AIDSVAX. VaxGen.

AIDSVAX, a bivalent vaccine consisting of a preparation of recombinant gp120 from two types of HIV, is being developed by VaxGen for the potential prevention of HIV infection [274573]. Two versions of the vaccine are in phase III trials: AIDSVAX B/E, in phase III trials in Thailand, stimulates production of antibodies to HIV subtypes common to South and Southeast Asia, and countries of the Pacific Rim; and AIDSVAX B/B, in phase III trials in the US and Europe, stimulates production of antibodies to HIV subtypes that are found in the Americas, Europe, the Caribbean and Australia [314365], [397186]. The bivalent vaccine is based on an earlier monovalent vaccine, MNrgp120 (Genetech Inc/VaxGen Inc) [274573], [356845]. In April 2001, the independent data and safety monitoring board (DSMB) reviewed data from the trials and indicated that the vaccine appeared safe and that the trials were being conducted appropriately. VaxGen has disclosed that an average of 95% of volunteers continue to participate in the phase III trials. The DSMB was scheduled to conduct its review in November 2001, to examine the efficacy of AIDSVAX in the prevention of HIV infection. VaxGen would halt the trial early if the interim analysis shows the vaccine to be effective. The company would then begin the process of applying for regulatory approval. If the interim analysis proves inconclusive, the trial would proceed to its scheduled conclusion in the fourth quarter of 2002 [386730], [405696]. In August 1998, a collaborative research agreement was signed with the National Institute of Allergy and Infectious Diseases (NIAID). This includes research on combinations of AIDSVAX and other NIAID vaccines and research for new formulations suitable for developing nations [295166].

Entecavir (Bristol-Myers Squibb).

Bristol-Myers Squibb (BMS) is developing entecavir, a viral replication inhibitor, for the potential treatment of hepatitis B virus (HBV) infection [220240]. The compound is a cyclopentyl guanosine analog and is in phase II trials in the US [383065]. Entecavir was originally developed as SQ-34676 for the treatment of herpes simplex virus infections [221992], but displayed only moderate activity which eventually led to discontinuation of development for this indication. However, Bristol-Myers Squibb later discovered that entecavir was extremely potent against HBV (ED50 = 3.0 nM, compared with 200 nM for lamivudine) with relatively low toxicity (CC50 = 30,000 nM) [221986] and acted through inhibition of DNA polymerase [220240]. The triphosphate form is a potent HBV polymerase inhibitor in both woodchuck and duck models [306056]. By September 2000, a large-scale clinical trial was underway in China for HBV infection [400209] and by October 2000 phase I trials were ongoing in Japan [384751]. In March 2001 SG Cowen predicted sales of US$25 million in 2002, US$50 million in 2003 and US$75 million in 2004 [403751].

Synthesis and biological evaluation of first N-alkyl syn dimeric 4-aryl-1,4-dihydropyridines as competitive HIV-1 protease inhibitors.

A first series of novel N-alkyl substituted syn dimeric 4-aryl-1,4-dihydropyridines 12--17 have been synthesised and evaluated as HIV-1 protease inhibitors in in vitro assays. While the N-methyl derivatives 12 and 13 were almost inactive, with IC(50)-values of about 225 microM, the N-benzyl compounds with varied ester groups all exhibited stronger activities, with IC(50)-values of 11--12 microM for the presently best compounds 16 and 17 with ethyl ester functions. The type of HIV-1 protease inhibition of the novel inhibitors was characterised as competitive. With the increase of observed activity from N-methyl derivatives to N-benzyl compounds the binding mode may correspond to that of cyclic ureas with hydrophobic interactions of the four aromatic residues to the S1/S1' and S2/S2' regions of HIV-1 protease.

Sanglifehrin A, a novel cyclophilin-binding compound showing immunosuppressive activity with a new mechanism of action.

