, Toxicity, and Contrast-Enhanced Cancer MRI of a Cancer-Targeting Contrast Agent, MT218.

OBJECTIVES: Preclinical assessments were performed according to the US Food and Drug Administration guidelines to determine the physicochemical properties, pharmacokinetics, clearance, safety, and tumor-specific magnetic resonance (MR) imaging of MT218, a peptidic gadolinium-based MR imaging agent targeting to extradomain B fibronectin for MR molecular imaging of aggressive tumors. MATERIALS AND METHODS: Relaxivity, chelation stability, binding affinity, safety-related target profiling, and effects on CYP450 enzymes and transporters were evaluated in vitro. Magnetic resonance imaging was performed with rats bearing prostate cancer xenografts, immunocompetent mice bearing murine pancreatic cancer allografts, and mice bearing lung cancer xenografts at different doses of MT218. Pharmacological effects on cardiovascular, respiratory, and central nervous systems were determined in rats and conscious beagle dogs. Pharmacokinetics were tested in rats and dogs. Biodistribution and excretion were studied in rats. Single and repeated dosing toxicity was evaluated in rats and dogs. In vitro and in vivo genotoxicity, in vitro hemolysis, and anaphylactic reactivity were also performed. RESULTS: At 1.4 T, the r1 and r2 relaxivities of MT218 were 5.43 and 7.40 mM -1 s -1 in pure water, 6.58 and 8.87 mM -1 s -1 in phosphate-buffered saline, and 6.54 and 8.70 mM -1 s -1 in aqueous solution of human serum albumin, respectively. The binding affinity of MT218 to extradomain B fragment is 3.45 µM. MT218 exhibited no dissociation of the Gd(III) chelates under physiological conditions. The peptide degradation half-life ( t1/2 ) of MT218 was 1.63, 5.85, and 2.63 hours in rat, dog, and human plasma, respectively. It had little effect on CYP450 enzymes and transporters. MT218 produced up to 7-fold increase of contrast-to-noise ratios in the extradomain B fibronectin-rich tumors with a dose of 0.04 mmol/kg for at least 30 minutes. MT218 had little pharmacological effect on central nervous, cardiovascular, or respiratory systems. MT218 had a mean plasma elimination half-life ( t1/2 ) of 0.31 and 0.89 hours in rats and dogs at 0.1 mmol/kg, respectively. No detectable Gd deposition was observed in the brain at 6 hours postinjection of MT218 at 0.1 mmol/kg in rats. MT218 was not mutagenic and had no mortality or morbidity in the rats or dogs up to 1.39 and 0.70 mmol/kg/d, respectively. The no observed adverse effect level of MT218 in Sprague-Dawley rats was 1.39 mmol/kg for single dosing and 0.46 mmol/kg/d for repeated dosing. The no observed adverse effect level in dogs was 0.07 mmol/kg/d. MT218 exhibited no genotoxicity, hemolysis, and anaphylactic reactivity. CONCLUSION: The preclinical assessments showed that the targeted contrast agent MT218 has high r1 and r2 relaxivities, satisfactory physicochemical properties, pharmacokinetic, and safety profiles and produces effective tumor enhancement in multiple cancer types in rats and mice at reduced doses.

Peptide Derivatives of Retinylamine Prevent Retinal Degeneration with Minimal Side Effects on Vision in Mice.

