Targeted alpha therapy using short-lived alpha-particles and the promise of nanobodies as targeting vehicle

ABSTRACT

Introduction: The combination of a targeted biomolecule that specifically defines the target and a radionuclide that delivers a cytotoxic payload offers a specific way to destroy cancer cells. Targeted radionuclide therapy (TRNT) aims to deliver cytotoxic radiation to cancer cells and causes minimal toxicity to surrounding healthy tissues. Recent advances using α-particle radiation emphasizes their potential to generate radiation in a highly localized and toxic manner because of their high level of ionization and short range in tissue.

Areas covered: We review the importance of targeted alpha therapy (TAT) and focus on nanobodies as potential beneficial vehicles. In recent years, nanobodies have been evaluated intensively as unique antigen-specific vehicles for molecular imaging and TRNT.

Expert opinion: We expect that the efficient targeting capacity and fast clearance of nanobodies offer a high potential for TAT. More particularly, we argue that the nanobodies’ pharmacokinetic properties match perfectly with the interesting decay properties of the short-lived α-particle emitting radionuclides Astatine-211 and Bismuth-213 and offer an interesting treatment option particularly for micrometastatic cancer and residual disease.     

Studies of pancreatic alpha cell function in normal and diabetic subjects

The development of a glucagon radioimmunoassay with a relatively high degree of specificity for pancreatic glucagon made possible studies of alpha cell function in healthy nondiabetic subjects and in patients with diabetes mellitus. In the former group mean fasting plasma glucagon averaged 108 mumug/ml (SEM +/-10). In 12 juvenile-type diabetics fasting glucagon averaged 110 (+/-9) and in 33 adult-type diabetics the average was 114 (+/-8). The diabetic averages did not differ significantly from the nondiabetic subjects; however, when hyperglycemia was induced by glucose infusion in the nondiabetic subjects so as to simulate the fasting hyperglycemia of the diabetics, mean glucagon fell to 57 mumug (+/-8), which was significantly below the diabetic mean.In 28 healthy subjects the infusion of arginine elicited a rise in glucagon of at least 100 mumug/ml with a peak level averaging 331 mumug/ml (+/-22) at 40 min. This response to arginine was diminished but not abolished during hyperglycemia induced by simultaneous glucose infusion. In everyone of 45 diabetic subjects tested the infusion of arginine elicited a rise in glucagon of at least 140 mumug/ml to levels significantly greater than in nondiabetics. The peak glucagon level in juvenile-type diabetics averaged 458 mumug/ml (SEM +/-36) and in adult-type diabetics averaged 452 mumug/ml (SEM +/-38). The glucagon response to arginine was unrelated to duration of diabetes, to body weight, type of diabetic treatment, or to other known factors. Marked hyperresponsiveness of glucagon to arginine infusion was observed in two patients with advanced Kimmelsteil-Wilson disease. Glucagon levels were markedly elevated in certain patients with severe diabetic ketoacidosis before treatment with insulin.The findings suggest that alpha cell function is inappropriately increased in diabetes mellitus and could play a significant role in the diabetic syndrome.     

Conditional gene targeting in the mouse nervous system: Insights into brain function and diseases

Conditional gene knockout represents an extremely powerful approach to study the function of single genes in the nervous system. The Cre-LoxP system is the most advanced technology for spatial and temporal control of genetic inactivation, and there is rapid progress using this methodology in neuroscience research. In this approach, mice with LoxP sites flanking the gene of interest (floxed mice) are bred with transgenic mice expressing Cre recombinase under the control of a selected promoter (Cre mice). This promoter is critical in that it determines the time and site of Cre expression. Cre enzyme, in turn, recombines the floxed gene and produces gene knockout. Here we review Cre mouse lines that have been developed to target either the entire brain, selected brain areas, or specific neuronal populations. We then summarize phenotypic consequences of conditional gene targeting in the brain for more than 40 genes, as reported to date. For many broadly expressed genes, brain-restricted knockout has overcome lethality of conventional knockout (KO) and has highlighted a specific role of the encoded protein in some aspect of brain function. In the case of neural genes, data from null mutants in specific brain sites or neurons has refined our understanding of the role of individual molecules that regulate complex behaviors or synaptic plasticity within neural circuits. Among the many developing functional genomic approaches, conditional gene targeting in the mouse has become an excellent tool to elucidate the function of the approximately 5000 known or unknown genes that operate in the nervous system.     