We report here on the characterization of the novel immunosuppressant Sanglifehrin A (SFA). SFA is a representative of a class of macrolides produced by actinomycetes that bind to cyclophilin A (CypA), the binding protein of the fungal cyclic peptide cyclosporin A (CsA). SFA interacts with high affinity with the CsA binding side of CypA and inhibits its peptidyl-prolyl isomerase activity. The mode of action of SFA is different from known immunosuppressive drugs. It has no effect on the phosphatase activity of calcineurin, the target of the immunosuppressants CsA and FK506 when complexed to their binding proteins CypA and FK binding protein, respectively. Moreover, its effects are independent of binding of cyclophilin. SFA inhibits alloantigen-stimulated T cell proliferation but acts at a later stage than CsA and FK506. In contrast to these drugs, SFA does not affect IL-2 transcription or secretion. However, it blocks IL-2-dependent proliferation and cytokine production of T cells, in this respect resembling rapamycin. SFA inhibits the proliferation of mitogen-activated B cells, but, unlike rapamycin, it has no effect on CD154/IL-4-induced Ab synthesis. The activity of SFA is also different from that of other known late-acting immunosuppressants, e.g., mycophenolate mofetil or brequinar, as it does not affect de novo purine and pyrimidine biosynthesis. In summary, we have identified a novel immunosuppressant, which represents, in addition to CsA, FK506 and rapamycin, a fourth class of immunophilin-binding metabolites with a new, yet undefined mechanism of action.

Comparison of human skin or epidermis models with human and animal skin in in-vitro percutaneous absorption.

For the study of in-vitro skin penetration of candidate drugs, excised animal skin is frequently used as a replacement for human skin. Reconstructed human skin or epidermis equivalents have been proposed as alternatives. We compared the penetration properties of human, pig and rat skin with the Graftskin LSE (living skin equivalent) and the Skinethic HRE (human reconstructed epidermis) models using four topical dermatological drugs (salicylic acid, hydrocortisone, clotrimazole and terbinafine) with widely varying polarity. In agreement with published data, pig skin appeared as the most suitable model for human skin: the fluxes through the skin and concentrations in the skin were of the same order of magnitude for both tissues, with differences of at most two- or fourfold, respectively. Graftskin LSE provided an adequate barrier to salicylic acid, but was very permeable for the more hydrophobic compounds (e.g. about 900-fold higher flux and 50-fold higher skin concentrations of clotrimazole as compared to human skin), even more than rat skin. In the case of the Skinethic HRE, we found similar concentrations of salicylic acid as in human skin and an approximately sevenfold higher flux. In contrast, the permeation of hydrophobic compounds through the epidermal layer was vastly higher than through split-thickness human skin (up to a factor of about 800). To conclude, currently available reconstituted skin models cannot be regarded as generally useful for in-vitro penetration studies.

Steroid sulfatase in the human hair follicle concentrates in the dermal papilla.

5 alpha-dihydrotestosterone is known to play a crucial part in the regulation of hair growth and in the development of androgenetic alopecia. 5 alpha-dihydrotestosterone is formed locally within the hair follicle from the systemic precursor testosterone by cutaneous steroid 5 alpha-reductase. Moreover, adrenal steroids such as dehydroepiandrosterone are converted to 5 alpha-dihydrotestosterone by isolated hair follicles, which may provide an additional source of intrafollicular 5 alpha-dihydrotestosterone levels. Elevated urinary dehydroepiandrosterone and serum dehydroepiandrosterone sulfate have been reported to be present in balding young men. These reports suggest that dehydroepiandrosterone sulfate may act as an important endocrine factor in the development of androgenetic alopecia. Hence the question arises whether the dehydroepiandrosterone sulfate can be metabolized within the hair follicles to yield dehydroepiandrosterone by the microsomal enzyme steroid sulfatase, and where steroid sulfatase might be localized. We therefore performed immunostaining for steroid sulfatase on human scalp biopsies as well as analysis of steroid sulfatase enzyme activity in defined compartments of human beard and occipital hair follicles ex vivo. Using both methods steroid sulfatase was primarily detected in the dermal papilla. Steroid sulfatase activity was inhibited by estrone-3-O-sulfamate, a specific inhibitor of steroid sulfatase, in a concentration-dependent way. Furthermore, we show that dermal papillae are able to utilize dehydroepiandrosterone sulfate to produce 5 alpha-dihydrotestosterone, which lends further support to the hypothesis that dehydroepiandrosterone sulfate contributes to androgenetic alopecia and that steroid sulfatase inhibitors could be novel drugs to treat androgen-dependent disorders of the hair follicle such as androgenetic alopecia or hirsutism.

Stimulation of MCF-7 breast cancer cell proliferation by estrone sulfate and dehydroepiandrosterone sulfate: inhibition by novel non-steroidal steroid sulfatase inhibitors.