Safe and effective molecular therapeutics for prophylactic treatment of retinal degenerative diseases are greatly needed. Disruptions in the clearance of all-trans-retinal (atRAL) by the visual (retinoid) cycle of the retina can lead to the accumulation of atRAL and its condensation products known to initiate progressive retinal dystrophy. Retinylamine (Ret-NH2) and its analogues are known to be effective in lowering the concentration of atRAL within the eye and thus preventing retinal degeneration in mouse models of human retinopathies. Here, we chemically modified Ret-NH2 with amino acids and peptides to improve the stability and ocular bioavailability of the resulting derivatives and to minimize their side effects. Fourteen Ret-NH2 derivatives were synthesized and tested in vitro and in vivo. These derivatives exhibited structure-dependent therapeutic efficacy in preventing light-induced retinal degeneration in Abca4-/-Rdh8-/- double-knockout mice, with the compounds containing glycine and/or L-valine generally exhibiting greater protective effects than Ret-NH2 or other tested amino acid derivatives of Ret-NH2. Ret-NH2-L-valylglycine amide (RVG) exhibited good stability in storage; and effective uptake and prolonged retention in mouse eyes. RVG readily formed a Schiff base with atRAL and did not inhibit RPE65 enzymatic activity. Administered by oral gavage, this retinoid also provided effective protection against light-induced retinal degeneration in Abca4-/-Rdh8-/- mice. Notably, the treatment with RVG had minimal effects on the regeneration of 11-cis-retinal and recovery of retinal function. RVG holds promise as a lead therapy for effective and safe treatment of human retinal degenerative diseases.

Formulation and efficacy of ECO/pRHO-ABCA4-SV40 nanoparticles for nonviral gene therapy of Stargardt disease in a mouse model.

It is still a challenge to develop gene replacement therapy for retinal disorders caused by mutations in large genes, such as Stargardt disease (STGD). STGD is caused by mutations in ABCA4 gene. Previously, we have developed an effective non-viral gene therapy using self-assembled nanoparticles of a multifunctional pH-sensitive amino lipid ECO and a therapeutic ABCA4 plasmid containing rhodopsin promoter (pRHO-ABCA4). In this study, we modified the ABCA4 plasmid with simian virus 40 enhancer (SV40, pRHO-ABCA4-SV40) for enhanced gene expression. We also prepared and assessed the formulations of ECO/pDNA nanoparticles using sucrose or sorbitol as a stablilizer to develop consistent and stable formulations. Results demonstrated that ECO formed stable nanoparticles with pRHO-ABCA4-SV40 in the presence of sucrose, but not with sorbitol. The transfection efficiency in vitro increased significantly after introduction of SV40 enhancer for plasmid pCMV-ABCA4-SV40 with a CMV promoter. Sucrose didn't affect the transfection efficiency, while sorbitol resulted in a fluctuation of the in vitro transfection efficiency. Subretinal gene therapy in Abca4-/- mice using ECO/pRHO-ABCA4 and ECO/pRHO-ABCA4-SV40 nanoparticles induced 36% and 29% reduction in A2E accumulation respectively. Therefore, the ECO/pABCA4 based nanoparticles are promising for non-viral gene therapy for Stargardt disease and can be expended for applications in a variety of visual dystrophies with mutated large genes.

Magnetic Resonance Molecular Imaging of Extradomain B Fibronectin Improves Imaging of Pancreatic Cancer Tumor Xenografts.

The survival of pancreatic cancer patients can be greatly improved if their disease is detected at an early, potentially curable stage. Magnetic resonance molecular imaging (MRMI) of oncoproteins is a promising strategy for accurate, early detection of the disease. Here, we test the hypothesis that MRMI of extradomain-B fibronectin (EDB-FN), an abundant oncoprotein in the tumor extracellular matrix, can overcome the stromal barriers of pancreatic cancer to facilitate effective molecular imaging and detection of small tumors. Specimens of normal, premalignant, and malignant human pancreatic tissues were stained with a peptide-fluorophore conjugate (ZD2-Cy5.5) to assess EDB-FN binding and expression. MRMI with ZD2-N3-Gd(HP-DO3A) (MT218) specific to EDB-FN and MRI with Gd(HP-DO3A) were performed in three murine models bearing human pancreatic cancer xenografts, including a Capan-1 flank model, a BxPC3-GFP-Luc and a PANC-1-GFP-Luc intrapancreatic xenograft model. Tumor enhancement of the contrast agents was analyzed and compared. Staining of human tissue samples with ZD2-Cy5.5 revealed high EDB-FN expression in pancreatic tumors, moderate expression in premalignant tissue, and little expression in normal tissue. MRMI with MT218 generated robust intratumoral contrast, clearly detected and delineated small tumors (smallest average size: 6.1 mm2), and out-performed conventional contrast enhanced MRI with Gd(HP-DO3A). Quantitative analysis of signal enhancement revealed that MT218 produced 2.7, 2.1, and 1.6 times greater contrast-to-noise ratio (CNR) than the clinical agent in the Capan-1 flank, BxPC3-GFP-Luc and PANC-1-GFP-Luc intrapancreatic models, respectively (p < 0.05). MRMI of the ECM oncoprotein EDB-FN with MT218 is able to generate superior contrast enhancement in small pancreatic tumors and provide accurate tumor delineation in animal models. Early, accurate detection and delineation of pancreatic cancer with high-resolution MRMI has the potential to guide timely treatment and significantly improve the long-term survival of pancreatic cancer patients.