Defensive applications of gene transfer technology in the face of bioterrorism: DNA-based vaccines and immune targeting

Gene transfer involves the introduction of an engineered gene into a person's cells with the expectation that the protein expressed from the gene will produce a therapeutic benefit. Strategies based on this principle have led to the approval of > 600 clinical trials and enrollment of approximately 3500 subjects worldwide in attempts to treat diseases ranging from cancer to AIDS to cystic fibrosis. While gene therapy has met with limited success and still has many hurdles to overcome before it sees wide application, it may be useful as a defensive strategy against bioterrorism agents including infectious microbes and toxins. Although many defensive strategies are possible, immunological strategies are currently the most developed and are being actively applied to the development of strategies against several of the most virulent potential bio-weapons. While most of these strategies are not yet ready for human application, DNA-based vaccines appear to be among the most promising in the fight against bioterrorism.     

Increased glucose tolerance and reduced adiposity in the absence of fasting hypoglycemia in mice with liver-specific Gs alpha deficiency

The G protein G(s)alpha is essential for hormone-stimulated cAMP generation and is an important metabolic regulator. We investigated the role of liver G(s)-signaling pathways by developing mice with liver-specific G(s)alpha deficiency (LGsKO mice). LGsKO mice had increased liver weight and glycogen content and reduced adiposity, whereas survival, body weight, food intake, and metabolic rates at ambient temperature were unaffected. LGsKO mice had increased glucose tolerance with both increased glucose-stimulated insulin secretion and increased insulin sensitivity in liver and muscle. Fed LGsKO mice were hypoglycemic and hypoinsulinemic, with low expression of hepatic gluconeogenic enzymes and PPARgamma coactivator-1. However, LGsKO mice maintained normal fasting glucose and insulin levels, probably due to prolonged breakdown of glycogen stores and possibly increased extrahepatic gluconeogenesis. Lipid metabolism was unaffected in fed LGsKO mice, but fasted LGsKO mice had increased lipogenic and reduced lipid oxidation gene expression in liver and increased serum triglyceride and FFA levels. LGsKO mice had very high serum glucagon and glucagon-like peptide-1 levels and pancreatic alpha cell hyperplasia, probably secondary to hepatic glucagon resistance and/or chronic hypoglycemia. Our results define novel roles for hepatic G(s)-signaling pathways in glucose and lipid regulation, which may prove useful in designing new therapeutic targets for diabetes and obesity.     

Transgenic mouse and gene targeting]

Transgenic mouse technology has proved to be a powerful tool for medical research. So far, a large number of transgenic mice have been generated expressing, e.g. oncogenes, viral genes, immunoglobulins, lymphokines and MHC antigens, and have provided much valuable information. Furthermore, gene targeting technology (homologous recombination between specific chromosomal DNA sequences and exogenously introduced DNA sequences) has been improved and applied to pluripotent, mouse embryonic stem (ES) cells, providing the means to create mice of specifically altered genotype. In this review, some of the background and recent advances of transgenic mouse technology and gene targeting in mouse ES cells are described.     

Differential regulation of mesolimbic alpha 3/alpha 6 beta 2 and alpha 4 beta 2 nicotinic acetylcholine receptor sites and function after long-term oral nicotine to monkeys

