Preclinical to clinical translation of CNS transporter occupancy of TD-9855, a novel norepinephrine and serotonin reuptake inhibitor.

BACKGROUND: Monoamine reuptake inhibitors exhibit unique clinical profiles that reflect distinct engagement of the central nervous system (CNS) transporters. METHODS: We used a translational strategy, including rodent pharmacokinetic/pharmacodynamic modeling and positron emission tomography (PET) imaging in humans, to establish the transporter profile of TD-9855, a novel norepinephrine and serotonin reuptake inhibitor. RESULTS: TD-9855 was a potent inhibitor of norepinephrine (NE) and serotonin 5-HT uptake in vitro with an inhibitory selectivity of 4- to 10-fold for NE at human and rat transporters. TD-9855 engaged norepinephrine transporters (NET) and serotonin transporters (SERT) in rat spinal cord, with a plasma EC50 of 11.7 ng/mL and 50.8 ng/mL, respectively, consistent with modest selectivity for NET in vivo. Accounting for species differences in protein binding, the projected human NET and SERT plasma EC50 values were 5.5 ng/mL and 23.9 ng/mL, respectively. A single-dose, open-label PET study (4-20mg TD-9855, oral) was conducted in eight healthy males using the radiotracers [(11)C]-3-amino-4- [2-[(di(methyl)amino)methyl]phenyl]sulfanylbenzonitrile for SERT and [(11)C]-(S,S)-methylreboxetine for NET. The long pharmacokinetic half-life (30-40 h) of TD-9855 allowed for sequential assessment of SERT and NET occupancy in the same subject. The plasma EC50 for NET was estimated to be 1.21 ng/mL, and at doses of greater than 4 mg the projected steady-state NET occupancy is high (>75%). After a single oral dose of 20mg, SERT occupancy was 25 (±8)% at a plasma level of 6.35 ng/mL. CONCLUSIONS: These data establish the CNS penetration and transporter profile of TD-9855 and inform the selection of potential doses for future clinical evaluation.

In vivo pharmacological characterization of TD-4208, a novel lung-selective inhaled muscarinic antagonist with sustained bronchoprotective effect in experimental animal models.

Tiotropium is currently the only once-daily, long-acting muscarinic antagonist (LAMA) approved in the United States and other countries for the treatment of chronic obstructive pulmonary disease (COPD). Glycopyrronium has shown promise as a LAMA and was recently approved for once-daily maintenance treatment of COPD in the European Union. Here, we describe the in vivo preclinical efficacy and lung selectivity of a novel inhaled muscarinic antagonist, TD-4208 (biphenyl-2-ylcarbamic acid 1-(2-{[4-(4-carbamoylpiperidin-1-ylmethyl)benzoyl]methylamino}ethyl)piperidin-4-yl ester) and compare its profile to tiotropium and glycopyrronium. In anesthetized dogs, TD-4208, along with tiotropium and glycopyrronium, produced sustained inhibition of acetylcholine-induced bronchoconstriction for up to 24 hours. In anesthetized rats, inhaled TD-4208 exhibited dose-dependent 24-hour bronchoprotection against methacholine-induced bronchoconstriction. The estimated 24-hour potency (expressed as concentration of dosing solution) was 45.0 µg/ml. The bronchoprotective potencies of TD-4208 and tiotropium were maintained after 7 days of once-daily dosing, whereas glycopyrronium showed a 6-fold loss in potency after repeat dosing. To assess systemic functional activity using a clinically relevant readout, the antisialagogue effect of compounds was also evaluated. The calculated lung selectivity index (i.e., ratio of antisialagogue and bronchoprotective potency) of TD-4208 was superior to glycopyrronium after both single and repeat dosing regimens and was superior to tiotropium after repeat dosing. In conclusion, the in vivo preclinical profile suggests that TD-4208 has the potential to be a long-acting bronchodilator for once-daily treatment of respiratory diseases. Its greater functional selectivity for the lung in preclinical models may translate to an improved tolerability profile compared with marketed muscarinic receptor antagonists.

