Pharmacokinetic and biodistribution studies of a bone-targeting drug delivery system based on N-(2-hydroxypropyl)methacrylamide copolymers

Osteotropicity of novel bone-targeted HPMA copolymer conjugates has been demonstrated previously with bone histomorphometric analysis. The pharmacokinetics and biodistribution of this delivery system were investigated in the current study with healthy young BALB/c mice. The 125I-labeled bone-targeted and control (nontargeted) HPMA copolymers were administered intravenously to mice, and their distribution to different organs and tissues was followed using gamma counter and single photon emission computed tomography (SPECT). Both the invasive and noninvasive data further confirmed that the incorporation of D-aspartic acid octapeptide (D-Asp8) as bone-targeting moiety could favorably deposit the HPMA copolymers to the entire skeleton, especially to the high bone turnover sites. To evaluate the influence of molecular weight, three fractions (Mw of 24, 46, and 96 kDa) of HPMA copolymer-D-Asp8 conjugate were prepared and evaluated. Higher molecular weight of the conjugate enhanced the deposition to bone due to the prolonged half-life in circulation, but it weakened the bone selectivity. A higher content of bone-targeting moiety (D-Asp8) in the conjugate is desirable to achieve superior hard tissue selectivity. Further validation of the bone-targeting efficacy of the conjugates in animal models of osteoporosis and other skeletal diseases is needed in the future.     

Macrophage targeted N-(2-hydroxypropyl)methacrylamide conjugates for magnetic resonance imaging

This study describes the synthesis, characterization and in vitro evaluation of targetable N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-gadolinium (Gd) chelates for enhanced magnetic resonance imaging (MRI) of macrophages. Copolymers of HPMA, methacryloylglycylglycyl-mannosamine (MA-GG-ManN), aminopropylmethacrylamide-benzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (APMA-DOTA), and 5-(3-(methacryloylaminopropyl)thioureidyl) fluorescein (MA-AP-FITC) were synthesized and characterized. Gd was chelated to the polymeric precursors. The conjugates were characterized for gadolinium content by inductively coupled plasma optical emission spectrometry (ICP-OES) and T1 relaxivity (r1) at room temperature and 1.5 T. The effect of ManN content on mannose receptor mediated uptake of THP-1 human macrophages was evaluated as a function of time and temperature. The polymer conjugates showed relaxivities in the range of 21.8-24.9 s(-1) mM(-1) Gd. Relaxivities of the conjugates per mM Gd were up to 7 times higher than that of a commercially available MR contrast agent Gd-DOTA. Significantly (p < 0.042) higher uptake was observed for targeted conjugates compared to nontargeted conjugates. The uptake of polymeric conjugates was time and concentration dependent and appears to be mannose receptor mediated. The increased relaxivity coupled with the ability to target these carriers to cells containing ManN receptors shows promise for the application of these agents in clinical MR imaging of macrophage mediated malignancies.     

Clinical implications of N-(2-hydroxypropyl)methacrylamide copolymers

Different anticancer drugs, farmorubicin, doxorubicin, paclitaxel and cis-platin have been conjugated through a Gly-Phe-Leu-Gly tetrapeptide side chain to a water-soluble synthetic polymeric carrier based on N-(2-hydroxypropyl)methacryalmide (HPMA) non-targeted or targeted with galactosamine and/or human IVIg and used in Phase I clinical trials. Conjugation of the drugs to the polymeric carrier that is non-toxic and non-immunogenic in man significantly decreased their non-specific organ toxicities and increased maximum tolerated dose up to 5 times. Macromolecular therapeutics based on HPMA have radically different pharmacokinetics. Drugs are not released from their polymeric carrier and remain in the peripheral blood and urine of patients mostly in their polymer-bound form. A clinical response against some refractory cancers was recorded in Phase I clinical trials. It was also demonstrated that doxorubicin-HPMA copolymer conjugates containing an immunoglobulin moiety have both cytostatic and immunomobilizing activity.     