Synthesis and assessment of ZD2-(68Ga-NOTA) specific to extradomain B fibronectin in tumor microenvironment for PET imaging of pancreatic cancer.

Patients diagnosed with pancreatic cancer at a late stage have a dismal survival rate. Accurate early detection of pancreatic cancer with a size of 10 mm or less could dramatically improve patient survival after timely treatments. We have developed a new PET probe ZD2-(68Ga-NOTA) specific to extradomain-B fibronectin (EDB-FN), an oncoprotein in tumor microenvironment, for sensitive molecular imaging and early diagnosis of pancreatic cancer. A targeted ligand ZD2-NOTA is synthesized by conjugation of a macrocyclic ligand NOTA via a 6-aminohexanoic acid spacer to a linear ZD2 peptide (Thr-Val-Arg-Thr-Ser-Ala-Asp). ZD2-(68Ga-NOTA) is synthesized by relabeling of ZD2-NOTA with 68GaCl3 in a high purity under GMP conditions. The expression of EDB-FN is demonstrated in BxPC3 and Capan-1 human pancreatic cancer cells and tumor xenografts in mice. ZD2-(68Ga-NOTA) results in significantly higher uptake in the both BxPC3 and Capan-1 tumor xenografts than normal organs and tissues, including the brain, heart, liver and muscle, at 1 hr postinjection in mice. The tumor to muscle uptake ratio is at least 5 folds for the tracer in both tumors. ZD2-(68Ga-NOTA) is able to clearly delineate the PaCa tumors with a size of 10 mm or less with minimal background noise in normal tissues, including the liver. Substantial tumor uptake is still visible at 2 hr post-injection. The results suggest that the ZD2 peptide targeted PET probe has a potential for sensitive molecular imaging of EDB-FN and early detection of pancreatic cancer to improve healthcare of the patients diagnosed with the disease.

Preparation and Evaluation of ZD2 Peptide 64Cu-DOTA Conjugate as a Positron Emission Tomography Probe for Detection and Characterization of Prostate Cancer.

Positron emission tomography (PET) is a sensitive modality for cancer molecular imaging. We aim to develop a PET probe for sensitive detection and risk stratification of prostate cancer by targeting an abundant microenvironment oncoprotein, extradomain-B fibronectin (EDB-FN). The probe consists of a small ZD2 peptide specific to EDB-FN and a 64Cu-DOTA chelate. The probe was synthesized using standard solid-phase peptide chemistry and chelated to 64Cu prior to imaging. PET images were acquired at 4 and 22 h after intravenously injecting a 200 µCi probe into mice bearing human PC3 and LNCaP tumors, which represent highly aggressive and slow-growing prostate tumors, respectively. At 4 and 22 h postinjection, tumors could be clearly identified in the PET images. A significant higher signal was observed in PC3 tumors than in LNCaP tumors at 22 h (p = 0.01). Probe accumulation was also higher in PC3 tumors at 24 h. These data demonstrated that PET molecular imaging of EDB-FN in the tumor microenvironment of prostate cancer allows efficient differentiation of PC3 and LNCaP tumors in vivo. The ZD2 peptide-targeted PET probe shows potential in the detection and characterization of high-risk prostate cancer to improve the clinical management of prostate cancer.

