René Ouellet

Water-soluble aluminium phthalocyanine-polymer conjugates for PDT: photodynamic activities and pharmacokinetics in tumour-bearing mice.

SummaryThe potential use of unsubstituted aluminium phthalocyanine (AlClPc) as a sensitizer for photodynamic therapy (PDT) of cancer has not been fully exploited in spite of its higher efficiency as compared to the sulphonated derivatives. This is largely due to the strong hydrophobic character of AlClPc which renders the material difficult to formulate for in vivo administration. We prepared two water-soluble derivatives of AlClPc by axial coordination of polyethyleneglycol (PEG, MW 2000) or polyvinylalcohol (PVA, MW 13 000–23 000) to the central aluminium ion. Their photodynamic activities were evaluated in vitro against the EMT-6 mouse mammary tumour cells and in vivo against the EMT-6 and the colon carcinoma Colo-26 tumours implanted intradermally in Balb/c mice. Pharmacokinetics were studied in the EMT-6 tumour-bearing mice. After 1 h incubation, the light dose required to kill 90% of cells (LD90) was at least three times less for AlClPc (Cremophor emulsion) as compared to AlPc–PEG and AlPc–PVA, while after 24 h incubation all three preparations were highly phototoxic. All three dye preparations induced complete EMT-6 tumour regression in 75–100% of animals at a low drug dose (0.25 μmol kg–1) following PDT (400 J cm–2, 650–700 nm) at 24 h pi. Complete tumour regression in the Colo-26 tumour model was obtained in 30% of mice at a dose of 2 μmol kg–1. In the non-cured animals, AlPc–PVA induced the most significant tumour growth delay. This dye showed a prolonged plasma half-life (6.8 h) as compared to AlClPc (2.6 h) and AlPc–PEG (23 min), lower retention by liver and spleen and higher tumour-to-skin and tumour-to-muscle ratios. Our data demonstrate that addition of hydrophilic axial ligands to AlPc, while modifying in vitro and in vivo kinetics, does not reduce the PDT efficiency of the parent molecule. Moreover, in the case of the polyvinylalcohol derivative, axial coordination confers advantageous pharmacokinetics to AlPc, which makes this photosensitizer a valuable, water soluble candidate drug for clinical PDT of cancer.

PHTHALOCYANINE AND NAPHTHALOCYANINE PHOTOSENSITIZED OXIDATION OF 2′‐DEOXYGUANOSINE

Abstract— The photodynamic properties of the di‐and tetrasulfonated zinc and aluminium phthalocyanines and a tetrasulfonated aluminium napththalocyanine were studied using 2′‐deoxyguanosine as a DNA model compound. The major photooxidation products of this nucleoside were identified and classified according to their formation through a radical mechanism (type I) or a singlet oxygen mediated mechanism (type II). The major type I product was obtained and identified as 2,2‐diamino [(2‐deoxy‐β‐d‐erythropentofuranosyl)‐4‐amino]‐5(2H)‐oxazolone. Two major type II products were characterized as the 4R* and 4S* diastereomers of 9‐(2‐deoxy‐β‐d‐erythropentofuranosyl)‐7,8‐dihydro‐4‐hydroxy‐8‐oxoguanine. In addition a third product, also resulting from a type II photooxidation, was identified as 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine. Quantification of these products provided a means to estimate the contribution of type I and type II pathways during the phthalocyanine and naphthalocyanine mediated photooxidation of 2′‐deoxyguanosine, confirming the major role of singlet oxygen in these processes.