Poly[N-(2-hydroxypropyl)methacrylamide] conjugates of bovine pancreatic ribonuclease (RNase A) inhibit growth of human melanoma in nude mice

Recently hydrophilic poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) was used for BS-RNase modification to prevent its degradation in bloodstream or fast elimination. Polymer-conjugated BS-RNase preparations proved to be cytotoxic after intravenous or intraperitoneal application, whereas native BS-RNase was ineffective. Here RNase A unimer was conjugated with two HPMA polymers (classic and star) and their antitumor effects both in vitro and in vivo were compared with those of BS-RNase polymers. Surprisingly, the antitumor effect of RNase A conjugates was also pronounced. The RNase A conjugates (classic and star) injected intravenously to mice bearing melanoma tumor caused a significant reduction in tumor volume following ten doses of 5 and 1 mg/kg, respectively. Despite the antitumor activity observed in vivo, the in vitro tested cytotoxic activity of RNase A did not differ from that caused by native RNase A while native BS-RNase (50 microg/ml) totally inhibited DNA synthesis in treated cells. The experiments with 125I-labeled preparations demonstrated concentration-dependent internalization of native BS-RNase by tumor cells within an hour, whereas the polymer conjugate (S-BS) was not internalized. On the contrary, the in vivo experiments showed that whereas 40% of S-BS conjugate persisted in bloodstream for 24h after administration, 98% of the native BS-RNase was already eliminated. Improved antitumor activities of PHPMA-modified RNases in vivo might be ascribed to their prolonged retention in bloodstream, better proteolytic stability and resistance to the action of the ribonuclease inhibitor.     

Biodegradable multiblock poly(N-2-hydroxypropyl)methacrylamide gemcitabine and paclitaxel conjugates for ovarian cancer cell combination treatment

The synthesis, characterization, and in vitro evaluation of a combination delivery of multiblock poly(N-2-hydroxypropyl)methacrylamide (HPMA), gemcitabine (GEM) and paclitaxel (PTX) conjugates is described in this study. Multiblock copolymer conjugates of a large molecular weight (M_w > 200 kDa) were studied and compared to traditional, small molecular weight (M_w < 45 kDa) conjugates. Stability of the conjugates in different pH was assessed, and their cytotoxicity in combination toward A2780 human ovarian cancer cells was evaluated by combination index analysis. Treatment duration (4 and 72 h) and sequence of addition were explored. In addition, an HPMA copolymer conjugate with both GEM and PTX in the side chains was evaluated in a similar manner and compared to a physical mixture of individual conjugates. Conjugates with narrow molecular weight distribution (M_w/M_n < 1.1) were obtained via RAFT polymerization, and drug loadings of between 5.5 and 9.2 wt% were achieved. Conjugates demonstrated moderate stability with less than 65% release over 24 h at pH 7.4, and near complete drug release in the presence of the lysosomal enzyme cathepsin B in 3 h. In combination, the cytotoxic effects of a mixture of the conjugates were primarily additive. Synergistic effects were observed when A2780 human ovarian cancer cells were treated simultaneously for 4 h with multiblock conjugates (CI < 0.7). When both GEM and PTX were conjugated to the same copolymer backbone, moderate antagonism (CI 1.3–1.6) was observed. These results demonstrate that multiblock HPMA copolymer–GEM and –PTX conjugates, when delivered as a mixture of individual agents, are promising for the treatment of ovarian cancer.     

Improvement of anti-tumor abilities on human non-small cell lung carcinoma by micellization and cross-linking of N-(2-hydroxypropyl) methacrylamide copolymers

