A phase I combination study of olaparib with cisplatin and gemcitabine in adults with solid tumors.

PURPOSE: To determine the safety and tolerability of olaparib with cisplatin and gemcitabine, establish the maximum tolerated dose (MTD), and evaluate the pharmacodynamic and pharmacokinetic profile of the combination. EXPERIMENTAL DESIGN: We conducted a phase I study of olaparib with cisplatin and gemcitabine in patients with advanced solid tumors. Treatment at dose level 1 (DL1) consisted of olaparib 100 mg orally every 12 hours on days 1 to 4, gemcitabine 500 mg/m(2) on days 3 and 10, and cisplatin 60 mg/m(2) on day 3. PAR levels were measured in peripheral blood mononuclear cells (PBMC). RESULTS: Dose-limiting toxicities (DLT) in two of three patients at DL1 included thrombocytopenia and febrile neutropenia. The protocol was amended to enroll patients treated with ≤ 2 prior severely myelosuppressive chemotherapy regimens and treated with olaparib 100 mg once daily on days 1 to 4 (DL-1). No DLTs were seen in six patients at DL-1. Because of persistent thrombocytopenia and neutropenia following a return to DL1, patients received 100 mg olaparib every 12 hours on day 1 only. No hematologic DLTs were observed; nonhematologic DLTs included gastrointestinal bleed, syncope, and hypoxia. Of 21 patients evaluable for response, two had partial response. Olaparib inhibited PARP in PBMCs and tumor tissue, although PAR levels were less effectively inhibited when olaparib was used for a short duration. CONCLUSIONS: Olaparib in combination with cisplatin and gemcitabine is associated with myelosuppression even at relatively low doses. Modified schedules of olaparib in chemotherapy naive patients will have to be explored with standard doses of chemotherapy.

A phase I study of PF-04929113 (SNX-5422), an orally bioavailable heat shock protein 90 inhibitor, in patients with refractory solid tumor malignancies and lymphomas.

PURPOSE: To determine the maximum tolerated dose (MTD), toxicities, and pharmacokinetic/pharmacodynamic profile of the Hsp90 inhibitor PF-04929113 (SNX-5422) in patients with advanced solid tumors and lymphomas. METHODS: This was a single-institution, phase I, dose-escalation study of PF-04929113 administered twice weekly. Endpoints included determination of dose-limiting toxicities (DLT), MTD, the safety profile of PF-04929113, pharmacodynamic assessment of PF-04929113 on Hsp70 induction, pharmacokinetic analysis of PF-04928473 (SNX-2112) and its prodrug PF-04929113, and assessment of response. RESULTS: Thirty-three patients with advanced malignancies were treated. Dose escalation was continued up to 177 mg/m(2) administered orally twice a week. One DLT (nonseptic arthritis) was noted. No grade 4 drug-related adverse events were seen; grade 3 adverse events included diarrhea (9%), nonseptic arthritis (3%), aspartate aminotransferase elevation (3%), and thrombocytopenia (3%). No objective responses were seen in 32 evaluable patients. Fifteen patients (47%) had stable disease; 17 patients (53%) had progressive disease. Pharmacokinetic data revealed rapid absorption, hepatic, and extrahepatic clearance, extensive tissue binding, and almost linear pharmacokinetics of the active drug PF-04928473. Pharmacodynamic studies confirmed inhibition of Hsp90 and a linear correlation between pharmacokinetic parameters and Hsp70 induction. CONCLUSIONS: PF-04929113 administered orally twice a week is well tolerated and inhibits its intended target Hsp90. No objective responses were seen, but long-lasting stabilizations were obtained. Although no clinically significant drug-related ocular toxicity was seen in this study, the development of PF-04929113 has been discontinued because of ocular toxicity seen in animal models and in a separate phase I study.

A study of the specificity of lymphocytes in nevirapine-induced skin rash.

