by Chan Hung Chuen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 143-151). / Abstract also in Chinese. / Acknowledgments --- p.i / Abstract --- p.ii / Abstract (Chinese version) --- p.v / Abbreviations --- p.viii / Content --- p.ix / Chapter CHAPTER ONE --- INTRODUCTION / Chapter 1. --- Free radical and free radical-mediated antitumor drugs --- p.1 / Chapter 2. --- Mitomycin C (MC) / Chapter 2.1 --- Drug actions of MC --- p.2 / Chapter 2.2 --- Adverse side effects of MC --- p.5 / Chapter 3. --- Doxorubicin (DOX) / Chapter 3.1 --- Drug actions of DOX --- p.7 / Chapter 3.2 --- Adverse side effects of DOX --- p.8 / Chapter 4. --- Antioxidants --- p.14 / Chapter 5. --- Effects of exogenous ATP on the antitumor activity of Doxorubicin and Mitomycin C / Chapter 5.1 --- Glutathione (GSH) and related enzymes --- p.17 / Chapter 5.2 --- Glutathione (GSH) and Anticancer Quinones --- p.19 / Chapter 5.3 --- Glutathione and the cardiac toxicity of the anticancer drugs --- p.20 / Chapter 5.4 --- Glutathione depletion in tumor cells by exogenous ATP --- p.21 / Chapter 6. --- Aim of research --- p.24 / Chapter CHAPTER TWO --- THE EFFECT OF ANTIOXIDANTS ON DOXORUBICIN- OR MITOMYCIN C-INDUCED CYTOTOXICITY ON HUMAN TUMOR AND NORMAL CELL LINES / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Materials and Methods --- p.28 / Chapter 2.3 --- Results --- p.36 / Chapter 2.4 --- Discussion --- p.60 / Chapter CHAPTER THREE --- STUDY OF CARDIOPROTECTIVE EFFECTS OF ANTIOXIDANTS AGAINST DOXORUBICIN- OR MITOMYCIN C-INDUCED TOXICITY BY LANGENDORFF PERFUEED ISOLATED RAT HEART MODEL / Chapter 3.1 --- Introduction --- p.64 / Chapter 3.2 --- Materials and Methods --- p.67 / Chapter 3.3 --- Results --- p.75 / Chapter 3.4 --- Discussion --- p.76 / Chapter CHAPTER FOUR --- THE EFFECT OF ANTIOXIDANTS DURING CHEMOTHERAPY OF DOXORUBICIN OR MITOMYCIN C IN TUMOR-BEARING MICE / Chapter 4.1 --- Introduction --- p.78 / Chapter 4.2 --- Materials and Methods --- p.80 / Chapter 4.3 --- Results --- p.83 / Chapter 4.4 --- Discussion --- p.93 / Chapter CHAPTER FIVE --- HISTOLOGICAL STUDY AND LIPID PEROXIDATION STUDY OF PROTECTIVE EFFECT OF ANTIOXIDANTS IN TUMOR-BEARING MICE TREATED WITH DOXORUBICIN OR MITOMYCIN C / Chapter 5.1 --- Introduction --- p.95 / Chapter 5.2 --- Materials and Methods --- p.98 / Chapter 5.3 --- Results --- p.103 / Chapter 5.4 --- Discussion --- p.117 / Chapter CHAPTER SIX --- EFFECT OF EXOGENOUS ATP ON THE ANTITUMOR ACTIVITY OF DOXORUBICIN AND MITOMYCIN C ON CULTURED HUMAN HEPATOMA CELLS / Chapter 6.1 --- Introduction --- p.122 / Chapter 6.2 --- Materials and Methods --- p.124 / Chapter 6.3 --- Results --- p.126 / Chapter 6.4 --- Discussion --- p.136 / Chapter CHAPTER SEVEN --- CONCLUSION / Chapter 7.1 --- Conclusion --- p.139 / Chapter 7.2 --- Future perspective --- p.141 / Bibliography --- p.142

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This research initiated an investigation of the structural relationships between melanocyte stimulating hormone (α-MSH) and its melanin dispersion on lizard (Anolis carolinensis) and frog (Rana pipiens) skins bioassays as representing models for mammalian and amphibian melanocytes, respectively. From previous extensive structure-activity relationships of α -MSH together with the theoretical modeling we were able to design a group of linear and cyclic peptides related to "4-10" fragment analogues of α -MSH. The solid phase synthesis of α -MSH and its related analogues using the p-methyl-benzhydrylamine resin was accomplished. The C-terminal carboxamide and N-terminal acetylamide were maintained in all peptides synthesized. The cyclic peptides were prepared in solution phase using the linear peptides generated by solid phase. All the cyclization were done by using the hydrochloride salts of the peptide and DMF as solvent with diphenylphosphoryl azide (DPPA) as a coupling reagent in the presence of K₂HPO₄ as a base. The yields of the cyclic peptides were in the range of 30-40 percent. In all the synthesized peptides the replacement of D-Phe⁷ with L-Phe⁷ causes reduction in the potency of the peptide on lizard or frog skins bioassays. Also, the reduction or increase in ring size in the cyclic peptide from a 23 membered ring diminishes the biological effect of the peptide under testing.

