The synthetic peptide T-20 (enfuvirtide) represents the first of a new class of antiretroviral compounds to demonstrate in vivo potency by targeting a step in viral entry. T-20 inhibits a conformational change in the human immunodeficiency virus type 1 (HIV-1) transmembrane glycoprotein (gp41) that is required for fusion between HIV-1 and target cell membranes. The initial phase I clinical trial of T-20 treatment for HIV-infected patients thus provided a unique opportunity to evaluate the emergence of resistant virus in vivo to this novel class of antiretroviral agents. All four patients who received an intermediate dose of T-20 (30 mg twice daily) had an initial decline in plasma viral load over the first 10 days but a rising trend by day 14, suggestive of selection for resistant virus. Plasma virus derived from patients enrolled in all dosage groups of the phase I T-20 trial was analyzed by population sequencing before and after treatment. While no mutations were found within a highly conserved 3-amino-acid sequence (GIV) known to be critical for fusion at baseline, after 14 days of therapy, virus from one patient in the 30-mg dose group (30-1) developed a mutation in this motif, specifically an aspartic acid (D) substitution for glycine (G) at position 36. Multiple env clones were derived from the plasma virus of all four patients in the 30-mg dosage group. Sequence analysis of 49 clones derived from the plasma of patient 30-1 on day 14 revealed that 25 clones contained the G36D mutation, while 8 contained the V38A mutation. Dual mutations involving G36D and other residues within the HR1 domain were also identified. In 5 of the 49 env clones, other mutations involving residues 32 (Q32R or Q32H) and 39 (Q39R) were found in combination with G36D. Cloned env sequences derived from the plasma virus of subject 30-3 also had single mutations in the GIV sequence (V38M and I37V) detectable following therapy with T-20. The plasma virus from subjects 30-2 and 30-4 did not contain changes within the GIV sequence. To analyze the biological resistance properties of these mutations, we developed a novel single-cycle HIV-1 entry assay using JC53BL cells which express ␤-galactosidase and luciferase under control of the HIV-1 long terminal repeat. Full-length env clones were derived from the plasma virus of patients 30-1 and 30-3 and used to generate pseudotyped virus stocks. The mean 50% inhibition concentrations (IC 50 s) for mutants G36D and V38A (patient 30-1) were 2.3 g/ml and 11.2 g/ml, respectively, statistically significant increases of 9.1-and 45-fold, respectively, compared with those of wild-type Env. The IC 50 for the V38 M mutation (patient 30-3) was 7.6 g/ml, an 8-fold increase compared with that of the wild type. The I37V mutation resulted in an IC 50 3.2-fold greater than that of the wild type. Envs with double mutations (Q32R plus G36D and Q32H plus G36D) exhibited a level of resistance similar to that of G36D alone. These findings provide the first evidence for the rapid emergence of clinical resistance to a...

Fathers' total and percentage body fat were predictors of changes in body fat of premenarcheal girls during a 2. 7-y period.

Pain is the most frequent and disabling complication of herpes zoster. The analysis of pain severity data is complicated by the nonlinear rate of resolution. Further, three distinct phases characterize pain resolution--acute, subacute, and chronic. Using two clinical trial datasets as the bases for analyses, the rates of baseline pain resolution were computed across each of three phases and compared for age, severity of pain at onset, and number of lesions at baseline. The results defined transition points of 24.4 +/- 3.34 for the subacute phase and 110.3 +/- 11.9 days for the chronic phase. The model demonstrated a treatment effect of valiciclovir (VACV) during the subacute phase as compared to acyclovir (ACV) (P = 0.006) and supports effects of age, baseline pain and number lesions on pain cessation rates in the acute phase. This model verifies three phases of zoster pain and delineates the impact of treatment and other factors on the phase-specific rates of pain cessation.

Tumor-targeted drug delivery is an attractive strategy in cancer treatment. We have previously reported a paclitaxel model conjugate using a bombesin receptor-recognizing peptide in which the drug cytotoxicity against H1299 human nonsmall cell lung cancer was enhanced compared to unconjugated taxol. In an effort to expand the development of tumor-recognizing taxanes, paclitaxel (PTX, taxol) was conjugated to the anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody (MAb) Erbitux (C225) to serve as a model MAb-mediated drug delivery compound. Thus, paclitaxel was derivatized at its 2'-hydroxy function by introduction of a succinate linker, and the carboxyl group of the latter was covalently attached to C225 through amide bond formation. The final product conjugate (PTXC225) was analyzed mass spectrometrically for assessment of the drug-to-antibody ratios. Cytotoxicity screening of the drug-antibody conjugate against A431, UM-SCC-1, and UM-SCC-6 cells indicated an enhancement in cytocidal effect of paclitaxel as compared to those of the free drug, the intact antibody, and a physical mixture of the two (the controls). In A431 cells, the conjugate showed 25.2% +/- 2.2% of apoptosis induction as compared to little or no apoptosis caused by the controls. Biodistribution analysis of the PTXC225 in tumor-implanted nude mice and a tyrosine-kinase assay showed that conjugation of the drug did not interfere with the immunoreactivity of the antibody. The 24-h tumor uptake of C225 and PTXC225 were 11.7% +/- 6.0% and 7.1% +/- 3.6% of the injected dose per gram of tissue (%ID/g), respectively, which were not significantly different. Also, in A431-implanted nude mice, the conjugate and C225 showed tumor growth inhibition effects of 57.2% and 41.2%, respectively, against a saline-treated control, which were not significantly different from each other. This lack of difference in the in vivo antitumor activity of the MAb-delivered drug and free PTX may be due to either a relatively low dose of the antibody-delivered drug (346 microg/kg), or an untimely release of it, or both. The tumor growth inhibition pattern of the conjugate, however, was identical to that of C225, indicating that the attachment of PTX did not affect the antigen-binding and growth inhibitory features of the MAb. These preliminary results demonstrate the potential of tumor-targeted delivery of taxol as a promising strategy in cancer treatment and warrant further work to develop more suitable drug-MAb linkers as well as improved dosage and treatment protocols.

The purpose of this phase I study was to determine the potential efficacy of adenoviral-mediated suicide gene therapy in women with recurrent ovarian cancer. Fourteen patients were treated intraperitoneally with herpes simplex virus-thymidine kinase (HSV-TK)-encoding adenovirus (AdHSV-TK) in dosages ranging between 1x10(9) and 1x10(11) pfu. Beginning 2 days later, ganciclovir (GCV) was administered intravenously at a dose of 5 mg/kg bid for 14 days. Transient vector-associated fever was experienced by 4 of 14 (29%) treated patients. Other possible vector-associated constitutional symptoms, abdominal pain, and gastrointestinal symptoms were experienced by 6 of 14 (43%) treated patients. No other dose-limiting vector-specific side effects were noted. Of the 13 patients evaluable for response, 5 (38%) had stable disease and 8 (62%) had evidence of progressive disease. Molecular analysis of evaluable ascites samples demonstrated the presence of transgene DNA and RNA in most patients 2 days following Ad HSV-TK administration. Ten of 11 evaluable patients had an increase in anti-adenovirus antibody titer. These results suggest that treatment with AdHSV-TK in combination with GCV is feasible in the context of human ovarian cancer and tolerated at the dosages studied.