Intra-tumor L-methionine level highly correlates with tumor size in both pancreatic cancer and melanoma patient-derived orthotopic xenograft (PDOX) nude-mouse models.

An excessive requirement for methionine (MET) for growth, termed MET dependence, appears to be a general metabolic defect in cancer. We have previously shown that cancer-cell growth can be selectively arrested by MET restriction such as with recombinant methioninase (rMETase). In the present study, we utilized patient-derived orthotopic xenograft (PDOX) nude mouse models with pancreatic cancer or melanoma to determine the relationship between intra-tumor MET level and tumor size. After the tumors grew to 100 mm3, the PDOX nude mice were divided into two groups: untreated control and treated with rMETase (100 units, i.p., 14 consecutive days). On day 14 from initiation of treatment, intra-tumor MET levels were measured and found to highly correlate with tumor volume, both in the pancreatic cancer PDOX (p<0.0001, R2=0.89016) and melanoma PDOX (p<0.0001, R2=0.88114). Tumors with low concentration of MET were smaller. The present results demonstrates that patient tumors are highly dependent on MET for growth and that rMETase effectively lowers tumor MET

Recombinant methioninase combined with doxorubicin (DOX) regresses a DOX-resistant synovial sarcoma in a patient-derived orthotopic xenograft (PDOX) mouse model.

Synovial sarcoma (SS) is a recalcitrant subgroup of soft tissue sarcoma (STS). A tumor from a patient with high grade SS from a lower extremity was grown orthotopically in the right biceps femoris muscle of nude mice to establish a patient-derived orthotopic xenograft (PDOX) mouse model. The PDOX mice were randomized into the following groups when tumor volume reached approximately 100 mm3: G1, control without treatment; G2, doxorubicin (DOX) (3 mg/kg, intraperitoneal [i.p.] injection, weekly, for 2 weeks; G3, rMETase (100 unit/mouse, i.p., daily, for 2 weeks); G4 DOX (3mg/kg), i.p. weekly, for 2 weeks) combined with rMETase (100 unit/mouse, i.p., daily, for 2 weeks). On day 14 after treatment initiation, all therapies significantly inhibited tumor growth compared to untreated control, except DOX: (DOX: p = 0.48; rMETase: p < 0.005; DOX combined with rMETase < 0.0001). DOX combined with rMETase was significantly more effective than both DOX alone (p < 0.001) and rMETase alone (p < 0.05). The relative body weight on day 14 compared with day 0 did not significantly differ between any treatment group or untreated control. The results indicate that r-METase can overcome DOX-resistance in this recalcitrant disease

ИСПОЛЬЗОВАНИЕ ПИРИДОКСИНА ДЛЯ ПОВЫШЕНИЯ ПРОТИВООПУХОЛЕВОЙ АКТИВНОСТИ МЕТИОНИН-ГАММАЛИАЗЫ НА МОДЕЛЯХ ПЕРЕВИВАЕМЫХ ОПУХОЛЕЙ МЫШЕЙ