Cardiotoxicity associated with many cancer drugs is a critical issue facing physicians these days and a huge hurdle that must be overcome for a side effects-free cancer therapy. Survival of cardiac myocytes is compromised upon the exposure to certain chemotherapeutic drugs. Unfortunately, the mechanisms implicated in cardiac toxicity and the pathways governing myocyte survival are poorly understood. The following thesis addresses the mechanisms underlying the cardiotoxicity of two anticancer drugs, doxorubicin (DOX) and Imatinib mesylate (Gleevec). Transcription factor GATA-4, has recently emerged as an indispensable factor in the adult heart adaptive response and cardiomyocyte survival. Therefore, the specific aim of this project was to determine the role of GATA-4, its upstream regulators, as well as partners in survival. A combination of cell and molecular techniques done on in vivo, and ex vivo models were utilized to tackle these issues. In this study, we confirmed the cardiotoxicity of the anticancer drug, Imatinib mesylate and found to be age dependent. GATA-4, already known to be implicated in DOX-induced toxicity, was confirmed as an Imatinib target. At the molecular level, we identified IGF-1 and AKT as upstream regulators of GATA-4. Moreover, we confirmed ZFP260 (PEX-1), a key regulator of the cardiac hypertrophic response, as a GATA-4 collaborator in common prosurvival pathways. Collectively, these results provide new insights on the mechanisms underlying drug-induced cardiotoxicity and raise the exciting possibility that cancer drugs are negatively affecting the same prosurvival pathway(s), in which GATA-4 is a critical component. Therapeutic interventions aimed at enhancing GATA-4 activity may be interesting to consider in the context of treatments with anticancer drugs.

A tumor targeted dendrimer based drug delivery system was designed and synthesized to carry chemotherapy drug doxorubicin. Polyamidoamine (PAMAM) dendrimer G4.5 was chosen as the underlying carrier. Anionic G4.5 is a good option for drug delivery as it consists of 128 surface groups, is less cytotoxic and favorably biodistributed. The delivery system was synthesized using a layer-by layer arrangement of three functional entities: chemotherapy drug doxorubicin, monoclonal antibody Cetuximab against EGF receptor, and polyethylene glycol (PEG). Doxorubicin was attached via an acid-sensitive hydrazon linkage to the dendrimer. Macromolecules are taken in by cells through endocytosis. pH inside the early endosomes to lysosomes ranges from pH 6 to 4.5. These acidic conditions are favorable for release of drug bound to the dendrimer vehicle through acid-sensitive linkage. 35% of all solid tumors of brain express exceptionally high EGF receptors whereas normal brain tumors express less EGFR. This makes the EGFR a potent targeting moiety for targeted drug delivery. Cetuximab will serve as a targeting ligand to help the delivery system target tumor cells. PEG was incorporated as a linker between Cetuximab and dendrimer to avoid reticuloendothelial system (RES) uptake of the system, increase biocompatibility, increase drug half-life and other shortcomings associated with nanomaterials. Nuclear magnetic resonance spectroscopy (NMR), fluorescence anisotropy, and western blotting were used to confirm the conjugation of PEG, doxorubicin and cetuximab to the dendrimer. The synthesized delivery system was characterized using ultraviolet-visible spectroscopy (UV-Vis) to approximate the number of doxorubicin attached. Dynamic light scattering (DLS) and zeta potential were used to analyze the change in size and surface properties of dendrimer during the synthesis. Doxorubicin release studies were conducted at different pHs. Maximum doxorubicin was released at pH 4.5 indicating the successful acid-sensitive linkage between the drug and dendrimer. Cytotoxicity studies indicated that the addition of PEG increased the biocompatibility as compared to free doxorubicin whereas; combination of doxorubicin and cetuximab exerted a significant toxic effect over a period of 72 hours. The cellular uptake of the delivery system was higher than that of free doxorubicin. Free DOX localized mainly in the nucleus whereas, CTX-G4.5-PEG-DOX conjugate localized within both cytoplasm and nucleus after 6 hour incubation. The synthesized delivery system represents a potential targeted drug delivery system.

