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Biomedical subjects

P Y Lam

Publications and source records attributed to P Y Lam.

At least 19 recordsLinked to original sources
PubMed2001

Discovery of 1-[3-(aminomethyl)phenyl]-N-3-fluoro-2'-(methylsulfonyl)-[1,1'-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC423), a highly potent, selective, and orally bioavailable inhibitor of blood coagulation factor Xa.

Factor Xa (fXa) plays a critical role in the coagulation cascade, serving as the point of convergence of the intrinsic and extrinsic pathways. Together with nonenzymatic cofactor Va and Ca2+ on the phospholipid surface of platelets or endothelial cells, factor Xa forms the prothrombinase complex, which is responsible for the proteolysis of prothrombin to catalytically active thrombin. Thrombin, in turn, catalyzes the cleavage of fibrinogen to fibrin, thus initiating a process that ultimately leads to clot formation. Recently, we reported on a series of isoxazoline and isoxazole monobasic noncovalent inhibitors of factor Xa which show good potency in animal models of thrombosis. In this paper, we wish to report on the optimization of the heterocyclic core, which ultimately led to the discovery of a novel pyrazole SN429 (2b; fXa K(i) = 13 pM). We also report on our efforts to improve the oral bioavailability and pharmacokinetic profile of this series while maintaining subnanomolar potency and in vitro selectivity. This was achieved by replacing the highly basic benzamidine P1 with a less basic benzylamine moiety. Further optimization of the pyrazole core substitution and the biphenyl P4 culminated in the discovery of DPC423 (17h), a highly potent, selective, and orally active factor Xa inhibitor which was chosen for clinical development.

Administration, Oral↗
PubMed2001

Delayed presentation of an ingested foreign body causing gastric perforation.

Ingested foreign bodies may lead to perforation of the gastrointestinal tract. We present a case of a 14-month-old boy who presented with an unusual abdominal mass secondary to ingesting a foreign body 4 months previously. Abdominal computerized tomography scan was valuable in making this diagnosis.

Foreign Bodies↗
PubMed2001

Potential of gene therapy for brain tumors.

Brain tumors comprise a broad spectrum of biological and clinical entities making it unlikely for any single therapeutic approach to be universally applicable. In particular, malignant glioblastoma multiforme have defied all current therapeutic modalities. Gene therapy offers the potential to augment current neurosurgical, radiation and drug treatments with little increase in morbidity. Many therapeutic transgenes have shown efficacy in experimental models, including generation of toxic compounds, enzymatic activation of pro-drugs, expression of tumor suppressor or apoptotic proteins, inhibition of angiogenesis and enhancement of immune responses to tumor antigens. Vectors have been used as gene delivery vehicles and as cytotoxic agents in their own right by selective replication and lysis of tumor cells, thereby also generating vectors on-site. Brain tumors appear to offer some "Achilles' heels" in that they are usually contained within the brain and represent a unique dividing cell population there. However, the heterogeneous and invasive characteristics of these tumor cells, as well as sequestration of tumor antigens within a relatively immune privileged location present serious problems for effective therapy. This review will focus on current transgene/vector strategies, including novel therapeutic genes, combinational therapies and new delivery modalities, the latter of which appears to be the rate limiting factor for gene therapy of brain tumors in humans.

Adenoviridae↗
PubMed2000

Expression of p19INK4d, CDK4, CDK6 in glioblastoma multiforme.

Deregulation of the G1/S checkpoint is a frequent event in the development of glioblastoma multiforme (GBM). Previous studies have shown more than 50% of primary GBM tumours contain either complete loss of the p16INK4a locus or amplification of the CDK4 gene. Moreover, many heterozygosity studies have shown deletion on human chromosome 19p13.2, where the p19INK4d gene has been localized. We examined the expression of p19INK4d and its two CDK substrates in a series of glioma-derived cell lines and tumours. No gene rearrangement or deletion was observed in the p19INK4d gene in these cell lines; however, expression of CDK4 and CDK6 was elevated relative to matched normal brain tissue in eight of 18 GBM tumours (44%). Furthermore, CDK6 expression level was increased in 12/14 glioblastomas, but undetectable in tumour samples of a previous lower grade tumour from the same patient. These data attest to the functional importance of both CDK4 and CDK6 in astrocytic tumourigenesis, particularly during the later stages of tumour progression.