Detection of human papilloma virus in paraffin-embedded tissue using the polymerase chain reaction.

Human papilloma virus (HPV) DNA sequences have been detected in paraffin-embedded tissue using an enzymatic in vitro amplification technique known as the polymerase chain reaction. Amplification of a HPV DNA sequence before its detection with a cDNA probe significantly increases the rapidity as well as the sensitivity of detection such that a single 5-10-micron thick paraffin-embedded tissue section can be analyzed within 24 h. The assay specifically detected HPV 16 or 18 without crossreactivity with HPV 6 or 11. As few as 20 viral copies could be detected. The rapid and sensitive analysis of HPV in normal and pathological tissues using this technique may contribute significantly to identifying the role of HPV as a risk factor in carcinoma.

Cell to cell interaction in the immune response. IV. Site of action of antilymphocyte globulin.

In this series of papers it has been shown that the immune response of mice to sheep erythrocytes requires the participation of two classes of lymphoid cells. Thymus-derived cells initially react with antigen and then interact with another class of cells, the antibody-forming cell precursors, to cause their differentiation to antibody-forming cells. Antilymphocyte globulin depressed the ability of mice to respond to sheep erythrocytes. This effect was more marked when the antigen was injected intraperitoneally than intravenously, and occurred only when the antilymphocyte globulin was given before or simultaneously with antigen. Injection of thymus cells restored to near normal the ability to respond to an intravenous injection of sheep erythrocytes. Spleen cells from antilymphocyte globulin-treated mice gave a weak adoptive immune response in irradiated recipients. The addition of thymus cells however enabled a response similar to that given by normal spleen cells. When thymectomized irradiated recipients were used, normal spleen cells continued to give a higher response to a challenge of sheep erythrocytes at 2 and 4 wk postirradiation than did spleen cells from ALG-treated donors. This result is more consistent with the notion that thymus-derived target cells are eliminated, rather than temporarily inactivated, by antilymphocyte globulin. These findings suggest that, in vivo, antilymphocyte globulin acts selectively on the thymus-derived antigen-reactive cells.

Lung tumor-associated derepressed alloantigen coded for by the K region of the H-2 major histocompatibility complex.

Transplacental induction of lung tumors in C3HfeB/HeN (C3Hf) strain mice can be readily achieved with the carcinogen 1-ethyl-1-nitrosourea. Several of these tumors express, as a tumor-associated transplantation antigen (TATA), a normal tissue alloantigen present in strain A and C3H/HeN (C3H) mice. In the present study it was shown that the tumor-associated alloantigen on the C3Hf-derived lung tumor 85 was present in all mice of H-2(a) and H-2(k) haplotypes tested and in CBA (532) strain mice (H-2(ka) haplotype). Studies using congenic-resistant and recombinant strains of mice indicated that the genetic locus controlling the expression of this antigen was either within or to the left of the H-2K region of the major histocompatibility complex (MHC). Thus the antigen was expressed in B10.A (4R) mice (kkbbbb MHC haplotype) but not in B10 (bbbbbb) or B10.AQR mice (qkkddd). The antigen was expressed in all tissues tested of C3H and A strain mice. It was not detected on any tissue tested including embryo tissue of C3Hf mice or mice of MHC haplotype other than H-2(k) or H-2(a). Because C3Hf strain mice were originally derived from C3H strain mice (H-2(k)), the MHC haplotype of C3Hf mice has been provisionally designated H-2(kb). The finding of a tumor-associated change in the expression of a H-2K region-coded antigen is consistent with the concept that MHC-coded antigens may act as targets for immunological surveillance of tumors.

Histocompatibility type and immune responsiveness in random bred Hartley strain guinea pigs.

Outbred Hartley strain guinea pigs capable of responding immunologically to 2,4-dinitrophenylated poly-L-lysine were shown to display a histocompatibility specificity in common with inbred strain 2 guinea pigs. This histocompatibility specificity was not detected in guinea pigs unable to respond immunologically to DNP-PLL. The result suggests that the poly-L-lysine specific immune response gene is very closely linked or even identical with a gene determining a major histocompatibility antigen in guinea pigs.