Steroid sulfatase (STS) regulates the formation of active steroids from systemic precursors, such as estrone sulfate and dehydroepiandrosterone sulfate (DHEAS). In breast tissues, this pathway is a source for local production of estrogens, which support the growth of endocrine-dependent tumours. Therefore, inhibitors of STS could have therapeutic potential. In this study, we report on substituted chromenone sulfamates as a novel class of non-steroidal irreversible inhibitors of STS. The compounds are substantially more potent (6- to 80-fold) than previously described types of non-steroidal inhibitors when tested against purified STS. In MCF-7 breast cancer cells, they inhibit STS activity with IC(50) below 100 pM. Importantly, the compounds also potently block estrone sulfate-stimulated growth of MCF-7 cells, again with IC(50) below 100 pM. For one compound, we also observed a lack of any estrogenic effect at high concentrations (1 microM). We also demonstrate for the first time that STS inhibitors can block the DHEAS-stimulated growth of MCF-7 cells. Interestingly, this cannot be achieved with specific inhibitors of the aromatase, suggesting that stimulation of MCF-7 cell growth by DHEAS follows an aromatase-independent pathway. This gives further justification to consider steroid sulfatase inhibitors as potential drugs in the therapy of breast cancer.

New fluorogenic substrate for the first continuous steroid sulfatase assay.

The screening for new inhibitors of steroid sulfatase requires an efficient test system. To overcome the shortcomings of the available discontinuous fluorimetric assay, several coumarin-type compounds were investigated as potential new substrates. 3,4-Benzocoumarin 7-O-sulfate was found to have appropriate substrate properties for the establishment of the first direct continuous assay of steroid sulfatase.

Technology evaluation: FluMist, University of Michigan.

FluMist is an intranasal influenza vaccine, which has been developed by Aviron. The genetically engineered, live, attenuated, cold-adapted virus vaccine produces influenza infection without the symptoms. The product has been field for approval in the US, where Merrill Lynch expects it to be marketed by mid-2000 [336561].

Pleconaril Sanofi Synthélabo/ViroPharma.

Pleconaril is an oral antiviral agent being developed by ViroPharma and Sanofi-Synthélabo (formerly Sterling Winthrop) for the potential treatment of several picornavirus-induced infections, including respiratory diseases and viral meningitis. A number of phase III clinical trials have been completed, and several others are ongoing [319499], [343187], [346302], [359231]. In early 1999, an NDA filing for viral meningitis was expected by the end of 1999 [313588], [319499]. However, an NDA had not been filed by February 2000 and, at this time, filing in the US was expected in 2000 for viral meningitis and 2001 for viral respiratory syndrome, while in Europe filing was expected in 2001 and 2002 for these indications, respectively [359231]. In July 2000, Salomon Smith Barney predicted a launch date of 2002 [387350]. In October 1999, Lehman Brothers predicted a 70% chance of the product reaching the market, with a possible launch date anticipated for 2001 and potential peak sales of US$50 million in 2009 [346267].

Cage dimeric N-acyl- and N-acyloxy-4-aryl-1,4-dihydropyridines as first representatives of a novel class of HIV-1 protease inhibitors.

The synthesis of a series of novel cage dimeric N-acyl and N-acyloxy-4-aryl-1,4-dihydropyridines starting either from solid-state synthetic ester dimers or from monomeric 4-aryl-1,4-dihydropyridines is presented. Their biological evaluation as novel HIV-1 protease inhibitors showed the most active compounds to be 5c and 5i with inhibitory activities of 52% (50 microM) and 49% (25 microM), respectively. Within each series of N-acyl- and N-acyloxy derivatives NCOBz and NBoc groups were found to be the best substituents. Although they exhibiting only moderate activities these cage dimers hold promise as a class of novel non-peptidic HIV-1 protease inhibitors.

Synthesis and biological evaluation of the first N-alkyl cage dimeric 4-aryl-1,4-dihydropyridines as novel nonpeptidic HIV-1 protease inhibitors.

A first series of novel NH and N-alkyl-substituted cage dimeric 4-aryl-1,4-dihydropyridines 3a-f has been synthesized and evaluated as HIV-1 protease inhibitors in in vitro assays. While the NH and N-methyl derivatives 3a,b,e,f were almost inactive with IC(50) values of about 200 microM, the N-Benzyl compounds exhibited stronger activity with an IC(50) value of 16.2 microM for the presently best compound 3c. The type of HIV-1 protease inhibition of these novel inhibitors was characterized as competitive. With the increase of observed activity from NH and N-methyl derivatives to N-benzyl compounds, respectively, the binding mode may correspond to that of cyclic and azacyclic ureas showing hydrophobic interactions of the four aromatic residues to the S1/S1' and S2/S2' regions of HIV-1 protease.