We presented results of monotherapy and combination therapy of transplantable murine tumor models using methionine-gamma-lyase (MGL) and pyridoxine hydrochloride. We studied MGL from Clostridium sporogenes and Citrobacter freundii. We used Lewis lung carcinoma (LLC), melanoma B16, leukemias P388 and L1210 and Fisher lymphadenosis L5178y. Neither monotherapy with MGL nor combination of MGL and pyridoxine demonstrated antitumor activity against P388 and L5178y. In the murine L1210 leukemia model, MGL C. sporogenes injected intraperitoneally in the dose of 2000 U/kg, 11 times with a 12-hour interval increased the life span of mice (ILS=22 %, р=0.035). In the LLC model, the combination of MGL C. sporogenes at a dose of 400 U/kg, i.p., 4 times with a 48-hour interval and pyridoxine at a dose of 250 mg/kg led to tumor growth inhibition (TGI=55 %, р<0.001) on the first day after the completion of treatment. Monotherapy with MGL or pyridoxine in the same regimens resulted in a 24 % TGI (р=0.263) or 21 % TGI (р=0.410), respectively. In a pair-wise comparison of treatments, MGL + pyridoxine was more effective compared to MGL used alone (р=0.061) and MGL + pyridoxine was more effective then pyridoxine alone (р=0.031). MGL from C. freundii at a dose of 200 U/kg, 4 times with a 48-hour interval plus pyridoxine at a dose of 500 mg/kg injected on day 9 after the completion of treatment led to 50 % TGI, whereas MGL monotherapy at a single dose of 400 U/kg or pyridoxine monotherapy in the same regimen showed 5 % TGI (р=0.991) and 4 % TGI (р=0.998), respectively. The pair-wise comparison showed that MGL (200 U/kg) + pyridoxine was more effective than MGL (400 U/ kg) alone (р<0.001) and pyridoxine alone (р=0.003). In the B16 model, the combination of MGL injected i.p at a dose of 2000 U/kg and pyridoxine at a dose of 300 mg/kg showed 56 % TGI on day 1after the completion of treatment (р=0.045) and 35 % TGI on day 3 (р=0.038). Pyridoxine significantly increased the anticancer effect of MGL: MGL 1000 U/kg i.p and MGL 1000 U/kg i.p. + pyridoxine 300 mg/kg led to TGI=45 % (р=0.034) on day 3 after the completion of treatment. Single maximum tolerated dose after multiple i.p. administration was defined as 2000 U/kg, simultaneous administration of pyridoxine did not increase the toxicity of MGL. In conclusion, LLC and B16 are sensitive to MGL treatment, and pyridoxine may increase the efficacy of MGL.Приведены экспериментальные данные монотерапии и комбинированной терапии моделей перевиваемых опухолей мышей препаратами метионин-γ-лиазы (МГЛ) и пиридоксина гидрохлорида. Изучены МГЛ Clostridium sporogenes и Citrobacter freundii. Использованы перевиваемые модели опухолей мышей: карцинома легкого Льюис (LLC), меланома В16, лимфолейкоз P388, лимфолейкоз L1210, лимфаденоз Фишера L5178y. На моделях P388, L5178y МГЛ не показала противоопухолевой активности ни в монорежиме, ни в сочетании с пиридоксином. На модели L1210 было получено пограничное увеличение продолжительности жизни (УПЖ) 22 %, р=0,035 при применении МГЛ C. sporogenes в дозе 2000 Е/кг 11-кратно внутрибрюшинно с интервалом 12 ч. На LLC показано, что на 1-е сут после окончания лечения одновременное внутрибрюшное (в/б) введение МГЛ C. sporogenes 400 Е/кг 4-кратно с интервалом 48 ч и пиридоксина в дозе 250 мг/кг вызывало ТРО=55 % (р<0,001), монотерапия МГЛ или пиридоксином в аналогичных дозах и режимах применения вызывала ТРО=24 % (р=0,263) и 21 % (р=0,410) соотетственно. При попарном сравнении: комбинированная терапия МГЛ + пиридоксин против монотерапии МГЛ в аналогичном режиме р=0,061, против монотерапии пиридоксином р=0,031. На LLC МГЛ C. freundii 200 Е/кг 4-кратно с интервалом 48 ч и пиридоксина в дозе 500 мг/кг одновременно на 9-е сут после окончания лечения вызывало ТРО=50 % (р=0,001), при этом монотерапия МГЛ в разовой дозе 400 Е/кг или пиридоксином в аналогичном режиме применения вызывала ТРО=+5 % (р=0,991) и 4 % (р=0,998) соответственно. При попарном сравнении: комбинированная терапия МГЛ 200 Е/кг + пиридоксин против монотерапии МГЛ 400 Е/кг в аналогичном режиме р<0,001, против монотерапии пиридоксином р=0,003. На модели меланома B16 МГЛ 2000 в/б + пиридоксин 300 мг/кг вызывает ТРО 56 % на 1-е сут (р=0,045) и 35 % на 3-и сут (р=0,038). При комбинированной терапии МГЛ + пиридоксин последний значимо повышал противоопухолевую активность МГЛ в сочетаниях: МГЛ 1000 Е/кг в/б и МГЛ 1000 Е/кг в/б + пиридоксин 300 мг/кг ТРО=45 % (р=0,034) на 3-и сут после окончания лечения. При внутривенном введении МГЛ 500 Е/кг и МГЛ 500 Е/кг + пиридоксин последний повышал эффективность лечения: максимальное ТРО 50 % на 1-е сут после окончания лечения (р=0,085, различие не достоверно) и 21 % на 3-и сут после окончания лечения ТРО 22 % (р=0,965, различие не достоверно).Разовая максимальная переносимая доза при многократном в/б введении составила 2000 Е/кг, одновременное применение пиридоксина не усугубляло токсичности МГЛ. Таким образом, LLC и меланома В16 обладают чувствительностью к терапии МГЛ. Одновременное введение пиридоксина на модели LLC и В16 достоверно повышает её эффективность

