Health sector reform and reproductive health in Latin America and the Caribbean: strengthening the links.

Many countries in Latin America and the Caribbean (LAC) are currently reforming their national health sectors and also implementing a comprehensive approach to reproductive health care. Three regional workshops to explore how health sector reform could improve reproductive health services have revealed the inherently complex, competing, and political nature of health sector reform and reproductive health. The objectives of reproductive health care can run parallel to those of health sector reform in that both are concerned with promoting equitable access to high quality care by means of integrated approaches to primary health care, and by the involvement of the public in setting health sector priorities. However, there is a serious risk that health reforms will be driven mainly by financial and/or political considerations and not by the need to improve the quality of health services as a basic human right. With only limited changes to the health systems in many Latin American and Caribbean countries and a handful of examples of positive progress resulting from reforms, the gap between rhetoric and practice remains wide.

Effects of SCH 59228, an orally bioavailable farnesyl protein transferase inhibitor, on the growth of oncogene-transformed fibroblasts and a human colon carcinoma xenograft in nude mice.

The products of the Ha-, Ki-, and N-ras proto-oncogenes comprise a family of 21 kDa guanine nucleotide-binding proteins which play a crucial role in growth factor signal transduction and in the control of cellular proliferation and differentiation. Activating mutations in the ras oncogenes occur in a wide variety of human tumors. Ras proteins undergo a series of posttranslational processing events. The first modification is addition of the 15-carbon isoprene, farnesyl, to a Cys residue near the carboxy-terminus of Ras. Prenylation allows the Ras oncoprotein to localize to the plasma membrane where it can initiate downstream signalling events leading to cellular transformation. Inhibitors of the enzyme which catalyzes this step, farnesyl protein transferase (FPT), are a potential class of novel anticancer drugs which interfere with Ras function. SCH 59228 is a tricyclic FPT inhibitor which inhibits the farnesylation of purified Ha-Ras with an IC50 of 95 nM and blocks the processing of Ha-Ras in Cos cells with an IC50 of 0.6 microM. SCH 59228 has favorable pharmacokinetic properties upon oral dosing in nude mice. The in vivo efficacy of SCH 59228 was evaluated using a panel of tumor models grown in nude mice. These included several rodent fibroblast lines expressing mutationally-activated (val12) forms of the Ha-Ras oncogene. In some cases, these proteins contain their native C-terminal sequence (CVLS) which directs farnesylation. In one model, the C-terminal sequence was altered to CVLL, making the expressed protein a substrate for a distinct prenyl transferase, geranylgeranyl protein transferase-1. When dosed orally at 10 and 50 mg/kg (four times a day, 7 days a week) SCH 59228 significantly inhibited tumor growth of cells expressing farnesylated Ha-Ras in a dose-dependent manner; over 90% growth inhibition was observed at the 50 mg/kg dose. Tumor growth of cells expressing the geranylgeranylated form of Ha-Ras was less potently inhibited. Growth of tumors derived from a rodent fibroblast line expressing activated Ki-Ras containing its native C-terminal sequence (CVIM), which preferentially directs farnesylation, was also inhibited by SCH 59228. Inhibition in the Ki-Ras model was less than that observed in the Ha-Ras model. In contrast, tumors derived from cells transformed with the mos oncogene were not significantly inhibited even at the highest dose level. SCH 59228 also significantly and dose-dependently inhibited the growth of human colon adenocarcinoma DLD-1 xenografts (which express activated Ki-ras). These results indicate that SCH 59228 possesses in vivo antitumor activity upon oral dosing in tumor models expressing activated ras oncogenes. This is the first report of oral antitumor activity with an FPT inhibitor. These results are discussed in light of recent observations on alternative prenylation of some Ras isoforms.

Antitumor activity of SCH 66336, an orally bioavailable tricyclic inhibitor of farnesyl protein transferase, in human tumor xenograft models and wap-ras transgenic mice.