Blotting, Western↗
PubMed1999

The oncogenic potential of the Pax3-FKHR fusion protein requires the Pax3 homeodomain recognition helix but not the Pax3 paired-box DNA binding domain.

The chimeric transcription factor Pax3-FKHR, produced by the t(2;13)(q35;q14) chromosomal translocation in alveolar rhabdomyosarcoma, consists of the two Pax3 DNA binding domains (paired box and homeodomain) fused to the C-terminal forkhead (FKHR) sequences that contain a potent transcriptional activation domain. To determine which of these domains are required for cellular transformation, Pax3, Pax3-FKHR, and selected mutants were retrovirally expressed in NIH 3T3 cells and evaluated for their capacity to promote anchorage-independent cell growth. Mutational analysis revealed that both the third alpha-helix of the homeodomain and a small region of the FKHR transactivation domain are absolutely required for efficient transformation by the Pax3-FKHR fusion protein. Surprisingly, point mutations in the paired domain that abrogate sequence-specific DNA binding retained transformation potential equivalent to that of the wild-type protein. This finding suggests that DNA binding mediated through the Pax3 paired box is not required for transformation. Our results demonstrate that the integrity of the Pax3 homeodomain recognition helix and the FKHR transactivation domain is necessary for efficient cellular transformation by the Pax3-FKHR fusion protein.

3T3 Cells↗
PubMed1998

Stereoisomers of cyclic urea HIV-1 protease inhibitors: synthesis and binding affinities.

We have synthesized stereoisomers of cyclic urea HIV-1 protease inhibitors to study the effect of varying configurations on binding affinities. Four different synthetic approaches were used to prepare the desired cyclic urea stereoisomers. The original cyclic urea synthesis using amino acid starting materials was used to prepare three isomers. Three additional isomers were prepared by synthetic routes utilizing L-tartaric acid and D-sorbitol as chiral starting materials. A stereoselective hydroxyl inversion of the cyclic urea trans-diol was used to prepare three additional isomers. In all 9 of the 10 possible cyclic urea stereoisomers were prepared, and their binding affinities are described.

Anti-HIV Agents↗
PubMed1998

Counteracting HIV-1 protease drug resistance: structural analysis of mutant proteases complexed with XV638 and SD146, cyclic urea amides with broad specificities.

The long-term therapeutic benefit of HIV antiretroviral therapy is still threatened by drug-resistant variants. Mutations in the S1 subsite of the protease are the primary cause for the loss of sensitivity toward many HIV protease inhibitors, including our first-generation cyclic urea-based inhibitors DMP323 and DMP450. We now report the structures of the three active-site mutant proteases V82F, I84V, and V82F/I84V in complex with XV638 and SD146, two P2 analogues of DMP323 that are 8-fold more potent against the wild type and are able to inhibit a broad panel of drug-resistant variants [Jadhav, P. K., et al. (1997) J. Med. Chem. 40, 181-191]. The increased efficacy of XV638 and SD146 is due primarily to an increase in P2-S2 interactions: 30-40% more van der Waals contacts and two to four additional hydrogen bonds. Furthermore, because these new interactions do not perturb other subsites in the protease, it appears that the large complementary surface areas of their P2 substituents compensate for the loss of P1-S1 interactions and reduce the probability of selecting for drug-resistant variants.

Amino Acid Substitution↗
PubMed1998

Cyclic HIV protease inhibitors: design and synthesis of orally bioavailable, pyrazole P2/P2' cyclic ureas with improved potency.

Highly potent HIV-1 protease (HIVPR) inhibitors have been designed and synthesized by introducing bidentate hydrogen-bonding oxime and pyrazole groups at the meta-position of the phenyl ring on the P2/P2' substituents of cyclic ureas. Nonsymmetrical cyclic ureas incorporating 3(1H)-pyrazolylbenzyl as P2 and hydrophilic functionalities as P2' show potent protease inhibition and antiviral activities against HIV and have good oral bioavailabilities. The X-ray structure of HIVPR.10A complex confirms that the two pyrazole rings of 10A form bidentate hydrogen bonds with the side-chain oxygen (C=O) and backbone nitrogen (N-H) of Asp30/30' of HIVPR.