Non-small cell lung carcinoma is one of the most frequently occurred cancers with a very high rate of recurrence. Self-assembly N-(2-hydroxypropyl) methacrylamide (HPMA) micelles and cross-linked micelles were developed to improve antitumor ability of linear HPMA copolymer. The characters of HPMA micelles were investigated and compared using human non-small cell lung carcinoma 3-D culture model and nude mice xenograft model. Cross-linked micelles showed highest cytotoxicity on A549 cell monolayers after a short time treatment in vitro. Moreover, both of the two micelles exhibited better in vitro anti-tumor activity on A549 tumor spheroids than linear HPMA conjugates especially the cross-linked micelles. On BALB/c nude mice bearing A549 xenograft tumors, the cross-linked micelles exhibited the greatest tumor accumulation and the best anti-tumor activity due to the highly improved stabilities and the more pronounced enhanced permeability and retention (EPR) effect, which were followed by the non-cross-linked micelles. Meanwhile, neither the two micelles nor the linear HPMA copolymers showed significant toxicity on the main organs of mice while free doxorubicin (DOX) showed obvious cardiac toxicity. All the results suggested that micellization improved the anti-tumor activity of HPMA copolymers on A549 human non-small cell lung carcinoma, furthermore, cross-linked HPMA copolymer micelles with pH-sensitivity and biodegradability showed more excellent anti-tumor activity.     

Gamma Scintigraphy of the Biodistribution of 123I-Labelled N-(2-Hydroxypropyl)methacrylamide Copolymer-Doxorubicin Conjugates in Mice with Transplanted Melanoma and Mammary Carcinoma

Abstract

An N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin conjugate is currently under clinical evaluation as a new antitumour agent. It has been shown previously that such conjugates exhibit selective tumour accumulation. In this study HPMA copolymer doxorubicin conjugates of low (LMW) or high (HMW) molecular weight were synthesised (which had a weight average molecular weight (Mw) of 25,000 and 94,000 respectively) and additionally contained a small amount (1 mol%) of the comonomer methacryloyltyrosinamide to permit labelling with [¹²³I or ¹²⁵I]iodide. Gamma camera imaging using the I-labelled probes was used to follow time-dependent biodistribution after intraperitoneal (i.p.) or intravenous (i.v.) administration to mice bearing subcutaneously either B16F10 melanoma or a mammary carcinoma. Imaging showed more rapid clearance of LMW conjugate from the peritoneal cavity than HMW conjugate. The images of mice given the LMW conjugate revealed rapid urinary excretion of radioactivity after both i.p. and i.v. injection with an early high concentration of tracer in the bladder, and subsequently a very high concentration in the kidneys, which came to dominate the views. Dissection analysis 2 days after administration of the LMW conjugate revealed a kidney level of radioactivity corresponding to 25-40 %dose/g tissue in mice bearing the two tumour models. Following administration of the HMW conjugate kidney accumulation at 2 days was less due to retention of the higher molecular weight polymer molecules in the circulation, and spleen and liver displayed the highest concentrations of radioactivity. The tumour accumulation of LMW and HMW conjugates was; mammary carcinoma 3.18 and 5.29 % dose/g respectively; B16F10 melanoma 3.23 and 8.82 %dose/g although these levels of tracer enabled visualisation in the images of the mammary carcinoma with HMW conjugate at later time points. The smaller size of the B16F10 tumour masses did not permit clear visualisation.     

Effect on in vitro cell response of the statistical insertion of N-(2-hydroxypropyl) methacrylamide on linear pro-dendronic polyamine’s gene carriers

Statistical copolymers of N-(2-hydroxypropyl) methacrylamide (HPMA) and the dendronic methacrylic monomer 2-(3-(Bis(2-(diethylamino)ethyl)amino)propanamido)ethyl methacrylate (TEDETAMA, derived from N,N,N′,N′-tetraethyldiethylenetriamine, TEDETA), were synthesized through radical copolymerization and evaluated in vitro as non-viral gene carriers. Three copolymers with nominal molar percentages of HPMA of 25%, 50% and 75% were prepared and studied comparatively to the positive controls poly-TEDETAMA and hyperbranched polyethyleneimine (PEI, 25 kDa). Their ability to complex DNA at different N/P molar ratios, from 1/1 up to 8/1, was determined through agarose gel electrophoresis and Dynamic Light Scattering. The resulting complexes (polyplexes) were characterized and evaluated in vitro as possible non-viral gene carriers for Swiss-3T3 fibroblasts, using luciferase as reporter gene and a calcein cytocompatibility assay. All the copolymers, except the one with highest HPMA proportion (75 molar %) at the lowest N/P ratio, condensed DNA to a particle size between 100 and 300 nm. The copolymers with 25 and 50 molar % of HPMA displayed higher transfection efficiency and cytocompatibility than the positive controls poly-TEDETAMA and PEI. A higher proportion of HPMA (75 molar %) led to copolymers that displayed very low transfection efficiency, despite their full cytocompatibility even at the highest N/P ratio. These results indicate that the statistical combination of TEDETAMA and HPMA and its fine compositional tuning in the copolymers may fulfill the fine balance of transfection efficiency and cytocompatibility in a superior way to the control poly-TEDETAMA and PEI.     