Development and Preclinical Evaluation of Enzyme Prodrug Therapies Targeted to the Tumor Vasculature

Breast cancer is a global health concern of high prevalence that lacks safe and effective therapies for advanced cases. A targeted enzyme prodrug therapy aims to address this issue using an enzyme localized to the tumor to convert a systemically administered nontoxic prodrug into a toxic anticancer agent exclusively in the tumor. The target of the presented enzyme prodrug systems, phosphatidylserine, exists on cancer cells and the cells of the tumor vasculature. Annexin V binds to phosphatidylserine with high affinity and was successfully fused to three enzymes for the targeted delivery of the enzyme prodrug systems to the tumor. Development of the purine nucleoside phosphorylase fusion with annexin V is described, and results showing strong in vitro binding and promising cytotoxicity are presented. This system is compared in vivo with targeted cytosine deaminase and targeted methioninase enzyme prodrug systems. The methioninase system produced the strongest antitumor results showing tumor regression for the duration of treatment. Further engineering of the system resulted in the generation of a mammalian cystathionine-γ-lyase protein with methioninase activity to prevent the immune response anticipated against foreign methioninase. Successful transition to immune competent models without incurring an immune response led to studies with combination therapies to achieve an enhanced therapeutic effect. Antitumor synergism was observed when the enzyme prodrug therapy was combined with rapamycin to address the hypoxic response. Combination with immunostimulatory levels of cyclophosphamide produced an anti-metastatic response and enhanced survival. Combination of the enzyme prodrug therapy with both rapamycin and cyclophosphamide effectively reduced tumor volumes, inhibited metastatic progression, and enhanced survival

Preclinical developments of enzyme-loaded red blood cells

Therapeutic enzymes are currently used in the treatment of several diseases. In most cases, the benefits are limited due to poor in vivo stability, immunogenicity, and drug-induced inactivating antibodies. A partial solution to the problem is obtained by masking the therapeutic protein by chemical modifications. Unfortunately, this is not a satisfactory solution because frequent adverse events, including anaphylaxis, can arise