There are many different types of targeted therapy for cancer treatment. The method of light mediated targeted therapy that we have developed uses photocaged molecules and photoswitchable peptides. In photocaging, a biologically active molecule is made inactive by the attachment of a photocleavable blocking group. On exposure to UV radiation the photocleavable entity is removed and the biologically active molecule is released. Using this concept we have designed a prodrug that consists of a cell impermeable hydrophilic molecule attached to a photocaged doxorubicin. Upon irradiation with UV light the photosensitive group is removed and cytotoxic doxorubicin is released at the tumor site. This concept has been further modified by attaching receptor binding molecules to the photocaged entity to increase its specificity. A peptide which consists of an azobenzene photoswitch has been used which, in the dark state is randomly coiled and cell impermeable but upon illumination becomes helical and cell permeable and can be used to deliver drugs into the cells. Upon illumination with UV light of suitable wavelength the azobenzene linker will change from a trans to a cis form and this will convert the randomly coiled cell impermeable peptide into an α helical permeable form. Thus a series of peptides have been designed with different arginine mutations which develop an arginine patch in the helical form. This arginine patch would help in cell permeability by interacting with cell surface glycans. The method could potentially be used to deliver drugs into cells in presence of light.

Our recent studies have shown that that erectile dysfunction (ED) drugs including Sildenafil (Viagra), Vardenafil (Levitra) and Tadalafil (Cialis) enhance killing of several types of cancer cells by anticancer drug, Doxorubicin (DOX). We observed increased cell death by apoptosis in response to the combined treatment with ED drugs and DOX. However, the mechanism of such enhancement of cell death by combined treatment of ED drugs and DOX is not fully understood. Nuclear factor-κB (NF-κB) is an oxidant-sensitive transcription factor that plays a critical role in the immediate-early activation of a multitude of genes that have been documented to play critical role in programmed cell death (apoptosis). NF-κB activation has been shown to block apoptosis and its inhibition improves existing anti-oncogenic therapy such as chemotherapy. In the present study, we tested the hypothesis whether combined treatment of prostate cancer cells, PC3, with Sildenafil plus DOX would attenuate the activation of NFκB by inhibiting translocation of the p65 and p50 subunits to the nucleus and by phosphorylation of cytosolic IκB In addition, we investigated the effect of DOX and DOX plus Sildenafil on the expression of BCL family of proteins which play critical role in apoptosis. We treated PC3 cells with 1.5 μM DOX with or without 10 µM Sildenafil for 6 hours and 72 hours. The nuclear translocation of p65 and p50 and expression of BCL family of proteins was determined by western blot analysis. Our results show that combined treatment of DOX and Sildenafil significantly reduced the nuclear translocation of p65 and p50 as compared with DOX alone (P < 0.05). This correlated with the significant reduction in the expression of Bcl-2, BclxL and phosphorylation of BAD. These data provide an important mechanism by which Sildenafil treatment augments the apoptotic potential of DOX in PC3 cancer cells.