We have been developing a series of nonpeptidic, small molecule farnesyl protein transferase inhibitors that share a common tricyclic nucleus and compete with peptide/protein substrates for binding to farnesyl protein transferase. Here, we report on pharmacological and in vivo studies with SCH 66336, a lead compound in this structural class. SCH 66336 potently inhibits Ha-Ras processing in whole cells and blocks the transformed growth properties of fibroblasts and human tumor cell lines expressing activated Ki-Ras proteins. The anchorage-independent growth of many human tumor lines that lack an activated ras oncogene is also blocked by treatment with SCH 66336. In mouse, rat, and monkey systems, SCH 66336 has excellent oral bioavailability and pharmacokinetic properties. In the nude mouse, SCH 66336 demonstrated potent oral activity in a wide array of human tumor xenograft models including tumors of colon, lung, pancreas, prostate, and urinary bladder origin. Enhanced in vivo efficacy was observed when SCH 66336 was combined with various cytotoxic agents (cyclophosphamide, 5-fluorouracil, and vincristine). In a Ha-Ras transgenic mouse model, prophylactic treatment with SCH 66336 delayed tumor onset, reduced the average number of tumors/mouse, and reduced the average tumor weight/animal. In a therapeutic mode in which gavage treatment was initiated after the transgenic mice had developed palpable tumors, significant tumor regression was induced by SCH 66336 in a dose-dependent fashion. This was associated with increased apoptosis and decreased DNA synthesis in tumors of animals treated with SCH 66336. Enhanced efficacy was also observed in this model when SCH 66336 was combined with cyclophosphamide. SCH 66336 is presently being evaluated in Phase I clinical trials.

Human smooth muscle alpha-actin gene is a transcriptional target of the p53 tumor suppressor protein.

Smooth muscle (sm) alpha-actin is expressed in vascular smooth muscle cells and fibroblast cells. Its expression is regulated by cell proliferation and repressed during oncogenic transformation. In this study, we demonstrate that p53 activation is associated with a dramatic increase in organized microfilament bundles and an increase in sm alpha-actin mRNA level. Wild-type p53, but not mutant p53, strongly stimulated human sm alpha-actin promoter activity in p53 null cell lines. The sequences homologous to the p53 consensus sequence and to the p53 binding sequence from the muscle creatine kinase, were found within a specific region of the sm alpha-actin promoter. This sequence was sufficient to confer p53-dependent activation to a heterologous promoter and p53 was capable of binding to this sequence as assessed by gel shift analysis. Ionizing irradiation of colorectal tumor cells caused an increase in alpha-actin mRNA level in a p53-dependent manner. Taken together, these results demonstrate that human sm alpha-actin gene is a transcriptional target for p53 tumor suppressor protein and represents the first example of a cytoskeletal gene with a functionally defined p53 response element.

Inhibitors of farnesyl protein transferase. 4-Amido, 4-carbamoyl, and 4-carboxamido derivatives of 1-(8-chloro-6,11-dihydro-5H-benzo[5,6]- cyclohepta[1,2-b]pyridin-11-yl)piperazine and 1-(3-bromo-8-chloro-6,11- dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl)piperazine.

The synthesis of a variety of novel 4-amido, 4-carbamoyl and 4-carboxamido derivatives of 1-(8-chloro-6,11-dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl) piperazine to explore the SAR of this series of FPT inhibitors is described. This resulted in the synthesis of the 4- and 3-pyridylacetyl analogues 45a and 50a, respectively, both of which were orally active but were found to be rapidly metabolized in vivo. Identification of the principal metabolites led to the synthesis of a variety of new compounds that would be less readily metabolized, the most interesting of which were the 3- and 4-pyridylacetyl N-oxides 80a and 83a. Novel replacements for the pyridylacetyl moiety were also sought, and this resulted in the discovery of the 4-N-methyl and 4-N-carboxamidopiperidinylacetyl derivatives 135a and 160a, respectively. All of these derivatives exhibited greatly improved pharmacokinetics. The synthesis of the corresponding 3-bromo analogues resulted in the discovery of the 4-pyridylacetyl N-oxides 83b (+/-) and 85b [11S(-)] and the 4-carboxamidopiperidinylacetamido derivative 160b (+/-), all of which exhibited potent FPT inhibition in vitro. All three showed excellent oral bioavailability in vivo in nude mice and cynomolgus monkeys and exhibited excellent antitumor efficacy against a series of tumor cell lines when dosed orally in nude mice.