Administration, Oral↗
PubMed1998

Molecular recognition of cyclic urea HIV-1 protease inhibitors.

As long as the threat of human immunodeficiency virus (HIV) protease drug resistance still exists, there will be a need for more potent antiretroviral agents. We have therefore determined the crystal structures of HIV-1 protease in complex with six cyclic urea inhibitors: XK216, XK263, DMP323, DMP450, XV638, and SD146, in an attempt to identify 1) the key interactions responsible for their high potency and 2) new interactions that might improve their therapeutic benefit. The structures reveal that the preorganized, C2 symmetric scaffolds of the inhibitors are anchored in the active site of the protease by six hydrogen bonds and that their P1 and P2 substituents participate in extensive van der Waals interactions and hydrogen bonds. Because all of our inhibitors possess benzyl groups at P1 and P1', their relative binding affinities are modulated by the extent of their P2 interactions, e.g. XK216, the least potent inhibitor (Ki (inhibition constant) = 4.70 nM), possesses the smallest P2 and the lowest number of P2-S2 interactions; whereas SD146, the most potent inhibitor (Ki = 0.02 nM), contains a benzimidazolylbenzamide at P2 and participates in fourteen hydrogen bonds and approximately 200 van der Waals interactions. This analysis identifies the strongest interactions between the protease and the inhibitors, suggests ways to improve potency by building into the S2 subsite, and reveals how conformational changes and unique features of the viral protease increase the binding affinity of HIV protease inhibitors.

Anti-HIV Agents↗
PubMed1998

Nonpeptide cyclic cyanoguanidines as HIV-1 protease inhibitors: synthesis, structure-activity relationships, and X-ray crystal structure studies.

Comparison of the high-resolution X-ray structures of the native HIV-1 protease and its complexes with the inhibitors suggested that the enzyme flaps are flexible. The movement at the tip of the flaps could be as large as 7 A. On the basis of this observation, cyclic cyanoguanidines have been designed, synthesized, and evaluated as HIV-1 protease (PR) inhibitors. Cyclic cyanoguanidines were found to be very potent inhibitors of HIV-1 protease. The choice of cyclic cyanoguanidines over cyclic guanidines was based on the reduced basicity of the former. X-ray structure studies of the HIV PR complex with cyclic cyanoguanidine demonstrated that in analogy to cyclic urea, cyclic cyanoguanidines also displace the unique structural water molecule. The structure-activity relationship of the cyclic cyanoguanidines is compared with that of the corresponding cyclic urea analogues. The differences in binding constants of the two series of compounds have been rationalized using high-resolution X-ray structure information.

Anti-HIV Agents↗
PubMed1998

Telomerase expression in gliomas including the nonastrocytic tumors.

Telomerase is a ribonucleoprotein that consists of a RNA component for synthesizing telomeric DNA repeats onto chromosome ends, so that telomere length can be maintained; telomerase activation in general signifies immortalization of cells. Because the telomerase activity of only a few cases of nonastrocytic gliomas and low-grade astrocytic gliomas have been examined before, telomerase activity from a broad spectrum of astrocytic and nonastrocytic gliomas were examined in this study. A total of 119 brain tumor samples, including 16 pilocytic astrocytomas, one dysembryoplastic neuroepithelial tumor (DNT), two pleomorphic xanthoastrocytomas (PXA), 15 ependymomas, 21 oligodendrogliomas, 13 grade II astrocytomas, 13 anaplastic astrocytomas, and 38 glioblastoma multiforme tumors (GBM), were studied, using a polymerase chain reaction (PCR)-based telomeric repeat amplification protocol assay. All pilocytic astrocytomas, DNT, PXA, and myxopapillary ependymomas were telomerase negative. Three of 13 (15.4%) classical ependymomas, 2 of 14 (14.3%) grade II oligodendrogliomas, and three of seven (42.9%) anaplastic oligodendrogliomas had detectable telomerase activity. The frequency of telomerase expression in ependymomas and oligodendrogliomas was lower than those observed in astrocytic tumors: fibrillary astrocytoma, 23.1%; anaplastic astrocytoma, 23.1%; and GBM, 26.3%. The mean age of telomerase-positive GBM patients (61.7 years) was significantly higher than that of telomerase-negative GBM patients (47.8 years, P=.002). These results suggest that telomerase activation may occur early in glial tumorigenesis, and astrocytomas may have mechanisms of immortalization other than telomerase activation.