Nevirapine treatment can cause a skin rash. We developed an animal model of this rash and determined that the 12-hydroxylation metabolic pathway is responsible for the rash, and treatment of animals with 12-OH-nevirapine also leads to a rash. In the present study, we investigated the specificity of lymphocytes in nevirapine-induced skin rash. Brown Norway rats were treated with nevirapine or 12-OH-nevirapine to induce a rash. Lymph nodes were removed, and the response of lymphocytes to nevirapine and its metabolites/analogs was determined by cytokine production (enzyme-linked immunosorbent assay, enzyme-linked immunosorbent spot assay, and Luminex) and proliferation (alamar blue assay). Subsets of lymphocytes were depleted to determine which cells were responsible for cytokine production. Lymphocytes from animals rechallenged with nevirapine proliferated to nevirapine, but not to 12-OH-nevirapine or 4-chloro-nevirapine. They also produced interferon-gamma (IFN-gamma) when exposed to nevirapine, significantly less when exposed to 4-chloro-nevirapine, and very little when exposed to 12-OH-nevirapine, even though oxidation to 12-OH-nevirapine is required to induce the rash. Moreover, the specificity of lymphocytes from 12-OH-nevirapine-treated rats was the same, i.e., responding to nevirapine more than to 12-OH-nevirapine, even though these animals had never been exposed to nevirapine. A Luminex immunoassay showed that a variety of other cytokines/chemokines were also produced by nevirapine-stimulated lymphocytes. CD4(+) cells were the major source of IFN-gamma. The specificity of lymphocytes in activation assays cannot be used to determine what initiated an immune response. This has significant implications for understanding the evolution of an immune response and the basis of the pharmacological interaction hypothesis.

Bioactivation of minocycline to reactive intermediates by myeloperoxidase, horseradish peroxidase, and hepatic microsomes: implications for minocycline-induced lupus and hepatitis.

Of the tetracyclines, minocycline is unique in causing a significant incidence of a lupus-like syndrome and autoimmune hepatitis. It is also unique among the tetracyclines in having a para-N,N-dimethylaminophenol ring. Many drugs that cause autoimmune reactions are oxidized to reactive metabolites by the myeloperoxidase (MPO) system of macrophages. In this study, we showed that minocycline is oxidized to reactive intermediates by MPO/H(2)O(2)/Cl(-), HOCl, horseradish peroxidase/H(2)O(2), or hepatic microsomes. When trapped with N-acetylcysteine (NAC), two adducts with protonated molecular ions at m/z 619 were isolated and analyzed by NMR. One represents attack of the aromatic D ring by NAC meta to the N,N-dimethylamino group, which implies that the reactive intermediate was a quinone iminium ion. The NMR of the other adduct, which was not observed when minocycline was oxidized by hepatic microsomes, indicates that the NAC is attached at the junction of the B and C rings. In the oxidation by HOCl, we found an intermediate with a protonated molecular ion of m/z 510 that represents the addition of HOCl to minocycline. The HOCl presumably adds across the double bond of the B ring, and reaction of this intermediate with NAC led to the second NAC adduct. We were surprised to find that the same NAC adduct was not observed after oxidation of tetracycline with HOCl, even though this part of the tetracycline structure is the same as for minocycline. We propose that one or more of these reactive metabolites are responsible for the idiosyncratic drug reactions that are specific to this tetracycline.

Covalent binding of penicillamine to macrophages: implications for penicillamine-induced autoimmunity.

Idiosyncratic drug reactions (IDRs) represent a major clinical problem, and at present, the mechanisms involved are still poorly understood. One animal model that we have used for mechanistic studies of IDRs is penicillamine-induced autoimmunity in Brown Norway (BN) rats. Previous work in our lab found that macrophage activation preceded the clinical autoimmune syndrome. It is thought that one of the interactions between T cells and macrophages involves reversible Schiff base formation between an amine on T cells and an aldehyde on macrophages, but the identity of the molecules involved is unknown. It is also known that penicillamine reacts with aldehyde groups to form a thiazolidine ring, which unlike a Schiff base, is essentially irreversible. Such binding could lead to macrophage activation. Generalized macrophage activation could lead to the observed autoimmune reaction. Hydralazine and isoniazid also react with aldehydes to form stable hydrazones, and they also cause an autoimmune lupus-like syndrome. In this study, isolated spleen cells from male BN rats were incubated with biotin-aldehyde-reactive probe (ARP, a hydroxylamine), biotin-hydrazide, or D-penicillamine. At all concentrations, ARP, hydrazide, and penicillamine preferentially "stained" macrophages relative to other spleen cells. In addition, preincubation of cells with penicillamine or hydralazine decreased ARP staining of macrophages, which further indicates that most of the ARP binding to macrophages involves binding to aldehyde groups. This provides support for the hypothesis that the interaction between aldehyde-containing signaling molecules on macrophages and penicillamine could be the initial event of penicillamine-induced autoimmunity. Several of the proteins to which ARP binds were identified, and some such as myosin are attractive candidates to mediate macrophage activation.