Protective Effect of a Locked Retinal Chromophore Analog against Light-Induced Retinal Degeneration.

Continuous regeneration of the 11-cis-retinal visual chromophore from all-trans-retinal is critical for vision. Insufficiency of 11-cis-retinal arising from the dysfunction of key proteins involved in its regeneration can impair retinal health, ultimately leading to loss of human sight. Delayed recovery of visual sensitivity and night blindness caused by inadequate regeneration of the visual pigment rhodopsin are typical early signs of this condition. Excessive concentrations of unliganded, constitutively active opsin and increased levels of all-trans-retinal and its byproducts in photoreceptors also accelerate retinal degeneration after light exposure. Exogenous 9-cis-retinal iso-chromophore can reduce the toxicity of ligand-free opsin but fails to prevent the buildup of retinoid photoproducts when their clearance is defective in human retinopathies, such as Stargardt disease or age-related macular degeneration. Here we evaluated the effect of a locked chromophore analog, 11-cis-6-membered ring-retinal against bright light-induced retinal degeneration in Abca4-/-Rdh8-/- mice. Using in vivo imaging techniques, optical coherence tomography, scanning laser ophthalmoscopy, and two-photon microscopy, along with in vitro histologic analysis of retinal morphology, we found that treatment with 11-cis-6-membered ring-retinal before light stimulation prevented rod and cone photoreceptor degradation and preserved functional acuity in these mice. Moreover, additive accumulation of 11-cis-6-membered ring-retinal measured in the eyes of these mice by quantitative liquid chromatography-mass spectrometry indicated stable binding of this retinoid to opsin. Together, these results suggest that eliminating excess of unliganded opsin can prevent light-induced retinal degeneration in Abca4-/-Rdh8-/- mice.

Optimization of ZD2 Peptide Targeted Gd(HP-DO3A) for Detection and Risk-Stratification of Prostate Cancer with MRI.

The aim of this work is to optimize a peptide targeted macrocyclic MRI contrast agent for detection and risk-stratification of aggressive prostate cancer. The optimized agent was prepared using click chemistry in the presence of CuSO4 and ascorbate at room temperature. The T1 and T2 relaxivities of ZD2-N3-Gd(HP-DO3A) are 5.44 and 7.10 mM-1 s-1 at 1.4 T, and 5.53 and 7.81 mM-1 s-1 at 7 T, respectively, higher than the previously reported ZD2-Gd(HP-DO3A). The specific tumor enhancement of the agent was investigated in male nude mice bearing aggressive PC3 human prostate cancer xenografts and slow-growing LNCaP tumor xenografts. Contrast enhanced MR images were acquired using a 2D spin-echo sequence and a 3D FLASH sequence with a 7 T small animal scanner. ZD2-N3-Gd(HP-DO3A) produced robust contrast enhancement in aggressive PC3 tumors and little enhancement in slow-growing LNCaP tumors. It produced 400% and 100% CNR increases in the T1-weighted 2D spin-echo MR images and 3D FLASH images of PC3 tumors, respectively, for at least 30 min at a dose of 0.1 mmol/kg. In contrast, less than 20% CNR increase was observed in the LNCaP tumors with both sequences. The optimized targeted contrast agent has higher relaxivities and are effective to detect aggressive PC3 tumors and differentiate the aggressive cancer from the slow-growing LNCaP prostate cancer in contrast enhanced MRI. ZD2-N3-Gd(HP-DO3A) has the promise for accurate detection and risk-stratification of aggressive prostate cancer.

A novel small molecule chaperone of rod opsin and its potential therapy for retinal degeneration.