Abnormal in vivo myocardial energy substrate uptake in diet-induced type 2 diabetic cardiomyopathy in rats

The purpose of this study was to determine in vivo myocardial energy metabolism and function in a nutritional model of type 2 diabetes. Wistar rats rendered insulin-resistant and mildly hyperglycemic, hyperinsulinemic, and hypertriglyceridemic with a high-fructose/high-fat diet over a 6-wk period with injection of a small dose of streptozotocin (HFHFS) and control rats were studied using micro-PET (microPET) without or with a euglycemic hyperinsulinemic clamp. During glucose clamp, myocardial metabolic rate of glucose measured with [(18)F]fluorodeoxyglucose ([(18)F]FDG) was reduced by approximately 81% (P < 0.05), whereas myocardial plasma nonesterified fatty acid (NEFA) uptake as determined by [(18)F]fluorothia-6-heptadecanoic acid ([(18)F]FTHA) was not significantly changed in HFHFS vs. control rats. Myocardial oxidative metabolism as assessed by [(11)C]acetate and myocardial perfusion index as assessed by [(13)N]ammonia were similar in both groups, whereas left ventricular ejection fraction as assessed by microPET was reduced by 26% in HFHFS rats (P < 0.05). Without glucose clamp, NEFA uptake was approximately 40% lower in HFHFS rats (P < 0.05). However, myocardial uptake of [(18)F]FTHA administered by gastric gavage was significantly higher in HFHFS rats (P < 0.05). These abnormalities were associated with reduced Glut4 mRNA expression and increased Cd36 mRNA expression and mitochondrial carnitine palmitoyltransferase 1 activity (P < 0.05). HFHFS rats display type 2 diabetes complicated by left ventricular contractile dysfunction with profound reduction in myocardial glucose utilization, activation of fatty acid metabolic pathways, and preserved myocardial oxidative metabolism, suggesting reduced myocardial metabolic efficiency. In this model, increased myocardial fatty acid exposure likely occurs from circulating triglyceride, but not from circulating plasma NEFA.

Receptor‐Mediated Targeting of Phthalocyanines to Macrophages Via Covalent Coupling to Native or Maleylated Bovine Serum Albumin

Abstract— Targeted delivery of aluminum tetrasulfophthalocyanine (AlPcS4) to the scavenger receptor of macrophages, via coupling to maleylated bovine serum albumin (mal‐BSA), was explored as a means to improve photodynamic efficacy. The AlPcS4 was covalently coupled to BSA (9:1 molar ratio) via one or two sulfonamide‐hexanoic‐amide spacer chains, followed by treatment with maleic anhydride to yield the mal‐BSA‐phthalocyanine conjugates. The latter were tested for singlet oxygen production, receptor‐mediated cell uptake and phototoxicity toward J774 cells of macrophage origin and nonphagocytic EMT‐6 cells. Cell uptake of 125I‐mal‐BSA showed specific binding for J774 cells but not for EMT‐6 cells. Competition studies of the conjugates with 125I‐mal‐BSA showed that coupling of AlPcS4 to BSA resulted in recognition of the conjugate by the scavenger receptor, whereas coupling to mal‐BSA further enhanced its binding affinity. This suggests that affinity for the scavenger receptor is related to the overall negative charge of the protein. Phototoxicity of the conjugates toward J774 cells paralleled their relative affinity, with mal‐BSA‐AlPcS4 coupled via two spacer chains showing the highest activity. The conjugates were less phototoxic toward the EMT‐6 cell line. The activities in both cell lines of all conjugated AlPcS4 preparations were, however, lower than that of the free disulfonated AlPcS2. Possible implications for the in vivo use of protein‐photosensitizer conjugates to target selectively various macrophage‐associated disorders is discussed.

THE EFFECTS OF PHOTODYNAMIC THERAPY USING DIFFERENTLY SUBSTITUTED ZINC PHTHALOCYANINES ON VESSEL CONSTRICTION, VESSEL LEAKAGE AND TUMOR RESPONSE