Demonstration of the metabolic pathway responsible for nevirapine-induced skin rash.

The reverse transcriptase inhibitor, nevirapine (NVP), causes skin rashes and hepatotoxicity. We used a rat model to determine if the rash is caused by the parent drug or a reactive metabolite. By manipulation of metabolic pathways and testing analogues, we eliminated all but one pathway, 12-hydroxylation, which involves the oxidation of an exocyclic methyl group, as being responsible for the rash. Treatment with 12-OH-NVP caused a rash, and an analogue in which the methyl hydrogens were replaced by deuterium to inhibit the 12-OH pathway did not cause a rash; however, quite unexpectedly, blood levels of the deuterated analogue were very low. This is due to partitioning of the benzylic free radial intermediate between oxygen rebound to form 12-OH-NVP and loss of another hydrogen atom to form a reactive quinone methide, which inactivates P450. Cotreatment with the P450 inhibitor, 1-aminobenzotriazole, led to comparable levels of NVP and the deuterated analogue, and the deuterated analogue still caused a lower rash incidence. These data clearly point to the 12-hydroxy pathway being responsible for NVP skin rash. We propose that the hepatotoxicity of NVP in humans is due to the quinone methide formed by P450 in the liver, while the skin rash may be due to the quinone methide formed in the skin by sulfation of 12-OH metabolite followed by loss of sulfate. This is the first example in which a valid animal model of an idiosyncratic drug reaction was used to determine the metabolic pathway responsible for the reaction.

Study of the sequence of events involved in nevirapine-induced skin rash in Brown Norway rats.

Nevirapine, used for the treatment of HIV infection, is associated with development of skin rash and liver toxicity. The mechanism of these idiosyncratic reactions is unknown. We have previously reported the discovery of a new animal model of nevirapine-induced skin rash in rats. When treated with nevirapine, Brown Norway rats developed red ears on about day 7 and skin rash on about day 21. On rechallenge, ears turn red within 24 h, and skin lesions develop by day 9. In the current study, we analyzed the time course of the sequence of events involved in the development of skin rash. Rats were treated with nevirapine for 7, 14, or 21 days or rechallenged with it for 0, 1, or 9 days. This treatment led to an increase in the total number of auricular lymph node T, B, and macrophage cells. There was also an increase in the activation/infiltration marker ICAM-1 and activation/antigen presentation marker MHC II in these cells compared with those from control rats. Immunohistochemistry analysis showed macrophage infiltration and ICAM-1 expression in the ears of treated rats as early as day 7 of treatment. Macrophage infiltration preceded T cell infiltration, which was not apparent until the onset of rash. Both MHC I and MHC II expression increased in the skin of nevirapine-treated rats that developed rash. A major inducer of MHC is IFNgamma. Although rechallenge with nevirapine led to a large increase in serum levels of IFNgamma, this was not observed during the treatment of naïve rats with nevirapine. These observations provide further clues to the mechanism of nevirapine-induced skin rash.

Engineering d-amino acid containing novel protease inhibitors using catalytic site architecture.

The mechanism of proteolysis by serine proteases is a reasonably well-understood process. Typically, a histidine residue acting as a general base deprotonates the catalytic serine residue and the hydrolytic water molecule. We disclose here, the use of an unnatural d-amino acid as a strategic residue in P1 position, designed de novo based on the architecture of the protease catalytic site to impede the catalytic histidine residue at the stage of acyl-enzyme intermediate. Several probe molecules containing d-homoserine or its derivatives at P1 position are evaluated. Compounds 1, 6, and 8-10 produced up to 57% loss of activity against chymotrypsin. More potent and specific inhibitors could be designed with structure optimization as this strategy is completely general and can be used to design inhibitors against any serine or cysteine protease.