O hemangiossarcoma é uma neoplasia maligna de origem endotelial vascular de ocorrência comum em cães. Nessa espécie, o órgão primário mais acometido é o baço. O hemangiossarcoma de partes moles em cães é caracterizado por ter um comportamento biológico muito agressivo e metástases, principalmente para o pulmão e fígado, são frequentes e ocorrem de maneira rápida no início do curso da doença. O óbito ocorre na maioria dos casos devido a hemorragia interna aguda secundária à ruptura do tumor e/ou devido ao processo metastástico. A cirurgia continua a ser o principal método de tratamento para quase todos os cães com hemangiossarcoma, mas a quimioterapia adjuvante é indicada em razão do alto índice metastático e do prognóstico ruim associado com o procedimento cirúrgico sozinho. Um estudo retrospectivo foi realizado para determinar o tempo de sobrevida e potenciais fatores de risco em cães diagnosticados com hemangiossarcoma visceral. Prontuários de 42 cães foram revisados. Dados como idade e peso no momento do diagnóstico, raça, sexo, localização do tumor, estágio clínico da doença, tipo de tratamento, e tempo mediano de sobrevida foram analisados. Vinte e três cães foram tratados apenas com cirurgia, enquanto que 19 cães foram tratados com cirurgia e quimioterapia adjuvante. Houve diferença estatística na sobrevida dos cães tratados com cirurgia e Doxorrubicina (274 dias) em comparação com cães tratados apenas com cirurgia (66 dias). Cães com hemangiossarcoma esplênico tiveram um tempo mediano de sobrevida mais longo do que os cães com hemangiossarcoma localizados em outros sítios primários e com metástase (274 versus 117 versus 38 dias, respectivamente, p = 0,013). O tempo mediano global de sobrevida para esses 42 cães foi de 237 dias, e a taxa de sobrevida de um ano foi estimada em 26,32%. Conclui-se que a localização primária do hemangiossarcoma teve associação com o prognóstico e que o uso da Doxorrubicina após o tratamento cirúrgico aumentou a sobrevida dos cães diagnosticados com essa doença. Neste estudo, o estadiamento clínico dos cães não influenciou o prognóstico / Hemangiosarcoma is a very common canine neoplasm of vascular endothelial origin. In the dog, the most frequent primary site for hemangiosarcoma is the spleen. Typically, canine hemangiosarcoma has a very aggressive biologic behavior with metastases, especially to lung and liver, occurring early in the course of the disease. In the majority of cases, death is related to acute hemoabdomen secondary to tumor rupture and/or metastases. Surgery remains the main method of treatment for most dogs with this type o cancer, but adjuvant chemotherapy is highly recommended due to the high risk of metastasis and the poor outcome associated with surgery alone. A retrospective study was performed to determine survival times and potential risk factors in dogs diagnosed with visceral hemangiosarcoma. Medical records of 42 dogs were reviewed. Age and baseline weight at the time of diagnosis, breed, sex, tumor location, clinical stage of the disease, treatment type and median survival time were evaluated. Twenty-three dogs were treated with surgery alone, while 19 dogs were treated with surgery and adjuvant chemotherapy. There was significant difference in survival between dogs treated with surgery alone (66 days) and with surgery followed by Doxorubicin (274 days). Dogs with splenic hemangiosarcoma had a longer median survival time than dogs with hemangiosarcoma of other sites, and with metastasis (274 versus 117 versus 38 days, respectively, p = 0,013). The overall median survival time for these 42 dogs was 237 days, and the one-year survival rate was estimated to be 26.32%. In conclusion, primary tumor location was associated with prognosis and the addition of doxorubicin after surgery did improve survival. In this study, clinical stage had no association with prognosis