Synthetic macromolecular drug carriers: biodistribution of poly[(N-2-hydroxypropyl)methacrylamide] copolymers and their accumulation in solid rat tumors.

To optimize polymer design for tumor directed drug delivery, the fate and the total body distribution of soluble synthetic macromolecules, derived from copolymers of [(N-2-(hydroxypropyl)methacrylamide] (HPMA) were monitored scintigraphically after radiolabeling with 131I during a seven day time window. Equimolar concentrations of radioiodinated copolymers of HPMA with small amounts of methacryloyltyrosinamide (pHPMA) differing in molecular weight (23.4 kD, 27.3 kD, 30.5 kD, 44 kD, 58.4 kD, 60.1 kD) were injected intravenously into Copenhagen rats bearing Dunning prostate carcinomas (subline R3327-AT1). Scintigraphic data were validated by determining absolute amounts of [131I]pHPMA in both tumor tissue and normal organs after sacrificing the animals. Copolymers were cleared from blood circulation in a molecular-weight dependent manner, either via excretion or by extravasation into normal and neoplastic tissues. While distribution patterns for pHPMAs in normal organs were quite similar, absolute amounts of copolymer uptake differed. The higher the molecular weight, the more radioactivity was taken up by the organs. Highest amounts of radioactivity were seen in the lung, liver, and spleen. In solid tumors, kinetics of pHPMA accumulation was clearly dependent on molecular weight. pHPMAs below the renal threshold peaked at 24 hours p.i. and then remained constant. In contrast, copolymers above the renal clearance threshold displayed a continuous accumulation reaching a significantly higher tumor uptake, presumably due to the very small or non existent polymer release from tumor tissue. Absolute amounts of tumor uptake determined by dissection analysis were 0.5 +/- 0.1% of injected dose/g tissue for the 27.3 kD pHPMA and 1.2 +/- 0.1% for the 60.1 kD pHPMA, respectively. In conclusion, our results demonstrate the influence of the molecular weight of the synthetic polymer pHPMA on plasma circulation time, excretion and organ clearance. While pHPMAs are cleared from all normal tissues except the spleen quite effectively, these polymers accumulate in solid tumors in a size dependent manner, due to the well known "enhanced permeability and retention" (EPR) effect. These data are of fundamental interest for ongoing studies on the pharmacokinetics of synthetic polymers, especially when these molecules are conjugated with targeting moieties and therapeutic or diagnostic agents.     

Stereoselective pharmacokinetic analysis and antiepileptic activity of N-2-hydroxypropyl valpromide, a central nervous system--active chiral valproylamide

The purpose of this study was to evaluate the anticonvulsant activity and pharmacokinetics (PK) of a novel chiral CNS-active 2-hydroxypropyl valpromide (HP-VPD), a derivative of valproic acid (VPA). The individual enantiomers, R, S, and racemic (R,S)-HP-VPD were synthesized and evaluated for their pharmacokinetics and pharmacodynamics in a stereoselective manner. A stereoselective gas chromatography (GC) assay for simultaneous quantification of HP-VPD enantiomers in plasma and urine was developed and used to investigate the pharmacokinetics of HP-VPD in dogs. Pharmacodynamic analysis in rats showed that (S)-HP-VPD was 2.5 times more potent as an anticonvulsant in the maximal electroshock seizure (MES) test than its enantiomer and approximately 10 times more potent than VPA. No significant differences were observed in major PK parameters (clearance, volume of distribution, and half-life) between S and (R)-HP-VPD, and this suggested that pharmacodynamic differences could be attributed to the intrinsic pharmacodynamics of each enantiomer rather than to a preferable pharmacokinetic profile. The pharmacokinetic (metabolic) analysis showed that the fraction metabolized to HP-VPD-glucuronide ranged from 5% to 7% and no biotransformation of HP-VPD to VPA and 2-ketopropyl valpromide was observed. This is the first report of significant stereoselectivity in the anticonvulsant activity of a valproylamide with a chiral carbon situated on the alkyl chain of the amine moiety.     

