Orally administered endoxifen inhibits tumor growth in melanoma-bearing mice.

Endoxifen, an active metabolite of tamoxifen, has been shown to be an effective anti-estrogenic agent in estrogen receptor-positive breast cancer patients. In melanoma, estrogen receptor expression is shown to be associated with disease progression. However, the therapeutic benefit of endoxifen in melanoma has not yet been evaluated. Here, we present the first demonstration of the anti-melanogenic activity of endoxifen in vitro and in vivo. The in vitro cytotoxic effect of endoxifen was tested using a cell viability assay. The in vivo anti-melanogenic activity was evaluated in B16F10 cell-bearing C57BL/6 mice, a mouse melanoma model. The general toxicity was tested in Swiss albino mice. Endoxifen exhibited greater activity against melanoma cell lines. Treatment of B16F10 mouse and SK-MEL-5 human melanoma cell lines with 10 µM of endoxifen for 48 h respectively resulted in 93.6 and 92.5% cell death. Orally administered endoxifen, at dose levels of 4 and 8 mg/kg body weight/day for 20 consecutive days, respectively reduced metastatic melanoma nodules in the lungs by 26.7 and 82.7%. Endoxifen was found to be a safe and effective anti-melanogenic agent in animal studies.

Carbohydrate mediated drug delivery: Synthesis and characterization of new lipid-conjugates.

A new synthetic methodology for cationic glycolipids using p-aminophenyl-α-d-mannopyranoside (PAPM), p-aminophenyl-α-d-galactopyranoside (PAPG) was developed. PAPM-lipids and PAPG-lipids conjugates were also synthesized for targeting drugs to receptors. A binding inhibition study of synthesized p-(dimethylamino butylamido) phenyl-α-d-mannopyranoside (1a) with Concanavalin A was performed using invertase enzyme. In addition, transfection of pSV-ß-gal reporter gene with was investigated in A549 cells.

Carbohydrate mediated drug delivery: synthesis and characterization of new lipid-conjugates.

A new synthetic methodology for cationic glycolipids using p-aminophenyl-α-D-mannopyranoside (PAPM) and p-aminophenyl-α-D-galactopyranoside (PAPG) with spacer in between the quaternary nitrogen atom and the sugar unit is developed. In addition, a new class of neutral glycolipid conjugates, such as PAPM-lipids or PAPG-lipids conjugates was also synthesized for targeting drugs to receptors. The precipitation-inhibition assay showed that conjugate of PAPM inhibited the concanavalin A and invertase aggregation. This binding inhibition study of a synthesized compound suggests that conjugates of PAPM can be potentially used to target mannose receptors. In addition, a higher transfection was obtained by mixing PAPM with pSV-ß-gal reporter gene and incubating with mannose binding protein/receptor expressing A549 cells. The coexistence of both mannose group and a net positive charge may result in improved transfection efficiency in cells expressing mannose binding proteins/receptors.

Nanosomal Amphotericin B is an efficacious alternative to Ambisome for fungal therapy.

Amphotericin B was formulated in lipids (Nanosomal Amphotericin B) without using any detergent or toxic organic solvents during the preparation. Electron microscopy and particle size determination of Nanosomal Amphotericin B showed a homogeneous population of nanosized particles below 100 nm. Hemolysis assay indicated that Nanosomal Amphotericin B causes significantly less lysis of red blood cells than Amphotericin B deoxycholate and was comparable to Ambisome. A maximum daily dose of Nanosomal Amphotericin B at 5 mg/kg in rabbits and 10 mg/kg in mice for 28 days showed no symptoms of toxicity, mortality or significant body weight reduction. Hematological and gross pathological analysis of tissues revealed no abnormalities attributable to the drug treatment. Nanosomal Amphotericin B and Ambisome were injected (iv) at 2 mg/kg consecutively for 5 days into mice infected with Aspergillus fumigatus. The treatment resulted in 90% survival with Nanosomal Amphotericin B and only 30% survival with Ambisome after 10 days of fungal infection. However, all of the 10 control mice which were not treated with Amphotericin B died within 5 days of fungal infection. Nanosomal Amphotericin B is safe, cost effective and provides an alternative option for treatment of fungal disease.

Endoxifen is a new potent inhibitor of PKC: a potential therapeutic agent for bipolar disorder.

Protein kinase C (PKC) plays a major role in regulation of both pre and postsynaptic neurotransmission. Excessive activation of PKC results in symptoms related to bipolar disorder. Tamoxifen, a widely used breast cancer drug is known to inhibit PKC and demonstrate antimanic properties in human. We describe herein the synthesis of endoxifen, a tamoxifen active metabolite and compared its PKC inhibitory activity with that of tamoxifen. Endoxifen exhibited fourfold higher potency compared to tamoxifen.

Guggullipid derivatives: synthesis and applications.