Rhodopsin homeostasis is tightly coupled to rod photoreceptor cell survival and vision. Mutations resulting in the misfolding of rhodopsin can lead to autosomal dominant retinitis pigmentosa (adRP), a progressive retinal degeneration that currently is untreatable. Using a cell-based high-throughput screen (HTS) to identify small molecules that can stabilize the P23H-opsin mutant, which causes most cases of adRP, we identified a novel pharmacological chaperone of rod photoreceptor opsin, YC-001. As a non-retinoid molecule, YC-001 demonstrates micromolar potency and efficacy greater than 9-cis-retinal with lower cytotoxicity. YC-001 binds to bovine rod opsin with an EC50 similar to 9-cis-retinal. The chaperone activity of YC-001 is evidenced by its ability to rescue the transport of multiple rod opsin mutants in mammalian cells. YC-001 is also an inverse agonist that non-competitively antagonizes rod opsin signaling. Significantly, a single dose of YC-001 protects Abca4 -/- Rdh8 -/- mice from bright light-induced retinal degeneration, suggesting its broad therapeutic potential.

New GABA modulators protect photoreceptor cells from light-induced degeneration in mouse models.

No clinically approved therapies are currently available that prevent the onset of photoreceptor death in retinal degeneration. Signaling between retinal neurons is regulated by the release and uptake of neurotransmitters, wherein GABA is the main inhibitory neurotransmitter. In this work, novel 3-chloropropiophenone derivatives and the clinical anticonvulsants tiagabine and vigabatrin were tested to modulate GABA signaling and protect against light-induced retinal degeneration. Abca4-/-Rdh8-/- mice, an accelerated model of retinal degeneration, were exposed to intense light after prophylactic injections of one of these compounds. Imaging and functional assessments of the retina indicated that these compounds successfully protected photoreceptor cells from degeneration to maintain a full-visual-field response. Furthermore, these compounds demonstrated a strong safety profile in wild-type mice and did not compromise visual function or damage the retina, despite repeated administration. These results indicate that modulating inhibitory GABA signaling can offer prophylactic protection against light-induced retinal degeneration.-Schur, R. M., Gao, S., Yu, G., Chen, Y., Maeda, A., Palczewski, K., Lu, Z.-R. New GABA modulators protect photoreceptor cells from light-induced degeneration in mouse models.

Multifunctional PEG retinylamine conjugate provides prolonged protection against retinal degeneration in mice.

A polyethylene glycol (PEG) retinylamine (Ret-NH2) conjugate PEG-GFL-NH-Ret with a glycine-phenylalanine-leucine (GFL) spacer was synthesized for controlled oral delivery of Ret-NH2 to treat retinal degenerative diseases, including Stargardt disease (STGD) and age-related macular degeneration (AMD). The peptide spacer was introduced for sustained release of the drug by digestive enzymes in the gastrointestinal tract. The pharmacokinetics experiments showed that the PEG conjugate could control the sustained drug release after oral administration and had much lower nonspecific liver drug accumulation than the free drug in wild-type female C57BL mice. In the mean time, the conjugate maintained the same concentration of Ret-NH2 in the eye as the free drug. Also, PEG-GFL-NH-Ret at a Ret-NH2 equivalent dose of 25 mg/kg produced complete protection of Abca4(-/-)Rdh8(-/-) mouse retinas against light-induced retinal degeneration for 3 days after oral administration, as revealed by OCT retina imaging, whereas free Ret-NH2 did not provide any protection under identical conditions. The polymer conjugate PEG-GFL-NH-Ret has great potential for controlled delivery of Ret-NH2 to the eye for effective protection against retinal degenerative diseases.

Antitumor efficacy of colon-specific HPMA copolymer/9-aminocamptothecin conjugates in mice bearing human-colon carcinoma xenografts.

The antitumor activity of a colon-specific N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer - 9-aminocamptothecin (9-AC) conjugate (P-9-AC) was assessed in orthotopic and subcutaneous animal (HT29 xenograft) tumor models. P-9-AC treatment of mice bearing orthotopic colon tumors, with a dose of 3 mg/kg of 9-AC equivalent every other day for 6 weeks, resulted in regression of tumors in 9 of 10 mice. A lower dose of P-9-AC (1.25 mg/kg of 9-AC equivalent) every other day for 8 weeks inhibited subcutaneous tumor growth in all mice. No liver metastases were observed. Colon-specific release of 9-AC from polymer conjugates enhanced antitumor activity and minimized the systemic toxicity.