Because the mesolimbic dopamine system plays a critical role in nicotine addiction/reinforcement and because nicotinic receptors regulate dopamine release, we initiated a study to evaluate the long-term effects of nicotine (>6 months at the final dose) on nicotinic acetylcholine receptor (nAChR) sites and function in the nucleus accumbens of nonhuman primates. Nicotine was given in the drinking water as this mode of administration is long-term but intermittent, thus resembling smoking in this aspect. We determined the effects of nicotine treatment on function and binding of the alpha3/alpha6beta2* and alpha4beta2* nAChRs subtypes in nucleus accumbens, a region directly implicated in the addictive effects of nicotine. To evaluate function, we measured nicotine and K+-evoked [3H]dopamine release from nucleus accumbens synaptosomes. Changes in alpha4beta2* and alpha3/alpha6beta2* nAChRs were measured using 125I-epibatidine, [125I]A85380 [5-[125I]iodo-3(2(S)-azetidinylmethoxy) pyridine] and 125I-alpha-conotoxin MII autoradiography. Chronic nicotine treatment, which led to plasma nicotine levels in the range of smokers, significantly increased nucleus accumbens alpha4beta2* nAChR sites and function compared with control. By contrast, this treatment did not significantly change alpha3/alpha6beta2* nAChR sites or evoked dopamine release in this region compared with control. Thus, these data are distinct from previous results in striatum in which the same nicotine treatment paradigm decreased striatal alpha3/alpha6beta2* nAChR sites and function. The finding that long-term nicotine treatment selectively modulates alpha4beta2* and not alpha3/alpha6beta2* nAChR expression in primate nucleus accumbens is consistent with the results of studies in nicotinic receptor mutant mice implicating the alpha4beta2* nAChR subtype in nicotine-mediated addiction.     

Decreased Gs alpha mRNA levels accompany the fall in Gs and adenylyl cyclase activities in compensated left ventricular hypertrophy. In heart failure, only the impairment in adenylyl cyclase activation progresses.

We have previously reported that there is a global reduction in adenylyl cyclase associated with a decrement in Gs functional activity in cardiac sarcolemma from animals with pressure overload-induced hypertrophy and heart failure. This study was performed to determine whether hypertrophy alone in the absence of heart failure is sufficient to promote these changes and whether the superimposition of heart failure intensified these changes. Basal and stimulated adenylyl cyclase and Gs activity, as determined in the S49 cyc- reconstitution assay, were measured in sarcolemma from normal (NL), left ventricular hypertrophy (LVH) and heart failure (HF) animals. Simultaneously, we measured the mRNA level encoding for the Gs alpha subunit. These studies indicate that Gs activity and Gs alpha mRNA are decreased by approximately 30% both in the failing heart and even in the heart with compensated hypertrophy before heart failure develops (Gs activity, pmol cyclic AMP/10 min per microgram, NL 4.2 +/- 0.4, LVH 3.0 +/- 0.2, HF 3.2 +/- 0.3; Gs alpha mRNA, pg/10 micrograms RNA, NL 131 +/- 9.0, LVH 104 +/- 7.4, HF 97.4 +/- 9.1; P less than 0.05 as compared with NL for LVH and HF). Accompanying this decrement in Gs activity is a fall in adenylyl cyclase, both basal and stimulated. However, we also identified a further decrease in adenylyl cyclase without any additional change in Gs or in its alpha subunit mRNA level. This is seen only in the sarcolemma from animals with heart failure as compared with those with compensated LV hypertrophy (e.g., NaF-stimulated activity, pmol cyclic AMP/min per mg, NL 420.2 +/- 17.5, LVH 347.1 +/- 29.6, HF 244.2 +/- 27.3; P less than 0.05 compared with NL for LVH and HF, P less than 0.05 compared with LVH for HF). In summary, these studies indicate that both Gs and adenylyl cyclase activities fall in parallel with the development of LV hypertrophy followed by a further decrement in adenylyl cyclase, independent of Gs, in the setting of heart failure.     