X-ray structure and conformational dynamics of the HIV-1 protease in complex with the inhibitor SDZ283-910: agreement of time-resolved spectroscopy and molecular dynamics simulations.

Based on the X-ray structure of the human immunodeficiency virus type-1 (HIV-1) protease in complex with the statine-derived inhibitor SDZ283-910, a 542 ps molecular dynamics trajectory was computed. For comparison with the 805 ps trajectory obtained for the uncomplexed enzyme, the theoretical fluorescence anisotropy decay of the unliganded protease and the inhibitor complex was calculated from the trajectories of the Trp6A/Trp6B and Trp42A/Trp42B transition dipole moments. This enabled us to directly compare the simulated data with the experimental picosecond time-resolved fluorescence data. Fitting both experimental and simulated data to the Kohlrausch-Williams-Watts (KWW) function exp(-t/tauk)beta revealed a very good agreement for the uncomplexed protease as well as for the SDZ283-910 complex. Binding of the inhibitor induced a faster decay of both the experimental and the computed protease fluorescence anisotropy decay. By this integrative approach, the atomic detail of inhibitor-induced changes in the conformational dynamics of the HIV-1 protease was experimentally verified and will be used for further inhibitor optimisation.

Amprenavir (Vertex Pharmaceuticals Inc).

Amprenavir is an oral, non-peptidic HIV-protease inhibitor undergoing phase III trials for the treatment of HIV and AIDS. In October 1998, Glaxo Wellcome submitted a new drug application (NDA) for amprenavir (Agenerase) in the US and Canada for which there is an April 1999 review deadline and in November 1998, filed an application for market approval in Europe. This product has been designated for fast-track approval. Merrill Lynch anticipates product launch in mid-1999 and estimates that Agenerase will reach peak sales of $500M after its launch. Amprenavir is a small molecule with significant aqueous solubility designed to combine antiviral potency with good oral bioavailability. The compound is a potent and specific inhibitor of isolated HIV-1 protease and of HIV-1 replication in T-cells and shows good absorption after oral dosage in man. The plasma half-life of amprenavir is approximately 10 h, allowing for twice daily dosing. The drug appears to cross the blood-brain barrier which may be an important feature in long-term treatment. Amprenavir selects for a unique mutation in the HIV-1 protease gene in vitro, and shows no or limited cross-resistance to other protease inhibitors. In January 1999 Paribas predicted Glaxo sales of pound sterling 75 million in 1999 rising to pound sterling 400 million in 2003.

Time-resolved fluorescence anisotropy of HIV-1 protease inhibitor complexes correlates with inhibitory activity.

The tryptophan time-resolved fluorescence intensity and anisotropy of the HIV-1 protease dimer is shown to be a quick and efficient method for the conformational characterization of protease inhibitor complexes. Four fluorescence lifetimes were needed to adequately describe the fluorescence decay of the two tryptophan residues, W6 and W42, per protease monomer. As a result of the wavelength dependence of the respective amplitudes, the 2.06 ns and the 4.46 ns decay constants were suggested to be the intrinsic fluorescence lifetimes of the more solvent-exposed W6 and the less exposed W42 residues, respectively. Analysis of the fluorescence anisotropy decay yielded a short correlation time of 250 ps corresponding to local chromophore motions, and a long correlation time of 12.96 ns resulting from overall rotation of the protease enzyme. Fluorescence lifetimes and rotational correlation times changed when inhibitors of the HIV-1 protease were added. The effects of 11 different inhibitors including statine-derived, hydroxyethylamine-derived, and 2 symmetrical inhibitors on the protease fluorescence dynamics were investigated. Inhibitor binding is shown to induce an increase of the mean fluorescence lifetime taumean, an increase of the short rotational correlation time phi1, as well as a decrease of the long rotational correlation time phi2. The mean rotational correlation time phimean was identified as the global dynamic parameter for a given molecular complex, which correlates with the inhibitor dissociation constant Ki, and therefore with the activity of the inhibitor.

GS-4104 Gilead Sciences Inc.