by Chu Chi Yuen, Andrew. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 140-153). / Abstract also in Chinese. / ABSTRACT --- p.i / Chapter 1 --- INTRODUCTION --- p.3 / Chapter 1.1 --- Using Low density lipoprotein (LDL) as a drug carrier --- p.4 / Chapter 1.1.1 --- The structure of Low density lipoprotein (LDL) --- p.4 / Chapter 1.1.2 --- The metabolic pathway of LDL in human bodies --- p.4 / Chapter 1.1.3 --- The rationale for using LDL as a drug carrier --- p.7 / Chapter 1.1.4 --- Reconstitution of LDL with cytotoxic drugs --- p.9 / Chapter 1.1.5 --- Up and down regulation of LDL receptors --- p.11 / Chapter 1.2 --- Doxorubicin (DOX) --- p.12 / Chapter 1.2.1 --- Characteristics of DOX --- p.12 / Chapter 1.2.2 --- Drug actions of DOX --- p.14 / Chapter 1.2.3 --- The adverse side effects of DOX --- p.15 / Chapter 1.3 --- Multidrug resistance phenomenon in tumor cells --- p.17 / Chapter 1.3.1 --- The possible mechanisms of multidrug resistance --- p.19 / Chapter 1.3.2 --- The structure of P-glycoprotein --- p.20 / Chapter 1.3.3 --- The mechanisms of the P-glycoprotein --- p.22 / Chapter 1.3.4 --- Our aim in dealing with multidrug resistance --- p.22 / Chapter 2 --- MATERIALS AND METHODS --- p.23 / Chapter 2.1 --- Materials --- p.23 / Chapter 2.1.1 --- Animals --- p.23 / Chapter 2.1.2 --- Buffers --- p.24 / Chapter 2.1.3 --- Culture media --- p.25 / Chapter 2.1.4 --- Chemicals --- p.26 / Chapter 2.1.5 --- Culture of cells --- p.27 / Chapter 2.2 --- Methods --- p.29 / Chapter 2.2.1 --- In vitro studies --- p.29 / Chapter 2.2.2 --- In vivo studies --- p.44 / Chapter 3 --- RESULTS --- p.51 / Chapter 3.1 --- In vitro studies --- p.51 / Chapter 3.1.1 --- Preparation of LDL-DOX --- p.51 / Chapter 3.1.2 --- Comparison of the cytotoxicity of DOX and LDL-DOX on HepG2 cells --- p.59 / Chapter 3.1.3 --- Modulation of LDL receptors on HepG2 cells and ECV304 cells… --- p.63 / Chapter 3.1.4 --- The effect of combined treatment of LDL-DOX and hyperthermia on HepG2 cells --- p.84 / Chapter 3.1.5 --- The effect of LDL-DOX on resistant cell line R-HepG2 cells --- p.90 / Chapter 3.2 --- In vivo studies --- p.105 / Chapter 3.2.1 --- The comparison of organ distribution of LDL-DOX and DOXin BALB-c mice after administration --- p.105 / Chapter 3.2.2 --- The comparison of organ distribution of LDL-DOX and DOX in nude mice bearing HepG2 cells after adminstration --- p.108 / Chapter 3.2.3 --- Histological studies of heart of nude mice bearing HepG2 cells treated with DOX and LDL-DOX --- p.111 / Chapter 3.2.4 --- Myocardial injury measured by Lactate dehydrogenase (LDH) activity in nude mice bearing HepG2 treated with DOX and LDL- DOX --- p.117 / Chapter 3.2.5 --- The comparison of DOX and LDL-DOX on reducing the tumor sizes and weight in nude mice bearing HepG2 cells --- p.119 / Chapter 4 --- DISCUSSION --- p.122 / Chapter 4.1 --- In vitro studies --- p.122 / Chapter 4.1.1 --- Preparation of LDL-DOX complex --- p.122 / Chapter 4.1.2 --- The cytotoxicity ofLDL-DOX --- p.125 / Chapter 4.1.3 --- The combined treatment of hyperthermia and LDL-DOX --- p.129 / Chapter 4.1.4 --- The ability of LDL-DOX to circumvent muiltidrug resistance --- p.131 / Chapter 4.2 --- In vivo studies --- p.134 / Chapter 5 --- CONCLUSION --- p.136 / Chapter 5.1 --- Conclusion --- p.136 / Chapter 5.2 --- Future pospective --- p.139 / BIBLIOGRAPHY --- p.140

O objetivo do presente estudo visa o desenvolvimento de um copolímero em bloco constituído por metacrilato de metila (MMA) e de dimetilaminoetila (DMAEMA), tendo como macroniciador poli--caprolactona dibromada (Br-PCL-Br), e que permite formar nanocápsulas sensíveis ao pH, contendo ou não o tripeptídeo leucina-ácido aspártico-valina (LDV) na superfície para a vetorização ativa de anti-neoplásicos. Os métodos envolveram diferentes abordagens sintéticas testadas, sendo que a técnica de transferência eletrônica por regeneração de ativadores (ATRP-ARGET) permitiu obter o copolímero PCL-P(MMA-DMAEMA)2 de forma mais prática e com rendimentos entre 30 e 70%. Por fim, o tripeptídeo LDV foi conjugado ao copolímero por meio do ligante metacrilato de 2-isocianato de etila (IEM). Um método por cromatografia líquida de alta eficiência (CLAE) foi adaptado para a quantificação da doxorrubicina e as nanopartículas foram