Therapeutic targeting the loss of the birt-hogg-dube suppressor gene

Brit-Hogg-Dubé (BHD) syndrome, an autosomal dominant familial cancer, is associated with increased risk of kidney cancer. BHD syndrome is caused by loss-of-function mutations in the folliculin (FLCN) protein. To develop therapeutic approaches for renal cell carcinoma (RCC) in BHD syndrome, we adopted a strategy to identify tumor-selective growth inhibition in a RCC cell line with FLCN inactivation. The COMPARE algorithm was used to identify candidate anticancer drugs tested against the NCI-60 cell lines that showed preferential toxicity to low FLCN expressing cell lines. Fifteen compounds were selected and detailed growth inhibition (SRB) assays were done in paired BHD RCC cell lines (UOK257 derived from a patient with BHD). Selective sensitivity of FLCN-null over FLCN-wt UOK257 cells was observed in seven compounds. The most selective growth-inhibitory sensitivity was induced by mithramycin, which showed an approximately 10-fold difference in GI(50) values between FLCN-null (64.2 ± 7.9 nmol/L, n = 3) and FLCN-wt UOK257 cells (634.3 ± 147.9 nmol/L, n = 4). Differential ability to induce caspase 3/7 activity by mithramycin was also detected in a dose-dependent manner. Clonogenic survival studies showed mithramycin to be approximately 10-fold more cytotoxic to FLCN-null than FLCN-wt UOK257 cells (200 nmol/L). Following mithramycin exposure, UOK257-FLCN-null cells were mainly arrested and blocked in S and G(2)-M phases of the cell cycle and low dose of rapamycin (1 nmol/L) potentiated mithramycin sensitivity (1.5-fold in G(2)-M population and 2-fold in G(2)-M period time, 2xGI(50), 48 hours). These results provide a basis for further evaluation of mithramycin as a potential therapeutic drug for RCC associated with BHD.     

A new liposome-based gene delivery system targeting lung epithelial cells using endothelin antagonist

We formulated a new gene delivery system based on targeted liposomes. The efficacy of the delivery system was demonstrated in in vitro and in vivo models. The targeting moiety consists of a high-affinity 7-amino-acid peptide, covalently and evenly conjugated to the liposome surface. The targeting peptide acts as an endothelin antagonist, and accelerates liposome binding and internalization. It is devoid of other biological activity. Liposomes with high phosphatidyl serine (PS) were specially formulated to help their fusion with the endosomal membrane at low pH and enable release of the liposome payload into the cytoplasm. A DNA payload, pre-compressed by protamine, was encapsulated into the liposomes, which directed the plasmid into the cell's nucleus. Upon exposure to epithelial cells, binding of the liposomes occurred within 5-10 min, followed by facilitated internalization of the complex. Endosomal escape was complete within 30 min, followed by DNA accumulation in the nucleus 2h post-transfection. A549 lung epithelial cells transfected with plasmid encoding for GFP encapsulated in targeted liposomes expressed significantly more protein than those transfected with plasmid complexed with Lipofectamine. The intra-tracheal instillation of plasmid encoding for GFP encapsulated in targeted liposomes into rat lungs resulted in the expression of GFP in bronchioles and alveoli within 5 days. These results suggest that this delivery system has great potential in targeting genes to lungs.     

Selective targeting of pentoxifylline to hepatic stellate cells using a novel platinum-based linker technology.

Targeting of antifibrotic drugs to hepatic stellate cells (HSC) is a promising strategy to block fibrotic processes leading to liver cirrhosis. For this purpose, we utilized the neo-glycoprotein mannose-6-phosphate-albumin (M6PHSA) that accumulates efficiently in HSC during liver fibrosis. Pentoxifylline (PTX), an antifibrotic compound that inhibits HSC proliferation and activation in vitro, was conjugated to M6PHSA. We employed a new type of platinum-based linker, which conjugates PTX via coordination chemistry rather than via covalent linkage. When incubated in plasma or in the presence of thiol compounds, free PTX was released from PTX-M6PHSA at a sustained slow rate. PTX-M6PHSA displayed pharmacological activity in cultured HSC as evidenced by changes in cell morphology and reduction of collagen I production. PTX-M6PHSA and platinum coupled PTX did not induce platinum-related toxicity (Alamar Blue viability assay) or apoptosis (caspase activation and TUNEL staining). In vivo distribution studies in fibrotic rats demonstrated specific accumulation of the conjugate in nonparenchymal cells in the fibrotic liver. In conclusion, we have developed PTX-M6PHSA employing a novel type of platinum linker, which allows sustained delivery of the drug to HSC in the fibrotic liver.     