Abstract— The effects of four different zinc phthalocyanines were studied during and after photodynamic therapy (PDT). Measurements of vessel constriction, vessel leakage, tumor interstitial pressure, eicosanoid release, and tumor response of chondrosarcoma were made in Sprague‐Dawley rats. Animals were injected intravenously with 1 μmol/ kg of mono‐, di‐, or tetrasulfonated zinc phthalocyanine, or 1 μmol/kg of a zinc phthalocyanine substituted with four tertiary butyl groups. Tissues were exposed to 400 J/cm2 670 nm light 24 h after photosensitizer injection. An additional group of animals was given indomethacin before treatment. The use of the monosulfonated and tertiary butyl substituted zinc phthalocyanines in PDT caused the release of specific eicosanoids, caused vessel constriction, and induced venule leakage and increases in tumor interstitial pressure. Tumor cures of 27% and 7% were observed. Photodynamic therapy using the disulfonated zinc phthalocyanine did not induce vessel constriction or the release ofeicosanoids, however; tumor cure was 43%. The use of thc tetrasulfonated zinc phthalocyanine caused intermediate effects between the mono‐ and disulfonated compounds. The administration of indornethacin to animals completely inhibited the effects of PDT using the monosulfonated compound but had minimal effects on treatment using the disulfonated compound. This suggests that the monosulfonated and disulfonated compounds act by different mechanisms of destruction.

Organ-specific dietary fatty acid uptake in humans using positron emission tomography coupled to computed tomography

A noninvasive method to determine postprandial fatty acid tissue partition may elucidate the link between excess dietary fat and type 2 diabetes. We hypothesized that the positron-emitting fatty acid analog 14(R,S)-[(18)F]fluoro-6-thia-heptadecanoic acid ((18)FTHA) administered orally during a meal would be incorporated into chylomicron triglycerides, allowing determination of interorgan dietary fatty acid uptake. We administered (18)FTHA orally at the beginning of a standard liquid meal ingested in nine healthy men. There was no significant (18)FTHA uptake in the portal vein and the liver during the 1st hour. Whole body PET/CT acquisition revealed early appearance of (18)FTHA in the distal thoracic duct, reaching a peak at time 240 min. (18)FTHA mean standard uptake value increased progressively in the liver, heart, quadriceps, and subcutaneous and visceral adipose tissues between time 60 and 240 min. Most circulating (18)F activity between time 0 and 360 min was recovered into chylomicron triglycerides. Using Triton WR-1339 treatment in rats that received (18)FTHA by gavage, we confirmed that >90% of this tracer reached the circulation as triglycerides. This novel noninvasive method to determine tissue dietary fatty acid distribution in humans should prove useful in the study of the mechanisms leading to lipotoxicity.

Photodynamic Therapy: Tumor Targeting with Adenoviral Proteins

A brief summary of the mechanisms involved in photo‐dynamic therapy (PDT) and the role of delivery vehicles for photosensitizer targeting is addressed. Phthalocya‐nines (Pc) have been coupled to adenovirus type 2 capsid proteins including the hexon, the penton base and the fiber to enhance their target selectivity. Adenovirus pen‐ton base proteins contain the arginine‐glycine‐aspartic acid peptidic sequence (RGD) motif known to bind with great affinity and high specificity to integrin receptors, expressed by several types of cancer. Tetrasulfonated aluminum phthalocyanine (AlPcS4) was covalently coupled to the various capsid proteins via one or two caproic acid spacer chains (A1 or A2) in 7:1 up to 66:1 molar ratios. The capacity of the bioconjugates for singlet oxygen production, as measured by an L‐tryptophan oxidation assay, was strongly reduced, likely reflecting scavenging by the carrier. Cell adsorption and in vitro pho‐tocytotoxicity assays were carried out using the A549 and HEp2 human cell lines expressing integrin receptors, and one murine, the EMT‐6 cell line, which lacks receptors for the RGD sequence. The AlPcS4A2‐protein complexes induced greater cytotoxicity as compared to the analogous AlPcS4A1 preparations. The penton base‐AIPcS4A2 derivative was the more phototoxic for all cell lines tested. Tumor response studies using Balb/c mice with EMT‐6 tumor implants demonstrated that the free AlPcS4A2 induced complete tumor regression at a dose of 1 μmol/kg and 400 J/cm2, which is comparable to the activity of the known AlPcS2adJ.A mixture of adenovirus type 2 soluble proteins covalently labeled with AlPcS4A2 required 0.5 μmol/kg to induce the same response with the same light dose, suggesting that the high affinity RGD/receptor complex is able to target Pc for PDT.