Hemagglutinin-specific cytotoxic T-cell response during influenza infection.

Specific cytotoxic thymus-derived (T) lymphocytes were detected in the cervical lymph nodes and spleen during influenza infection of mice. The cytotoxic T cells can distinguish target cells infected with different influenza A subtypes. Infection with parent viruses and their recombinant progeny possessing the hemagglutinin of one parent and the neuraminidase of the other demonstrated that significant cytotoxicity occurred only when the hemagglutinin of the immunizing viruses was the same as that of the virus used to infect the target cell. In addition to this specific cytotoxic response to the major surface antigen, a cross-reactive response could be detected when the relatively nonpermissive L cell was used as the target cell. These results indicate there is a specific cytotoxic T-cell response to the surface hemagglutinin, and a cross-reactive cytotoxic response, not directed to the hemagglutinin, during influenza infection. The cytotoxic T-cell response specific for the hemagglutinin antigen may play an important role in in vivo immunity to influenza.

Molecular characterization of the C3HfB/HeN H-2Kkm2 mutation. Implications for the molecular basis of alloreactivity.

The C3HfB/HeN (C3Hf) mouse strain expresses an H-2Kk molecule, previously denoted H-2Kkv1, that is structurally and functionally distinct from H-2Kk of the parental C3H strain. By molecular genetic analysis, we demonstrate that the C3Hf H-2K gene carries a homozygous coding region mutation relative to the C3H allele, revealing that C3Hf meets the requirements for assignment of a mutant haplotype, H-2km2. C3Hf H-2Kkm2 bears a single clustered substitution of four nucleotides within 14 contiguous nucleotides in exon 3. Since this sequence also is present intact at the homologous position in H-2Dk of both C3H and C3Hf, the origin of the H-2Kkm2 mutation is consistent with a nonreciprocal sequence transfer from the H-2Dk donor gene, analogous to the mechanism proposed for generation of the H-2Kb mutations. The H-2Kkm2 mutation encodes three clustered amino acid substitutions, at positions 95, 98, and 99, that map to one of the large beta strands at the bottom of the peptide antigen binding cleft of the H-2Kkm2 molecule. The nature and location of these amino acid substitutions are unique relative to any other known H-2 mutant or HLA variant, and underscore the importance of the beta-pleated sheet in influencing CTL recognition. These results indicate that H-2Kkm2 alloantigenicity may derive largely from altered presentation of self cellular peptides.

Markedly decreased expression of class I histocompatibility antigens, protein, and mRNA in human small-cell lung cancer.

We have found markedly deficient expression of the class I major histocompatibility antigens HLA-A,B,C and beta 2m on human small-cell lung cancer (SCLC) lines and fresh tumor samples. The deficit of HLA-A,B,C and beta 2-microglobulin (beta 2m) antigen expression was demonstrated with both radiobinding assays and indirect immunofluorescence assays. Immunoprecipitation of metabolically labeled cells with antibodies to class I antigens showed most SCLC lines to have synthesized almost no beta 2m and HLA-A,B,C proteins. Northern blot analysis, using human HLA-A,B, and beta 2m cDNA probes, showed that almost all SCLC lines tested had markedly decreased amounts of HLA and beta 2m mRNA, but both gene products could be induced with interferon treatment of SCLC lines. We conclude that human SCLC, in contrast to other lung cancer types, is characterized by greatly reduced transcription of HLA-A,B,C and beta 2m genes, which suggests the existence of a mechanism for evading the host immune response to the tumor and of an E1a-like product in this type of tumor cell.

Cell-to-cell interaction in the immune response. VII. Requirement for differentiation of thymus-derived cells.