The development of a quality information system: a case study of Mexico.

One of the primary obstacles in the implementation of continuous quality improvement (CQI) programmes in developing countries is the lack of timely and appropriate information for decentralized decision-making. The integrated quality information system (QIS) described herein demonstrates Mexico's unique effort to package four separate, yet mutually reinforcing, tools for the generation and use of quality-related information at all levels of the Mexican national health care system. The QIS is one element of the continuous quality improvement programme administered by the Secretariat of Health in Mexico. Mexico's QIS was designed to be flexible and capable of adapting to local needs, while at the same time allowing for the standardization of health care quality assurance indicators, and subsequent ability to measure and compare the quality performance of health facilities nationwide. The flexibility of the system extends to permit the optimal use of available data by health care managers at all levels of the health care system, as well as the generation of new information in important areas often neglected in more traditional information systems. Mexico's QIS consists of four integrated components: 1) a set of client and provider surveys, to assess specific issues in the quality of health services delivered; 2) client and provider national satisfaction surveys; 3) a sentinel health events strategy; and 4) a national Comparative Performance Evaluation System, for use by the Secretariate of Health for the quality assessment of state and provincial health care services (internal benchmarking). The QIS represents another step in Mexico's ongoing effort to use data for effective decision-making in the planning, monitoring and evaluation of services delivered by the national health care system. The design and application of Mexico's QIS provides a model for decentralized decision-making that could prove useful for developing countries, where the effective use of quality indicators is often limited. Further, the system could serve as a mechanism for motivating positive change in the way information is collected and used in the process of ensuring high quality health care service delivery.

Biochemical characterization of the binding of echistatin to integrin alphavbeta3 receptor.

Echistatin is a 49-amino-acid peptide belonging to the family of disintegrins that are derived from snake venoms and are potent inhibitors of platelet aggregation and cell adhesion. Integrin alphavbeta3 receptor plays a critical role in several physiological processes such as tumor-induced angiogenesis, tumor cell metastasis, osteoporosis and wound repair. In this study, we have characterized the binding of echistatin to purified integrin alphavbeta3 receptor and the form expressed on human embryonic kidney 293 cells. We show that both purified and membrane-bound integrin alphavbeta3 binds to echistatin with a high affinity, which can be competed efficiently by linear and cyclic peptides containing the RGD sequence. Previous studies have shown that alphavbeta3 binds to vitronectin in a nondissociable manner, whereas an RGD-containing peptide derived from vitronectin binds in a dissociable manner with a Kd of 9.4 x 10(-7) M. Our studies indicate that radiolabeled echistatin binds to alphavbeta3 in a nondissociable manner, similar to native echistatin. However, echistatin does not support the adhesion of 293 cells expressing alphavbeta3 receptor because of poor binding to plastic dishes and is a potent antagonist of the adhesion of these cells to vitronectin. These studies demonstrate that echistatin binding to alphavbeta3 is of high affinity and irreversible similar to vitronectin and provides an alternate ligand for high-throughput screening for alphavbeta3 antagonists.

K- and N-Ras are geranylgeranylated in cells treated with farnesyl protein transferase inhibitors.

The association of mutant forms of Ras protein with a variety of human cancers has stimulated intense interest in therapies based on inhibiting oncogenic Ras signaling. Attachment of Ras proteins to the plasma membrane is required for effective Ras signaling and is initiated by the enzyme farnesyl protein transferase. We found that in the presence of potent farnesyl protein transferase inhibitors, Ras proteins in the human colon carcinoma cell line DLD-1 were alternatively prenylated by geranylgeranyl transferase-1. When H-Ras, N-Ras, K-Ras4A, and K-Ras4B were expressed individually in COS cells, H-Ras prenylation and membrane association were found to be uniquely sensitive to farnesyl transferase inhibitors; N- and K-Ras proteins incorporated the geranylgeranyl isoprene group and remained associated with the membrane fraction. The alternative prenylation of N- and K-Ras has significant implications for our understanding of the mechanism of action of farnesyl protein transferase inhibitors as anti-cancer chemotherapeutics.

Efficacy of p53 adenovirus-mediated gene therapy against human breast cancer xenografts.