Adolescent↗
PubMed1998

Analysis of cell cycle regulators: p16INK4A, pRb, and CDK4 in low- and high-grade meningiomas.

Abnormalities in the p16INK4A, CDK4, and Rb genes, which regulate transition through G1 phase of the cell cycle, have been implicated in the progression of diverse types of cancer. To evaluate the involvement of p16INK4A, CDK4, and Rb in the tumorigenesis of meningiomas, the status of these genes or gene products were examined. The genetic alteration of the p16INK4A gene was examined by homozygous deletions and by mutation analysis. The methylation status of the p16INK4A was determined by Southern blotting. Neither homozygous deletions nor point mutations of the p16INK4A gene were observed in any of the 23 meningiomas. Partial rather than complete methylation of the p16INK4A gene at SacII or SmaI sites was shown in five (23.8%) meningiomas. The methylation status of the p16INK4A gene was not consistently associated with the expression of p16INK4A in meningiomas. These results suggest that the true relationship between methylation and expression of p16INK4A may be obscured in a complex manner by various mechanisms that regulate p16INK4A expression. Aberrant expressions of pRb and CDK4 were not observed in any of the meningiomas we examined, indicating that abnormalities of the pRb and CDK4 appear to be rare in meningiomas.

Adolescent↗
PubMed1998

Design and selection of DMP 850 and DMP 851: the next generation of cyclic urea HIV protease inhibitors.

BACKGROUND: Recent clinical trials have demonstrated that HIV protease inhibitors are useful in the treatment of AIDS. It is necessary, however, to use HIV protease inhibitors in combination with other antiviral agents to inhibit the development of resistance. The daunting ability of the virus to rapidly generate resistant mutants suggests that there is an ongoing need for new HIV protease inhibitors with superior pharmacokinetic and efficacy profiles. In our attempts to design and select improved cyclic urea HIV protease inhibitors, we have simultaneously optimized potency, resistance profile, protein binding and oral bioavailability. RESULTS: We have discovered that nonsymmetrical cyclic ureas containing a 3-aminoindazole P2 group are potent inhibitors of HIV protease with excellent oral bioavailability. Furthermore, the 3-aminoindazole group forms four hydrogen bonds with the enzyme and imparts a good resistance profile. The nonsymmetrical 3-aminoindazoles DMP 850 and DMP 851 were selected as our next generation of cyclic urea HIV protease inhibitors because they achieve 8 h trough blood levels in dog, with a 10 mg/kg dose, at or above the protein-binding-adjusted IC90 value for the worst single mutant--that containing the Ile84-->Val mutation. CONCLUSIONS: In selecting our next generation of cyclic urea HIV protease inhibitors, we established a rigorous set of criteria designed to maximize chances for a sustained antiviral effect in HIV-infected individuals. As DMP 850 and DMP 851 provide plasma levels of free drug that are sufficient to inhibit wild-type HIV and several mutant forms of HIV, they could show improved ability to decrease viral load for clinically significant time periods. The ultimate success of DMP 850 and DMP 851 in clinical trials might depend on achieving or exceeding the oral bioavailability seen in dog.

Animals↗
PubMed1998

Antisense epidermal growth factor receptor RNA transfection in human malignant glioma cells leads to inhibition of proliferation and induction of differentiation.

The epidermal growth factor receptor (EGFR) is a protooncogene that is frequently observed with alterations in late stage gliomas, suggesting an important role of this gene in glial tumorigenesis and progression. In this study we evaluated an antisense EGFR approach as an alternative therapeutic modality for glioblastomas. We transfected U-87MG cells with an antisense EGFR construct and obtained several clones stably expressing lower or undetectable levels of EGFR protein. These clones were found to have impaired proliferation as well as a reduced transforming potential to grow in soft agarose. The number of cells positive for the cell cycle-specific nuclear antigen Ki-67 was also significantly decreased (P < 0.05) in antisense EGFR-transfected clones compared with parental or empty vector-transfected cells. Flow cytometric analysis revealed that the proportion of cells in G0/G1 phases of the cell cycle in the antisense clones increased by up to 31% compared with control cells, whereas the proportion of cells in S phase decreased by up to 58%. In addition, the antisense EGFR-transfected cells showed higher expression of glial fibrillary acidic protein and a more differentiated form, with smaller cell bodies possessing fine tapering cell processes. These results suggest that EGFR plays a major role in modulating cell growth and differentiation in glioblastoma cells. Our experimental model of antisense EGFR provides a basis for future development of antisense EGFR oligodeoxynucleotides in treatment of glioblastomas.