Experiments were designed to test the possibility that thymus-derived (T) cells cooperate with nonthymus derived (B) cells in antibody responses by acting as passive carriers of antigen. Thoracic duct lymphocytes (TDL) from fowl gammaG-tolerant mice were incubated in vitro with fowl anti-mouse lymphocyte globulin (FALG), which was shown not to be immunosuppressive in mice. On transfer into adult thymectomized, irradiated, and marrow protected (TxBM) hosts together with a control antigen, horse RBC, a response to horse RBC but not to fowl gammaG was obtained. By contrast, TxBM recipients of nontolerant, FALG-coated TDL responded to both antigens and the antibody-forming cells were shown to be derived from the host, not from the injected TDL. These findings suggested that, under the conditions of the experiment, triggering of unprimed B cells in the spleens of TxBM hosts was not achieved with antigen-coated tolerant lymphocytes. Another model utilized the ability of B cells to bind antibody-antigen complexes. Spleen cells from TxBM mice, incubated in vitro with anti-fowl gammaG-fowl gammaG.NIP, were injected with or without normal TDL (a source of T cells) into irradiated hosts. Only mice given both cell types could produce an anti-NIP antibody response. In a further experiment, spleen cells from HGG.NIP-primed mice were injected together with NIP-coated B cells (prepared as above) into irradiated hosts. A substantial anti-NIP antibody response occurred. If, however, the T cells in the spleens of HGG.NIP-primed mice were eliminated by treatment with anti-theta serum and complement, the NIP response was abolished. It was concluded that antigen-coated B cells could not substitute for T cells either in the primary or secondary response. Treatment of T cells from unprimed or primed mice with mitomycin C impaired their capacity to collaborate with B cells on transfer into irradiated hosts. Taken together these findings suggest that before collaboration can take place T cells must be activated by antigen to differentiate and in so doing may produce some factor essential for triggering of B cells.

Derepressed alloantigen in transplacentally induced lung tumor coded for by H-2 linked gene.

A transplacentally induced lung tumor of strain C3Hf mice grows progressively when transplanted to (C3Hf X A)F1 hybrid mice but not when transplanted to C3Hf recipients. Progressive tumor growth occurs in [(C3Hf X A)F1 X C3Hf] backcross mice inheriting the H-2a haplotype from the F1 parent. Furthermore, radioresistant immunity to the lung tumor can be induced in C3Hf mice by immunization with normal tissue of B10.A and B10.A(2R) but not of B10 or B10.A(5R) strain mice.

Cell-adhesive motif in region II of malarial circumsporozoite protein.

The segment of the malarial circumsporozoite (CS) protein designated Region II is highly conserved among different malarial species. A similar sequence is also present in several other proteins, including thrombospondin, properdin, and a blood-stage antigen of Plasmodium falciparum. By means of peptides synthesized from sequences of the Plasmodium vivax CS protein in the vicinity of Region II, it was found that two overlapping 18- to 20-amino acid peptides promoted the adhesion of a variety of human hematopoietic cell lines. The amino acid sequence valine-threonine-cysteineglycine (VTCG), contained within this common motif, was shown to be the critical sequence for the observed cell-adhesive properties.

Impaired nociception and pain sensation in mice lacking the capsaicin receptor.

The capsaicin (vanilloid) receptor VR1 is a cation channel expressed by primary sensory neurons of the "pain" pathway. Heterologously expressed VR1 can be activated by vanilloid compounds, protons, or heat (>43 degrees C), but whether this channel contributes to chemical or thermal sensitivity in vivo is not known. Here, we demonstrate that sensory neurons from mice lacking VR1 are severely deficient in their responses to each of these noxious stimuli. VR1-/- mice showed normal responses to noxious mechanical stimuli but exhibited no vanilloid-evoked pain behavior, were impaired in the detection of painful heat, and showed little thermal hypersensitivity in the setting of inflammation. Thus, VR1 is essential for selective modalities of pain sensation and for tissue injury-induced thermal hyperalgesia.

Pain: nocistatin spells relief.

Tissue or nerve injury can dramatically alter the transmission of sensory stimuli by spinal cord neurons, so that a light touch produces pain. The discovery that peptide products of prepronociceptin processing either facilitate or inhibit these mechanisms suggests novel approaches to treating these conditions.