In response to DNA damage, p53 protein accumulates in the cell nucleus causing cells to undergo DNA repair or apoptosis, programmed cell death. Reintroduction of wild-type p53 into tumors with null or mutant p53 offers a novel strategy for controlling tumor growth, by inducing apoptotic death in neoplastic cells. The efficacy of a replication-deficient p53 adenovirus construct was tested against three human breast cancer cell lines expressing mutant p53, MDA-MB-231, -468, and -435. 231 and 468 cells were both highly transduced at a multiplicity of infection of 10. By contrast, 435 cells were rarely transduced. p53 adenovirus-mediated gene therapy was highly effective against 231 and 468 tumor xenografts in nude mice. At a total dose of 2.2 x 10(9) cellular infectious units (CIU), inhibition of 231 tumor growth was 86% (P < or = .01). Thirty-seven percent of that growth inhibition was due to p53, while 49% was adenovirus-specific. Inhibition of 468 tumor growth was 74% (P < or = .001). Forty-five percent of that inhibition was p53-specific, while 28% was adenovirus-specific. The ED50 values for 231 tumors and 468 tumor growth inhibition were 3 x 10(8) CIU and 2 x 10(8) CIU, respectively. Injection of p53 Ad into 231 or 468 tumors induced apoptosis. By contrast, growth inhibition in 435 tumors treated with p53 adenovirus was not significant, probably due to low adenovirus transduction. 231 and 435 cells both expressed high levels of alpha v, beta 1, beta 3, and beta 5 integrin subunits, ruling out lack of the appropriate integrins as the reason for the low infection rate in 435 cells. Our results demonstrate the ability of wild-type p53 to curtail cancerous cell growth in vivo in tumors expressing mutant p53. The ability of beta-gal Ad to infect tumor cells in vitro was generally predictive of in vivo p53 Ad efficacy.

Antitumor 8-chlorobenzocycloheptapyridines: a new class of selective, nonpeptidic, nonsulfhydryl inhibitors of ras farnesylation.

Ras farnesylation by farnesyl protein transferase (FPT) is an intracellular event that facilitates the membrane association of the ras protein and is involved in the signal transduction process. FPT inhibition could be a novel, noncytotoxic method of treating ras dependent tumor growth. We report here three structural classes of 8-chlorobenzocycloheptapyridines as novel, nonpeptidic, nonsulfhydryl FPT inhibitors having antitumor activity in mice when dosed orally. We discuss structural and conformational aspects of these compounds in relation to biological activities as well as a comparison to the conformation of a bound tetrapeptide FPT inhibitor.

Discovery of novel nonpeptide tricyclic inhibitors of Ras farnesyl protein transferase.

A comprehensive structure-activity relationship (SAR) study of novel tricyclic amides has been undertaken. The discovery of compounds that are potent FPT inhibitors in the nanomolar range has been achieved. These compounds are nonpeptidic and do not contain sulfhydryl groups. They selectively inhibit farnesyl protein transferase (FPT) and not geranylgeranyl protein transferase-1 (GGPT-1). They also inhibit H-Ras processing in Cos monkey kidney cells.

Ras oncoprotein inhibitors: the discovery of potent, ras nucleotide exchange inhibitors and the structural determination of a drug-protein complex.

The nucleotide exchange process is one of the key activation steps regulating the ras protein. This report describes the development of potent, non-nucleotide, small organic inhibitors of the ras nucleotide exchange process. These inhibitors bind to the ras protein in a previously unidentified binding pocket, without displacing bound nucleotide. This report also describes the development and use of mass spectrometry, NMR spectroscopy and molecular modeling techniques to elucidate the structure of a drug-protein complex, and aid in designing new ras inhibitor targets.

Structure-activity relationship of 3-substituted N-(pyridinylacetyl)-4- (8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene )- piperidine inhibitors of farnesyl-protein transferase: design and synthesis of in vivo active antitumor compounds.