Antibodies, Monoclonal↗
PubMed1998

The molecular genetics of central nervous system tumors.

Over the past few years, although much has been learned about the molecular genetics of central nervous system (CNS) tumors, researchers and pathologists are only beginning to understand the scientific basis of the development of these tumors. Data accumulated so far support the division of glioblastoma into two clinical and molecular subsets. Primary or de novo glioblastomas occur in older patients, are clinically aggressive and exhibit epidermal growth factor receptor amplification or overexpression. Secondary glioblastomas develop from pre-existing low-grade astrocytomas, have a more protracted clinical course, and frequently contain p53 mutations. Both types of tumors show deletions of chromosome 10 and possibly mutations of the PTEN/MMAC1 gene as an endstage event. Oligodendrogliomas have been shown to have genetic abnormalities distinct from those of the astrocytic tumors, commonly involving chromosomes 1p and 19q. As regards meningiomas, loss of chromosome 22q and mutations of the neurofibromatosis type 2 gene are frequent events and loss of chromosome 14q and 10q may be seen in atypical or malignant transformation. Such genetic findings, apart from providing a better understanding of neoplastic transformation in brain tumors, are beginning to form the basis of a new approach to neuro-oncology.

Astrocytoma↗
PubMed1997

Molecular basis of HIV-1 protease drug resistance: structural analysis of mutant proteases complexed with cyclic urea inhibitors.

In cell cultures, the key residues associated with HIV-1 resistance to cyclic urea-based HIV-1 protease (PR) inhibitors are Val82 and Ile84 of HIV-1 PR. To gain an understanding of how these two residues modulate inhibitor binding, we have measured the Ki values of three recombinant mutant proteases, I84V, V82F, and V82F/I84V, for DMP323 and DMP450, and determined the three-dimensional structures of their complexes to 2.1-1.9 A resolution with R factors of 18.7-19.6%. The Ki values of these mutants increased by 25-, 0.5-, and 1000-fold compared to the wild-type values of 0.8 and 0.4 nM for DMP323 and DMP450, respectively. The wild-type and mutant complexes overall are very similar (rms deviations of 0.2-0.3 A) except for differences in the patterns of their van der Waals (vdw) interactions, which appear to modulate the Ki values of the mutants. The loss of the CD1 atom of Ile84, in the I84V mutant complexes, creates a hole in the S1 subsite, reducing the number of vdw contacts and increasing the Ki values. The V82F mutant binds DMP323 more tightly than wild type because the side chain of Phe82 forms additional vdw and edge-to-face interactions with the P1 group of DMP323. The Ki values of the single mutants are not additive because the side chain of Phe82 rotates out of the S1 subsite in the double mutant (the chi 1 angles of Phe82 and -182 in the V82F and V82F/I84V mutants differ by 90 and 185 degrees, respectively), further reducing the vdw interactions. Finally, compensatory shifts in the I84V and V82F/ I84V complexes pick up a small number of new contacts, but too few to offset the initial loss of interactions caused by the mutations. Therefore, our data suggest that variants persist in the presence of DMP323 and DMP450 because of a decrease in vdw interactions between the mutant proteases and inhibitors.

Azepines↗
PubMed1996

HIV protease inhibitory bis-benzamide cyclic ureas: a quantitative structure-activity relationship analysis.

A series of N,N'-disubstituted cyclic urea 3-benzamides has been synthesized and evaluated for HIV protease inhibition and antiviral activity. Some of these benzamides have been shown to be potent inhibitors of HIV protease with Ki < 0.050 nM and IC90 < 20 nM for viral replication and, as such, may be useful in the treatment of AIDS. The synthesis and quantitative structure-activity relationship for this benzamide series will be discussed.

Anti-HIV Agents↗