preparadas por nanoprecipitação e avaliadas quanto à capacidade de expandir em diferentes pHs e citotoxicidade em células de câncer de mama. Os resultados do copolímero demonstram, por análises de infravermelho (IR-FT), sinais característicos em 2900 cm-1 e 1720 cm-1 correspondentes às funções –CH e –C=O. A análise de ressonância magnética nuclear de hidrogênio (RMN 1H) mostra a caracterização das cadeias hidrocarbônicas do copolímero, sendo que os deslocamentos químicos em 2,8 ppm e 3,8 ppm correspondem aos sinais dos grupamentos –CH2-N do DMAEMA e -OCH3 do MMA. As nanocápsulas preparadas a partir do copolímero expandiram de diâmetro quando expostas à pH ácido. Uma vez que o PMMA foi identificado como componente mais citotóxico, o copolímero foi otimizado por meio da redução da quantia de MMA. A quantificação da doxorrubicina encapsulada nas nanopartículas preparadas a partir dos copolímeros não otimizado (ARGET-A) e otimizado (ARGETB) foi de 61,42% e 64,88%, respectivamente. No estudo de citotoxicidade, as nanopartículas preparadas a partir do copolímero ARGET-B apresentaram-se eficazes no controle da proliferação celular de MCF-7. Conclui-se que o método de síntese ATRP-ARGET-B foi o mais apropriado para a produção do copolímero empregado no desenvolvimento de nanopartículas pH responsivas eficazes no 6 controle da proliferação de células tumorais. Ainda, existe a possibilidade do emprego do copolímero contendo o tripeptídeo LDV para alcançar uma vetorização ativa em células de câncer por meio da interação com integrinas específicas. Entretanto, até o presente, não foi realizada a avaliação das nanopartículas contendo LDV. / The objective of the present study looks for the development of a block copolymer constituted by methyl methacrylate (MMA) and dimethylaminoethyl methacrylate (DMAEMA), having poly--caprolactone dibromated (Br-PCL-Br) as a macroinitiator and, that could form pH sensible nanocapsules with or without the tripeptide leucineaspartic acid-valine (LDV) in its surface for active vectorization of anti-neoplasics. The methods employed different synthetic approaches tested, being that the activator regenerated by eletron transfer technique (ATRP-ARGET) allowed to obtain the copolymer PCL-P(MMA-DMAEMA)2 in a practicle way and with incomes between 30 and 70%. Finally, the tripeptide LDV was linked to the copolymer through the 2- isocyanatoethyl methacrylate (IEM). A high performance liquid chromatography method (HPLC) was adapted to doxorubicin quantification and, the nanopartircles were prepared by nanoprecipitation and evaluated conserning its ability to expand in different environments and citotoxycity in mammary cancer cells. The results from the copolymer demonstrated, by infrared (FT-IR), characteristic signals of 2900 cm-1 and 1720 cm-1 from the functions –CH and –C=O. And hydrogen nuclear magnetic resonance (RMN 1H) analysis allowed the characterization of the hydrogen-carbonic chains of the copolymer, being that the chemical displacement in 2,8 ppm and 3,8 ppm corresponds to the signals of the groups –CH2-N from DMAEMA and –O-CH3 from MMA. The nanocapsules prepared from the copolymer expanded its diameter when exposed to acidic pH. Once PMMA was identified as the most toxic component the copolymer was optimized by the reduction of MMA amount. Doxorubicin quantification in the nanocapsules prepared with the copolymers not optimized (ARGET-A) and optimized (ARGET-B) was 61,42% and 64,88%, respectively. In the cytotoxicity study, the nanocapsules prepared from copolymer ARGET-B showed to be efficient to control the cellular proliferation of MCF-7. It can be concluded that the ATRP-ARGET-B method was the more appropriate one for the copolymer production, which was employed in nanocapsules pH responsive effective to control 8 tumor proliferation. Besides, there is the possibility to use the copolymer functionalized with LDV to achieve an active delivery to cancer cells by it interaction with specific integrins. However, till the present, it was not realized the evaluation of the nanocapsules with LDV.