Novel tricyclic Ras farnesyl-protein transferase (FPT) inhibitors are described. A comprehensive structure-activity relationship (SAR) study of compounds arising from substitution at the 3-position of the tricyclic pyridine ring system has been explored. In the case of halogens, the chloro, bromo, and iodo analogues 19, 22, and 28 were found to be equipotent. However, the fluoro analogue 17 was an order of magnitude less active. Whereas a small alkyl substituent such as a methyl group resulted in a very potent FPT inhibitor (SCH 56580), introduction of bulky substituents such as tert-butyl, compound 33, or a phenyl group, compound 29, resulted in inactive FPT inhibitors. Polar groups at the 3-position such as amino 5, alkylamino 6, and hydroxyl 12 were less active. Whereas compound SCH 44342 did not show appreciable in vivo antitumor activity, the 3-bromo-substituted pyridyl N-oxide amide analogue 38 was a potent FPT inhibitor that reduced tumor growth by 81% when administered q.i.d. at 50 mpk and 52% at 10 mpk. These compounds are nonpeptidic and do not contain sulfhydryl groups. They selectively inhibit FPT and not geranylgeranyl-protein transferase-1 (GGPT-1). They also inhibit H-Ras processing in COS monkey kidney cells and soft agar growth of Ras-transformed cells.

Interleukin-10 inhibits tumor metastasis through an NK cell-dependent mechanism.

Interleukin-10 (IL-10) is a recently described pleiotropic cytokine secreted mainly by type 2 helper T cells. Previous studies have shown that IL-10 suppresses cytokine expression by natural killer (NK) and type 1 T cells, thus down-regulating cell-mediated immunity and stimulating humoral responses. We here report that injected IL-10 protein is an efficient inhibitor of tumor metastasis in experimental (B16-F10) and spontaneous (M27 and Lox human melanoma) metastasis models in vivo at doses that do not have toxic effects on normal or cancer cells. Histological characterization after IL-10 treatment confirmed the absence of CD8+ and CD4+ T cells and macrophages at the sites of tumor growth, but abundant NK cells were localized at these sites. This unexpected finding was confirmed by showing that IL-10 inhibits most B16-F10 and Lox metastases in mice deficient in T or B cells (SCID and nu/nu mice), but not in those deficient in NK cells (beige mice or NK cell-depleted mice). However, IL-10 downregulation of pro-inflammatory cytokine production and/or recruitment of additional effector cells may also be involved in the anti-tumor effect at higher local concentrations of IL-10, since transfected B16 tumor cells expressing high amounts of IL-10 were rejected by normal, nu/nu, or SCID mice at the primary tumor stage, and there was still a 33% inhibition of tumor metastasis in beige mice.

Novel tricyclic inhibitors of farnesyl protein transferase. Biochemical characterization and inhibition of Ras modification in transfected Cos cells.

Ras protooncogenes encode 21-kDa membrane-associated guanine nucleotide-binding proteins, which play a critical role in control of cellular proliferation and differentiation. Oncogenic, activated forms of Ras proteins are associated with a broad range of human cancers. The elucidation of the post-translational modifications that occur at the carboxyl terminus of Ras and the demonstration that farnesylation of Ras by farnesyl protein transferase is essential for Ras-induced cellular transformation has opened up a new and promising approach to the development of anti-Ras therapeutics. We report here a novel series of potent farnesyl protein transferase (FPT) inhibitors, represented by SCH 44342. This compound inhibits both rat brain and recombinant human FPT with an IC50 of approximately 250 nM, while it is only weakly active against rat brain geranylgeranyl protein transferase-1 (IC50 > 114 microM). FPT inhibition has been observed using both Ha-Ras protein and Ki-Ras-derived peptide substrates in two different assay formats. SCH 44342 and its analogs also inhibit farnesylation of Ras in Cos cells transiently expressing [Val12]Ha-Ras with IC50 values in the low micromolar range. At these concentrations they do not inhibit sterol biosynthesis or geranylgeranylation of protein. In addition, we observed that Cos cells undergo pronounced morphological changes upon overexpression of [Val12]activated forms of Ha-Ras containing COOH-terminal sequences allowing farnesylation (CVLS) or geranylgeranylation (CVLL) but not upon overexpression of activated Ras lacking the isoprenylated Cys (SVLS). Ras-induced morphological changes can be partially reverted with lovastatin. Importantly, SCH 44342 can block morphological changes induced by [Val12]Ha-Ras-CVLS but not [Val12]Ha-Ras-CVLL. Recently, a number of other FPT inhibitors have been reported. Most of the compounds reported to have cell-based activity are peptidomimetic analogs of the CAAX substrate. Our FPT inhibitors are novel in that although they compete with Ras protein in kinetic experiments they are entirely nonpeptidic in nature, they do not have oxidizable sulfhydryl functions, and they are active in cells at low micromolar concentrations.