Two guggullipid derivatives, Z-guggulsulfate [4,17(20)-pregnadiene-3-one-16beta-sulfate] sodium salt and Z-guggullaurate [4,17(20)-pregnadiene-3-one-16beta-laurate], have been synthesized and evaluated for liposomal drug delivery system. Its precursor, Z-guggulsterol [4,17(20)-pregnadiene-3-one-16beta-ol], is also synthesized in gram scale starting from guggulsterone using the novel combination of known reactions in fewer steps and with higher yield than previously reported synthesis. These new synthetic guggullipid derivatives were also used in the preparation of liposomes. This new class of lipid molecules will be a useful tool in the development of nanosomal or liposomal drug delivery system.

Orally administered endoxifen is a new therapeutic agent for breast cancer.

Endoxifen is the key active metabolite of tamoxifen, a widely used breast cancer drug. Orally administered tamoxifen, is extensively metabolized by cytochrome P450 (CYP) enzymes, namely CYP3A4 and CYP2D6, into active metabolites, especially endoxifen. Due to genetic polymorphism of CYP2D6, significant numbers of women metabolize tamoxifen to varying degree and may not receive the optimal benefit from tamoxifen treatment. We show that oral administration of endoxifen achieved the optimally effective systemic levels reliably, which may eliminate variability associated with tamoxifen metabolism that leads to unpredictability in efficacy. Furthermore, use of endoxifen may avoid a potential serious drug interaction found between tamoxifen and commonly used selective serotonin reuptake inhibitors, antidepressants. Endoxifen was active in inhibiting the growth of various breast tumor cell lines in NCI 60-Cell Line Screen. Orally administered endoxifen is rapidly absorbed and systemically available when tested in female rats. The endoxifen-treated rats showed 787% higher exposure (AUC(0-infinity)) and 1,500% higher concentration (C (max)) levels of endoxifen when compared with tamoxifen. Oral endoxifen administration once a day for 28 consecutive days at dosages 2, 4, and 8 mg/kg proved safe and resulted in progressive inhibition of the growth of the human mammary tumor xenografts in female mice. This is the first ever in vivo report on endoxifen as a potentially new therapeutic agent for breast cancer.

Polyoxyl 60 hydrogenated castor oil free nanosomal formulation of immunosuppressant Tacrolimus: pharmacokinetics, safety, and tolerability in rodents and humans.

OBJECTIVE: Develop Nanosomal formulation of Tacrolimus to provide safer alternative treatment for organ transplantation patients. Investigate safety, tolerability and pharmacokinetics of Nanosomal Tacrolimus formulation versus marketed Tacrolimus containing polyoxyl 60 hydrogenated castor oil (HCO-60) that causes side effects. METHODS: Nanosomal Tacrolimus was prepared in an aqueous system. The particle size was measured by Particle Sizing Systems and structure morphology was determined by freeze-fracture electron microscopy. Investigational safety studies were conducted in mice and rats. Safety and pharmacokinetics of Nanosomal Tacrolimus were also evaluated in healthy human subjects. RESULTS: The morphology of Nanosomal Tacrolimus showed a homogeneous population of nanosized particles with mean particle size of less than 100 nm. A 14 day consecutive administration of Nanosomal Tacrolimus up to 5 and 10mg/kg dose in rats and mice respectively, resulted in no mortality. Nanosomal Tacrolimus in human studies showed that it is safe and the pharmacokinetics profile is similar to the marketed HCO-60 based Tacrolimus. No significant change in peripheral blood lymphocyte percentage was noted in either mice or healthy human male subjects. CONCLUSIONS: Nanosomal Tacrolimus is well characterized product which provides a new treatment option. It contains no alcohol or surfactants like HCO-60. Thus, Nanosomal Tacrolimus presents a new and improved therapeutic approach for organ transplant patients compared to the marketed HCO-60 based Tacrolimus product.

Synthesis of 1,3- and 1,2-diphosphatidylglycerol.

A novel phosphonium salt methodology was utilized for the first time to synthesize 1,3-, and 1,2-diphosphatidylglycerol. Optically active 1,2-di-O-acyl-sn-glyceryl phosphate was coupled with unprotected glycerol in the presence of pyridiniumbromide perbromide and triethylamine to yield, after final removal of phosphate protecting group, the title compounds. The 1,2-diphosphatidylglycerol (1,2-isomer of cardiolipin) may be a member of a new class of phospholipids for industrial applications similar to other phosphocholines.

In-vitro and in-vivo anti-cancer activity of a novel gemcitabine-cardiolipin conjugate.