Gilead and Roche are codeveloping GS-4104, a neuraminidase inhibitor, for the potential treatment and prevention of influenza virus infections, type A and type B. The companies began international phase II/III trials in December 1997 [272638]. The phase II/III double-blind, placebo-controlled study involves centers in the US, Canada, Europe and Hong Kong [272638]. The program will consist of two treatment studies and two prophylaxis studies, each of which will recruit 750 patients [273331]. The trials involve over 1500 patients, who will receive either GS-4104 or placebo for 6 weeks, as a once- or twice-daily dosing schedule [276650], [276895]. The company has also registered 73 centers for the two treatment studies, and 55 of these had been activated by the end of January 1998. Patients in the treatment arm will receive either GS-4104 or placebo for 5 days. Roche is conducting similar trials in Europe, Canada and Hong Kong [276895]. In September 1997, results were announced from two phase II studies, initiated in May 1997, in which volunteers were exposed intranasally to influenza A. The patients received oral capsules of GS-4104, or placebo, once-daily or twice-daily. In the first study, GS-4104 decreased the duration of symptoms by nearly 50%. In the second study, all patients who were given GS-4104 prophylactically did not have detectable levels of virus, as compared to 50% in the placebo group [264362]. In phase I trials, GS-4104 was well-tolerated with good oral absorption and distribution in blood and tissue [248180].

The non-immunosuppressive cyclosporin A analogue SDZ NIM 811 inhibits cyclophilin A incorporation into virions and virus replication in human immunodeficiency virus type 1-infected primary and growth-arrested T cells.

SDZ NIM 811 is a cyclosporin A (CsA) analogue that is completely devoid of immunosuppressive capacity but exhibits potent and selective anti-human immunodeficiency virus type 1 (HIV-1) activity. Binding to cyclophilin A, the intracellular receptor for cyclosporins, is a prerequisite for HIV-1 inhibition by cyclosporins. Cyclophilin A was demonstrated to bind to HIV-1 p24gag and this cyclophilin-Gag interaction leads to the incorporation of cyclophilin A into HIV-1 virions. SDZ NIM 811 inhibits this protein interaction, and this is likely to be the molecular basis for its antiviral activity. Here, we show that in activated primary T cells SDZ NIM 811 interferes with two stages of the virus replication cycle: (i) translocation of pre-integration complexes into the nucleus and (ii) production of infectious virus particles. SDZ NIM 811 not only inhibits translocation of HIV-1 pre-integration complexes in primary T cells, but also in a growth-arrested T cell line. In vivo, most T lymphocytes are quiescent, but serve nevertheless as a major and inducible HIV-1 reservoir in infected individuals. Significant amounts of cyclophilin A were found to be associated with virus particles propagated in primary T cells. SDZ NIM 811 caused a strong reduction in the amount of incorporated cyclophilin A, thereby reducing infectivity. Thus, cyclophilin A seems to be necessary for HIV-1 replication in primary T cells.

Presence of cyclophilin A in synovial fluids of patients with rheumatoid arthritis.

Cyclophilins have been suggested to act as leukocyte chemotactic factors produced in the course of inflammation. Therefore we looked for the presence of cyclophilins in the synovial fluids (SF) from patients with rheumatoid arthritis (RA). Peptidyl prolyl cis-trans isomerase activity (PPIase) was measured in SF from knee punctures of 26 patients with RA and five patients with knee osteoarthritis (OA). PPIase was detected in SF from RA patients, but not in samples from OA patients. Enzyme activity was sensitive to inhibition by cyclosporin A (IC50 = 28-50 nM). Estimated concentrations of the SF-derived cyclophilin based on the enzyme activity were in the range of 11 to 705 nM. The presence of cyclophilin in the SF showed disease correlation; its concentration correlated with the number of cells in the SF (r = 0.91, P < 0.0001) and with the percentage of neutrophils in the cellular infiltrate and was higher in more acute cases of joint swelling. In immunoblots of partially purified preparations of SF from RA patients, an approximately 18-kD protein band reacted with polyclonal antibodies that recognize cyclophilin A and B, but not with antibodies specific for cyclophilin B. Sequencing of this protein revealed identity of the NH2-terminal amino acids with those of human cyclophilin A. The finding is unexpected since cyclophilin B rather than A is generally regarded as the secreted isoform, the presence of cyclophilin A being confined to the cytoplasm. Our data support the hypothesis that cyclophilins may contribute to the pathogenesis of inflammatory diseases, possibly by acting as cytokines. This may offer a possible explanation of the effectiveness of cyclosporin A in RA, in addition to the known immunosuppressive effects of the drug.