Activation of smooth muscle alpha-actin promoter in ras-transformed cells by treatments with antimitotic agents: correlation with stimulation of SRF:SRE mediated gene transcription.

Smooth muscle alpha-actin promoter is repressed in ras-transformed fibroblast cells and derepressed in revertant cells. We have recently shown that serum response factor (SRF) which can bind to the serum response elements (SREs) present in the alpha-actin promoter, can activate alpha-actin promoter activity in ras-transformed cells and suppress transformation by ras. Agents that stimulate SRF expression and alpha-actin promoter activity in ras-transformed cells are expected to be potential candidates as antitumor agents. In this study, we show that treatment of ras-transformed cells with antitumor agents such as taxol, vincristine, vinblastine, colchicine, and nocodazole leads to 5- to 7-fold activation of alpha-actin promoter driven CAT activity, whereas there was very little effect on thymidine kinase promoter driven CAT activity. This activation occurred at subcytotoxic concentrations of these agents and correlated with inhibition of cell cycle progression. Furthermore, these agents stimulated SRF expression in ras-transformed cells, as measured by its SRE binding activity. The increase in alpha-actin expression is accompanied by the restoration of actin filaments into organized bundles. These results suggest a novel mechanism by which antimitotic agents suppress the ras-transformed phenotype.

Activity of temozolomide in the treatment of central nervous system tumor xenografts.

The activity of 8-carbamoyl-3-methylimidazo[5,1-d]-1,2,3,5-tetrazin- 4(3H)-one (temozolomide) in the treatment of a panel of xenografts derived from ependymoma, medulloblastoma, and childhood and adult high-grade glioma was evaluated in athymic nude mice bearing s.c. and intracranial tumors. Temozolomide administered daily for a total of five doses demonstrated marked activity against a panel of Mer+ xenografts despite marginal to moderate activity of 1,3-bis(2-chloroethyl)-1-nitrosourea. The growth delays produced by temozolomide in these xenografts were 1.8-7.5-fold greater than those produced by procarbazine. Although temozolomide demonstrated marginal activity against the Mer+ cell line D341 Med when a 5-day schedule was used, a high-dose 1-day schedule resulted in moderate activity. Temozolomide produced increases in median survival of 1285% (adult glioma D-54 MG), 323% (childhood glioma D-456 MG), and 68% (ependymoma D612 EP). Pretreatment of mice with O6-benzylguanine increased temozolomide-induced mortality, requiring reduction of the dosage from 1200 to 750 mg/m2 on the single-day regimen. O6-Benzylguanine pretreatment of mice bearing Mer+ D341 Med increased the growth delay of temozolomide, in duplicate experiments, from -3.1 to 4.8 and 1.1 to 4.9 days. These studies suggest that temozolomide may be active in the treatment of a broad spectrum of central nervous system cancers, including Mer+ tumors resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea.

Two serum response elements mediate transcriptional repression of human smooth muscle alpha-actin promoter in ras-transformed cells.

The mechanism by which activated ras oncogene expression leads to repression of genes encoding specific actin filament proteins is not understood. However, these changes associated with loss of organized actin filaments, are necessary to maintain the transformed phenotype. The human smooth muscle (sm) alpha-actin promoter is repressed in ras-transformed fibroblast cells and derepressed in revertant cell lines. In this study, we demonstrate that two serum response elements (SREs) present in the alpha-actin promoter are required for transcriptional repression in ras-transformed cells and the two SREs act synergistically to repress heterologous promoters in a ras-transformation dependent manner. Serum response factor (SRF), which can bind to the sm alpha-actin SREs, restores alpha-actin promoter activity in ras-transformed cells. c-Fos, c-Jun and YY1 also repress alpha-actin promoter through SREs, suggesting that these transcription factors may play a role in repressing alpha-actin promoter in ras-transformed cells.