Syntheses of chiral N-(protected) tri- and tetrapeptide conjugates

Cbz-(protected)-tri- and tetrapeptide conjugates with steroids, sugars, terpenes, and heterocycles were prepared using Cbz-(protected)-tri- and tetrapeptidoylbenzotriazoles as active intermediates.     

Prolonged Blood Circulation in Rats of Nanospheres Surface-Modified with Semitelechelic Poly[N-(2-Hydroxypropyl)methacrylamide]

Semitelechelic poly[N-(2-hydroxypropyl)methacrylamide]s (ST-PHPMA) containing one amino end-group and differing in molecular weight were synthesized by radical polymerization in the presence of 2-aminoethanethiol (AET) as chain transfer agent. These polymers were covalently attached via amide bonds to the surface of nanospheres based on a copolymer of methyl methacrylate, maleic anhydride, and methacrylic acid. When compared to unmodified nanospheres, those with the surface modified with ST-PHPMA possessed a decreased protein (albumin, IgG, fibrinogen) adsorption in vitro, an increased intravascular half-life as well as a decreased accumulation in the liver after intravenous administration into rats. The higher the molecular weight of the ST-PHPMA, the more pronounced the changes in these properties. The results obtained have clearly demonstrated that covalently attached ST-PHPMA chains are efficient in decreasing the biorecognition of negatively charged (hydrophilic) polymer surfaces.     

Poly [N -(2-hydroxypropyl)methacrylamide] Conjugates of Bovine Pancreatic Ribonuclease (RNase A) Inhibit Growth of Human Melanoma in Nude Mice

Recently hydrophilic poly [N -(2-hydroxypropyl)methacrylamide] (PHPMA) was used for BS-RNase modification to prevent its degradation in bloodstream or fast elimination. Polymer-conjugated BS-RNase preparations proved to be cytotoxic after intravenous or intraperitoneal application, whereas native BS-RNase was ineffective. Here RNase A unimer was conjugated with two HPMA polymers (classic and star) and their antitumor effects both in vitro and in vivo were compared with those of BS-RNase polymers. Surprisingly, the antitumor effect of RNase A conjugates was also pronounced. The RNase A conjugates (classic and star) injected intravenously to mice bearing melanoma tumor caused a significant reduction in tumor volume following ten doses of 5 and 1 mg/kg, respectively. Despite the antitumor activity observed in vivo, the in vitro tested cytotoxic activity of RNase A did not differ from that caused by native RNase A while native BS-RNase (50 μ g/ml) totally inhibited DNA synthesis in treated cells. The experiments with 125 I-labeled preparations demonstrated concentration-dependent internalization of native BS-RNase by tumor cells within an hour, whereas the polymer conjugate (S-BS) was not internalized. On the contrary, the in vivo experiments showed that whereas 40% of S-BS conjugate persisted in bloodstream for 24 h after administration, 98% of the native BS-RNase was already eliminated. Improved antitumor activities of PHPMA-modified RNases in vivo might be ascribed to their prolonged retention in bloodstream, better proteolytic stability and resistance to the action of the ribonuclease inhibitor.     