Normal Postprandial Nonesterified Fatty Acid Uptake in Muscles Despite Increased Circulating Fatty Acids in Type 2 Diabetes

OBJECTIVE Postprandial plasma nonesterified fatty acid (NEFA) appearance is increased in type 2 diabetes. Our objective was to determine whether skeletal muscle uptake of plasma NEFA is abnormal during the postprandial state in type 2 diabetes. RESEARCH DESIGN AND METHODS Thigh muscle blood flow and oxidative metabolism indexes and NEFA uptake were determined using positron emission tomography coupled with computed tomography (PET/CT) with [11C]acetate and 14(R,S)-[18F]fluoro-6-thia-heptadecanoic acid (18FTHA) in seven healthy control subjects (CON) and seven subjects with type 2 diabetes during continuous oral intake of a liquid meal to achieve steady postprandial NEFA levels with insulin infusion to maintain similar plasma glucose levels in both groups. RESULTS In the postprandial state, plasma NEFA level was higher in type 2 diabetic subjects versus CON (P < 0.01), whereas plasma glucose was at the same level in both groups. Muscle NEFA fractional extraction and blood flow index levels were 56% (P < 0.05) and 24% (P = 0.27) lower in type 2 diabetes, respectively. However, muscle NEFA uptake was similar to that of CON (quadriceps femoris [QF] 1.47 ± 0.23 vs. 1.37 ± 0.24 nmol ⋅ g−1 ⋅ min−1, P = 0.77; biceps femoris [BF] 1.54 ± 0.26 vs. 1.46 ± 0.28 nmol ⋅ g−1 ⋅ min−1, P = 0.85). Muscle oxidative metabolism was similar in both groups. Muscle NEFA fractional extraction and blood flow index were strongly and positively correlated (r = 0.79, P < 0.005). CONCLUSIONS Postprandial muscle NEFA uptake is normal despite elevated systemic NEFA levels and acute normalization of plasma glucose in type 2 diabetes. Lower postprandial muscle blood flow with resulting reduction in muscle NEFA fractional extraction may explain this phenomenon.

Phthalocyanine and naphthalocyanine photosensitized oxidation of 2'-deoxyguanosine : distinct type I and type II products

Abstract

Mechanism of Reduced Myocardial Glucose Utilization During Acute Hypertriglyceridemia in Rats

PurposeThe purpose of the research is to study the effect of acute inhibition of intravascular lipolysis on myocardial substrate selection during hypertriglyceridemia using in vivo radiotracer analysis and positron emission tomography.ProceduresWe induced acute hypertriglyceridemia in vivo using an intravenous infusion of Intralipid 20% (IL) without and with acute inhibition of fatty acid delivery from circulating triglycerides with injection of Triton WR-1339 (TRI) during a euglycemic–hyperinsulinemic clamp in Wistar rats. We determined the effect of TRI on myocardial uptake of circulating triglycerides and free fatty acids using intravenous injection of [3H]-triolein and [14C]-bromopalmitate, respectively. Myocardial blood flow, oxidative metabolism, and metabolic rate of glucose (MMRG) were determined using micro-positron emission tomography (μPET) with [13N]-ammonia, [11C]-acetate, and 2-deoxy-2-[F-18]fluoro-d-glucose (FDG).ResultsTRI reduced myocardial incorporation of [3H]-triolein but not [14C]-bromopalmitate showing that it selectively reduces myocardial fatty acid delivery from circulating triglycerides but not from free fatty acids. IL reduced myocardial blood flow and MMRG by 37% and 56%, respectively, but did not affect myocardial oxidative metabolism. TRI did not abolish the effect of IL on myocardial blood flow and MMRG.ConclusionsHypertriglyceridemia acutely reduces myocardial blood flow and MMRG in rats, but this effect is not explained by increased myocardial fatty acid delivery through intravascular triglyceride lipolysis.