Pharmacokinetic modeling of absorption behavior of 9-aminocamptothecin (9-AC) released from colon-specific HPMA copolymer-9-AC conjugate in rats.

PURPOSE: To quantitate and predict colon-specific 9-aminocamptothecin (9-AC) release from the N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-9-AC conjugate and its absorption behavior after oral administration in rats. METHODS: Drug distribution in the gastrointestinal (GI) tract and the plasma concentration-time profile of 9-AC released from the HPMA copolymer conjugate were predicted using the degradation, transit, and absorption rate constants in cecum. The fate of 9-AC in cecum and liver was measured by in-situ cecum absorption and liver perfusion. RESULTS: Following oral administration of the conjugate, 9-AC was released rapidly in cecum. Based on the pharmacokinetic model, up to 60% of the dose was in the cecum at ~6 h, and 7% of the dose still remained there at 24 h. The predicted plasma concentration curve for released 9-AC after an oral dose of 3 mg/kg of 9-AC equivalent increased gradually and reached a peak of 98 nM at 7 h, then started decreasing slowly to 16 nM at 24 h. The bioavailability value was estimated as 0.31 after the first-pass elimination. CONCLUSIONS: A pharmacokinetic model delineated the impact of GI transit, drug absorption rate, and first-pass metabolism on drug disposition following oral administration of HPMA copolymer-9-AC conjugate in rats.

Biodistribution and pharmacokinetics of colon-specific HPMA copolymer--9-aminocamptothecin conjugate in mice.

A water soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-9-aminocamptothecin (9-AC) conjugate was designed for oral colon-specific drug delivery in the treatment of colon cancer. Comparative studies between the polymer conjugate and free drug have been performed to assess their biodistribution and pharmacokinetics in mice. After oral administration of equal doses of the polymer conjugate or free 9-AC, the drug concentrations in major organs at fixed time points were determined using an HPLC-fluorescence assay. Only 2+/-1% of 9-AC released from the polymer conjugate was detected in the small intestine (SI), and the mean peak concentration of free 9-AC was 45-fold higher than that from released drug. Colon-specific release of 9-AC produced high local concentrations. The mean peak concentration of released 9-AC in cecal contents, feces, cecal tissue, and colon tissue were, respectively, 3.2-fold, 3.5-fold, 2.2-fold and 1.6-fold higher than that using free 9-AC. In plasma, the high and sharp drug concentration profile from free drug was in contrast to the relatively low and flat pharmacokinetic profile obtained from drug released from the HPMA copolymer. There was no significant difference between released and free drug for the area under the concentration-time curve (AUC) and bioavailability values. As a consequence of the colon-specific release of unmodified 9-AC from the polymer conjugate, antitumor efficacy can be anticipated to be enhanced due to prolonged colon tumor exposure to higher and more localized drug concentrations.

Colon-specific 9-aminocamptothecin-HPMA copolymer conjugates containing a 1,6-elimination spacer.

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer-9-aminocamptothecin (9-AC) conjugate for oral colon-specific drug delivery was designed, synthesized, and characterized. The drug, 9-AC, was attached to the polymer carrier via a spacer containing a combination of an aromatic azo bond and a 4-aminobenzylcarbamate group. The design of the spacer ensured a fast and highly efficient release of unmodified 9-AC from the polymer in the colon by azo bond cleavage followed by a 1,6-elimination mechanism. An in vitro degradation study indicated that this conjugate was stable in simulated upper GI tract conditions, including small intestine (SI) contents, SI mucosa suspension, and in PBS (pH 1.5 and 7.4). A fast release of the unmodified drug (85+/-10% of 9-AC in 12 h) was detected in rat cecal contents. This drug delivery system has potential in the treatment of colon cancer.