Efficiency of chimeraplast gene targeting by direct nuclear injection using a GFP recovery assay

Traditional RNA-DNA chimeric oligonucleotides (chimeraplasts), composed of a continuous stretch of RNA and DNA residues in a duplex conformation, have been shown to correct single-base mutations in episomal and genomic DNA both in vitro and in vivo. In the current study, we have compared the efficiency of single-base pair correction between a traditionally designed chimeraplast (covalently linked duplex) and hybrid chimeraplasts (noncovalent duplexes formed from stretches of RNA and DNA nucleotides synthesized individually and hybridized in vitro). Six hybrid chimeraplasts of identical length were constructed with various lengths of target homology and strand location of the desired nucleotide change. These constructs were evaluated for their ability to correct a point mutation in the gene encoding recombinant enhanced green fluorescent protein (eGFP) that rendered the protein nonfluorescent. A plasmid encoding this mutant eGFP gene and a chimeraplast were co-introduced directly into the nuclei of primary fibroblasts by microinjection. As shown by the recovery of eGFP fluorescence, three of the six hybrid chimeraplasts demonstrated the ability to mediate gene correction (0.4–2.4%). Covalent joining of RNA and DNA strands in chimeraplasts was not necessary for correction of DNA mutations. However, the strand placement of the desired nucleotide change and the length of nonhomologous sequences flanking target nucleotides played a crucial role in the efficiency of chimeraplast-mediated gene correction. Despite the ability of certain chimeraplast designs to correct point mutations in episomal plasmids, targeted correction of integrated copies of the mutant eGFP transgene was unsuccessful in primary fibroblasts. These results demonstrate that, although chimeraplasts are fairly effective at targeting episomal DNA in primary cells, further optimization is required to increase the efficiency for targeting integrated genes.     

硫化氢与系统功能调节

硫化氢(H2S)在哺乳动物组织中大量产生,并且作为调节介质发挥多种理学效应.研究证明,H2S是继NO和CO之后发现的一种新的气体信号分子.H2S参与血管张力、心肌收缩力、神经递质和胰岛素分泌的调节,很多系统的功能调节与H2S有关.动脉高压、阿尔茨海默病、胃黏膜损伤和肝硬化的动物模型中均有H2S的缺乏,而外源性H2S可以改善与缺血/再灌注损伤相关的心功能不全,减少抗炎药物引起的胃黏膜损伤.本文对H2S在体内不同系统中的产生、功能调节及可能的作用机制进行综述,力求将H2S的系统功能调节作用展示出来.     

应用下一代测序技术对α地中海贫血进行胚胎植入前遗传学检测

目的 探讨下一代测序(next generation sequencing,NGS)技术在α地中海贫血胚胎植入前遗传学检测中的应用. 方法 选取2对α地中海贫血--SEA缺失型携带者夫妇体外受精胚胎活检后的6个胚胎样本应用全基因组扩增(whole genome amplification,WGA)技术、下一代测序技术进行胚胎植入前遗传学检测,同时采用跨越断裂点荧光PCR(gap-PCR)进行平行对照检测. 结果 2个家系6个胚胎样本的胚胎植入前遗传学诊断(preimplantation genetic diagnosis,PGD)结果分别为--SEA/αα母源携带、--SEA/--SEA、--SEA/αα母源携带、--SEA/--SEA、--SEA/αα母源携带和--SEA/--SEA;胚胎植入前遗传学筛查(preimplantation genetic screening,PGS)结果分别为45,XX,-5、46,XX、46,XY、47,XY,+1、46,XX和46,XY,+1,-2;Family 1 gap-PCR检测结果为父母均为SEA杂合子;E1为正常、E2为SEA纯合子、E3为正常;Family 2检测结果分别为:父母均为SEA杂合子;E4为SEA纯合子、E5为正常、E6为SEA纯合子. 结论 结果显示利用NGS不仅可以检测出23对染色体的核型,同时解决了单细胞扩增等位基因脱扣(allele drop-out,ADO)造成的假阳性和假阴性的风险,更具有市场潜力及应用前景.     

Normal indices of left ventricular function assessed by using computer technology

While analysing ventriculograms of 18 persons without any cardiovascular pathology 3 "independent" research workers (to exclude a subjective factor in the appraisal of the left ventricle contours) have developed normal indicators of the total and regional contractility of the left ventricle. The data obtained with the use of angiographic and computer-aided techniques of the recent generation and machine analysis employing unpaired statistics can be recommended for application as normal indicators during the performance of different hemodynamic studies.