TESTING OF NOVEL ENZYME PRODRUG AND PHOTOTHERMAL THERAPEUTICS FOR THE TREATMENT OF BREAST CANCER

The majority of this work characterizes two novel enzyme prodrug therapies using fusion proteins containing annexin V to target only tumor vascular endothelial cells and cancer cells, reducing the burden of systemic toxicity in healthy tissue. Methioninase enzyme will convert inert selenomethionine to toxic methylselenol and also deplete the cancer cells of methionine necessary for protein synthesis and continued growth. Cytosine deaminase converts 5-fluorocytosine into the well-known cancer therapeutic 5-fluorouracil. Additionally, continued work using single-walled carbon nanotubes targeted to cancer cells by the F3 peptide was done.Recombinant technology was used to express and purify methioninase-annexin V and cytosine deaminase-annexin V fusion proteins. In vitro testing of binding and cytotoxicity were completed. Studies of both fusion proteins binding to human endothelial cells and two breast cancer cell lines were done to obtain dissociation constants in the range of 0.6-6 nM, indicating relatively strong binding. Cytotoxicity studies revealed that methioninase-annexin V with selenomethionine can kill those same cell lines in only 3 days; cytosine deaminase-annexin V accomplished the same goal in 9 days using 5-fluorocytosine.The remaining enzyme prodrug work involved testing the methioninase-annexin V system in vivo. Pharmacokinetic testing revealed complete clearance of methioninase-annexin V from the bloodstream to occur within 8 hours following intraperitoneal injection. Selenomethionine levels up to 12 mg/kg were shown to cause no apparent toxicity, while higher levels were lethal. Tests were done with a maximum of 10 mg/kg. The enzyme prodrug system demonstrated a significant slowing of tumor growth compared to untreated mice or mice treated only with the prodrug or fusion protein. Using a fluorescent dye, it was shown that the blood flow through the treated tumor was significantly reduced. The results obtained in vivo with this enzyme prodrug treatment are promising.F3-targeted single-walled carbon nanotubes were tested for their ability to bind to and become internalized by endothelial cells and breast cancer cells. Following incubation with SWNT-F3, cells were irradiated with a near-infrared laser 980 nm. The irradiation increased the cell death as determined by a cell viability assay. The photothermal therapy produced promising results in vitro, and tests with mice are recommended

Engineered primate L-methioninase for therapeutic purposes

Methods and compositions relating to the engineering of an improved protein with methionine-γ-lyase enzyme activity are described. For example, in certain aspects there may be disclosed a modified cystathionine-γ-lyase (CGL) comprising one or more amino acid substitutions and capable of degrading methionine. Furthermore, certain aspects of the invention provide compositions and methods for the treatment of cancer with methionine depletion using the disclosed proteins or nucleic acids.Board of Regents, University of Texas Syste

Targeting fusion proteins containing L-methioninase to cancer cells.

The methioninase-annexin V fusion protein bound specifically to phosphatidylserine (PS) immobilized on plastic plates, as well as on the surface of MCF-7 cancer cells in which PS was induced to be on the surface by the addition of hydrogen peroxide.The influence of the fusion protein on the growth and motility of human breast cancer cells, SK-LU-1 human lung cancer cells, and PC-3 human prostate cancer cells was examined using a culture wounding assay. ATF-methioninase inhibited the proliferation and migration of all cancer cell lines. For MCF-7 breast cancer cells, the inhibition by ATF-methioninase was much greater than for either free L-methioninase or mutated ATF-methioninase (mutated to give inactive enzyme). TGF-methioninase inhibited the proliferation and migration in MCF-7 and PC-3 cancer cells. However, the inhibition was significantly greater for ATF-methioninase compared to TGF-methioninase, especially at treatment days 2 and 3.ATF-methioninase binding to MCF-7 cells was measured by immunocytochemical localization. MCF-7 tumor xenograft growth was measured in nude mice for the mice treated with ATF-methioninase and L-methioninase. Treatment of s.c. implanted MCF-7 breast cancer cells mouse xenografts with ATF-methioninase gave significantly more tumor regression when compared to mouse xenografts treated with L-methioninase alone or with the vehicle control.It has been shown that methionine depletion inhibits tumor cell growth and reduces tumor cell survival. The main purpose of this project is to examine three fusion proteins for targeting human cancer cells selectively and inhibiting the migration and proliferation of the cancer cells. The fusion proteins studied are ATF-methioninase (amino-terminal fragment of urokinase, amino acids 1-49, linked to the amino terminus of L-methioninase fro Pseudomonas putida ), TGF-methioinase (human transforming growth factor-alpha linked to L-methioninase), and methioninase-annexin V (L-methioninase linked to the amino terminus of human annexin V). The three fusion proteins were expressed as soluble proteins and purified to near homogeneity. Flash freezing and followed by lyophilization was found to be most effective way to store fusion protein samples

Targets in Gene Therapy

This book aims at providing an up-to-date report to cover key aspects of existing problems in the emerging field of targets in gene therapy. With the contributions in various disciplines of gene therapy, the book brings together major approaches: Target Strategy in Gene Therapy, Gene Therapy of Cancer and Gene Therapy of Other Diseases. This source enables clinicians and researchers to select and effectively utilize new translational approaches in gene therapy and analyze the developments in target strategy in gene therapy