Our objectives were to study the biological activity of a novel gemcitabine-cardiolipin conjugate (NEO6002) and compare that with gemcitabine. Cytotoxicity in vitro was determined against several gemcitabine-sensitive parental and gemcitabine-resistant cancer cell lines using the sulforhodamine B assay. The in vivo toxicity was examined by changes in body weight and hematologic indices of conventional mice. Immunodeficient SCID mice bearing P388 and BxPC-3 tumor xenografts were used to evaluate the in-vivo therapeutic efficacy. Both NEO6002 and gemcitabine showed pro-apoptotic and cytotoxic effects against all gemcitabine-sensitive cell lines tested. Unlike gemcitabine, the cytotoxicity of NEO6002 was independent of nucleoside transporter (NT) inhibitors, indicating a different internalization route of NEO6002. The conjugate demonstrated a favorable activity not only in ARAC-8C, a NT-deficient gemcitabine-resistant human leukemia cell line, but also in several other gemcitabine-resistant cell lines. At the in-vivo level, a comparative toxicity study showed a significant body weight loss and a decrease in white blood cell counts in gemcitabine-treated mice, whereas the influence of NEO6002 was mild. Treatment of NEO6002 at 27 micromol/kg increased the median survival of CD2F1 mice bearing P388 cells by up to 73%, while at the same doses and schedule of gemcitabine resulted in toxic deaths of all treated mice. At a dose of 18 micromol/kg, NEO6002 inhibited the growth of BxPC-3 xenografts by 52%, while only 32% of tumor inhibition was achieved with gemcitabine. We conclude that NEO6002 may be an effective chemotherapeutic agent with improved tolerability and can potentially circumvent NT-deficient, gemcitabine-resistant tumors.

Synthesis of cationic cardiolipin analogues.

An approach was developed to synthesize a new class of cationic cardiolipin analogues containing two quaternary ammonium groups with tetra alkyl groups retaining "glycerol" moiety, the central core of the molecule. Cationic cardiolipin analogues were modified via introduction of either two or four oxyethylene groups to enhance the solubility in polar solvents. These newly synthesized cationic cardiolipin analogues can be applied to a broad range of drug delivery systems such as transfection reagents.

Synthesis and biological evaluation of gemcitabine-lipid conjugate (NEO6002).

A novel gemcitabine-lipid conjugate 5 was synthesized and tested for its in vivo efficacy and toxicity. Compound 5 was tested in BxPC-3 human pancreatic tumor model in SCID mice and exhibited promising activity and lower toxicity when compared with Gemzar.

Novel cationic cardiolipin analogue-based liposome for efficient DNA and small interfering RNA delivery in vitro and in vivo.

Cationic liposomes have been successfully used as an alternative approach to viral systems to deliver nucleic acids. However, high toxicity and inconsistent transfection efficiency have been associated with the currently available liposomes. Therefore, a novel cationic liposome was developed based on a synthetic cationic cardiolipin analogue (CCLA) to test the DNA transfection efficiency. This CCLA-based liposome was also used to determine the therapeutic efficacy of c-raf small interfering RNA (siRNA) in mice. In this report, we showed that the CCLA-based liposome was less toxic and effectively transfected reporter genes in vitro and in vivo. The transfection efficiency in mice was seven-fold higher than the commercially available DOTAP-based liposome. In addition, c-raf siRNA in the presence of CCLA-based liposome induced up to 62% of growth inhibition in cancer cells. Treatment of c-raf siRNA/CCLA complex in SCID mice bearing human breast xenograft tumors resulted in 73% of tumor growth suppression as compared to free c-raf siRNA group. In conclusion, a novel CCLA-based liposome showed less toxicity and broad usage both in vitro and in vivo with DNA and siRNA.

A short, concise route to diphosphatidylglycerol (cardiolipin) and its variants.

A new approach is described for the synthesis of the cardiolipin family of phospholipids that uses phosphonium salt methodology. The method involves the reaction of 2-O-protected glycerol with a trialkyl phosphite derived from 1,2-diacylsn-glycerol in the presence of pyridinium bromide perbromide and triethylamine to afford the phosphoric triesters. The synthesis involves three steps and allows the preparation of a wide range of cardiolipins with different substitution patterns and chain lengths, including unsaturated derivatives. The use of inexpensive protecting groups and the ease of purification facilitate this synthetic route and allow its scale-up in a higher overall yield (72%) than the literature methods.

An efficient and novel method for the synthesis of cardiolipin and its analogs.

A novel synthetic method has been developed for cardiolipin and its analog via a chlorophosphoramidite coupling reaction followed by oxidation. The reagent, N,N-diisopropylmethylphosphoramidic chloride, couples effectively with 1,2-O-dimyristoyl-sn-glycerol in the presence of an amidite activator to form a phosphoamidite intermediate, which then reacts with 2-O-benzylglycerol in the presence of a basic catalyst followed by in situ oxidation to give the corresponding protected cardiolipin. Deprotection of the protecting groups provides tetramyristoyl cardiolipin in good overall yield of 60%. The synthetic method is applicable to large-scale synthesis of cardiolipin and various analogs with or without unsaturation for liposomal drug delivery.