Noninvasive Visualization of Pharmacokinetics,Biodistribution and Tumor Targeting of Poly[<Emphasis Type="BoldItalic">N</Emphasis>-(2-hydroxypropyl)methacrylamide] in Mice Using Contrast Enhanced MRI

Purpose To study a non-invasive method of using contrast enhanced magnetic resonance imaging (MRI) to visualize the real-time pharmacokinetics, biodistribution and tumor accumulation of paramagnetically labeled poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) copolymer conjugates with different molecular weights and spacers in tumor-bearing mice. Materials and Methods Paramagnetically labeled HPMA copolymer conjugates were synthesized by free radical copolymerization of HPMA with monomers containing a chelating ligand, followed by complexation with Gd(OAc)₃. A stable paramagnetic chelate, Gd-DO3A, was conjugated to the copolymers via a degradable spacer GlyPheLeuGly and a non-degradable spacer GlyGly, respectively. The conjugates with molecular weights of 28, 60 and 121 kDa and narrow molecular weight distributions were prepared by fractionation with size exclusion chromatography. The conjugates were injected into athymic nude mice bearing MDA-MB-231 human breast carcinoma xenografts via a tail vein. MR images were acquired before and at various time points after the injection with a 3D FLASH sequence and a 2D spin-echo sequence at 3T. Pharmacokinetics, biodistribution and tumor accumulation of the conjugates were visualized based on the contrast enhancement in the blood, major organs and tumor tissue at various time points. The size effect of the conjugates was analyzed among the conjugates. Results Contrast enhanced MRI resulted in a real-time, three-dimensional visualization of blood circulation, pharmacokinetics, biodistribution and tumor accumulation of the conjugates, and the size effect on these pharmaceutical properties. HPMA copolymer conjugates with high molecular weight had a prolonged blood circulation time and high passive tumor targeting efficiency. Non-biodegradable HPMA copolymers with molecular weights higher than the threshold of renal filtration demonstrated higher efficiency for tumor drug delivery than biodegradable poly(L-glutamic acid). Conclusions Contrast enhanced MRI is an effective method for non-invasive visualization of in vivo properties of the paramagnetically labeled polymer conjugates in preclinical studies.     

Technetium-99m-Labeled N-(2-Hydroxypropyl) Methacrylamide Copolymers: Synthesis,Characterization, and in Vivo Biodistribution

PURPOSE: To synthesize novel technetium-99m (99mTc)-labeled N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers and characterize the effect of charge and molecular weight on their biodistribution in SCID mice. METHODS: Electronegative and neutral 7-kDa, 21-kDa, and 70-kDa HPMA copolymers containing a 99mTc chelating comonomer, bearing N-omega-bis(2-pyridylmethyl)-L-lysine (DPK), were synthesized by free-radical precipitation copolymerization. The copolymers were labeled via 99mTc tricarbonyl chelation to DPK-bearing comonomer. They were characterized by side-chain content, molecular weight, molecular weight distribution, radiochemical purity, and labeling stability. Scintigraphic images were obtained during the first 90 min and at 24 h postintravenous injection in SCID mice. At 24 h, organ radioactivity was determined from necropsy tissue counting. RESULTS: 99mTc-labeled HPMA copolymers showed greater than 90% stability over a 24-h challenge with cysteine and histidine. Scintigraphic images and the necropsy data showed that the negatively charged copolymers were eliminated from the body significantly faster than the neutral copolymers in a size-dependent manner. CONCLUSIONS: To facilitate clinical scintigraphic imaging, stable chelation of 99mTc may be achieved by incorporation of a DPK-bearing comonomer into the HPMA backbone. Electronegative and neutral 99mTc-labeled HPMA copolymers of 7, 21, and 70 kDa show significant variation in organ biodistribution in SCID mice. 99mTc-labeled HPMA copolymers could be used as diagnostic agents and to study pharmacokinetics of delivery systems based on these copolymers.     

Synthesis and cytotoxic activity of N-(2-chloroethyl)-N-nitroureas and N-(2-chloroethyl)-N-nitrocarbamates

As analogs of the widely used anti-tumor agents, N-(2-chloroethyl)-N-nitrosoureas, N-(2-chloroethyl)-N-nitroureas and N-(2-chloroethyl)-N-nitrocarbamates were synthesized by nitration following the reaction of the appropriate amines or alcohols with 2-chloroethyl isocyanate. All tested compounds exert cytotoxic effect with IC50 values of 10(-4) to 10(-6) M and most of them show somewhat higher cytotoxicity in nitrogen than in air.