Role of fibronectin in Pneumocystis carinii attachment to cultured lung cells.

Attachment of pathogens to host cells is a prerequisite for the development of many infections. Pneumocystis carinii (PC) pneumonia is characterized by attachment of PC trophozoites to the alveolar epithelium. The mechanism of this process is unknown. Fibronectin (Fn) is a glycoprotein present in the alveolar space known to mediate cell-cell attachment, including the attachment of certain pathogens to host epithelial cells. In this study the binding of Fn to PC trophozoites has been characterized in vitro using 125I-Fn. Fn binds saturably and specifically to 6.4 x 10(5) binding sites per organism with an apparent binding constant, Kd, of 1.2 x 10(-8) M. Fn binding to PC was inhibited by the addition of Arg-Gly-Asp-Ser (RGDS), a tetrapeptide containing the active site of the cell-binding domain of Fn. PC attachment to an alveolar epithelial cell line was quantified using 51Cr-labeled PC trophozoites. Attachment was decreased from 24 +/- 1.9% to 12.1 +/- 1% (P less than 0.01) by the addition of an anti-Fn antibody, an effect that could be overcome by the addition of excess free Fn. It is concluded that binding of Fn to PC may be an important initial step in the attachment of the organism to alveolar epithelial cells. Furthermore, it appears that PC recognizes and binds to the RGDS cell attachment site of Fn.

Pneumocystis carinii: inhibition of lung cell growth mediated by parasite attachment.

Pneumocystis carinii pneumonia is a significant cause of mortality in immunocompromised patients. Current concepts suggest that attachment of P. carinii to alveolar epithelium is required for development of pneumonia. We examined the mechanism of P. carinii adherence to cultured A549 cells, a permanent cell line derived from human alveolar epithelium. P. carinii adherence was quantified by measuring attachment of 51Cr-labeled P. carinii to cultured A549 cells. After 8 h of incubation, 37.4 +/- 4.2% of P. carinii were adherent to A549 cells. In the presence of agents known to impair cytoskeletal function, including 10(-5) M cytochalasin B, 10(-5) M colchicine, and 10(-5) M trimethylcolchicinic acid (TMCA), adherence was decreased from 57.4 +/- 4.2% to 9.3 +/- 3.4%, 12.5 +/- 3.6%, and 21.5 +/- 3.6%, respectively (P less than 0.01, all comparisons). Secondly, we examined the effect of P. carinii on the function of A549 cells. P. carinii resulted in significant impairment of A549 cell growth, indicating P. carinii adversely affected the function of target lung cells. A P. carinii:A549 cell ratio of 50:1 resulted in 43.5 +/- 2.9% inhibition of A549 cell growth (P less than 0.001). Additionally, TMCA, which significantly prevented attachment of P. carinii, reversed the impairment of A549 cell growth. These data demonstrate that P. carinii attachment to cultured lung cells can be quantified, is dependent on intact cytoskeletal function and is necessary for impairment of lung cell replication.

Mechanism of Pneumocystis carinii attachment to cultured rat alveolar macrophages.

Pneumocystis carinii (PC) pneumonia begins as an intra-alveolar process resulting in injury to the alveolar epithelium with subsequent invasion of the lung interstitium. The clearance of PC organisms from the alveolar space is a critical function of alveolar macrophages (AM), the resident alveolar phagocytic cells. In this study the mechanism of PC attachment to AM was determined using 51Cr-labeled organisms, with PC attachment reaching a maximum of 18.9 +/- 2.5% after 4 h. Attachment was significantly decreased by preincubation of the AM with a monoclonal anti-fibronectin antibody directed against the cell attachment site of fibronectin (from 17.8 +/- 2.2% to 8.3 +/- 1.0%, P less than 0.01), or by addition of the fibronectin cell binding site analogue Arg-Gly-Asp-Ser (RGDS) (from 18.1 +/- 2.3% to 2.9 +/- 0.8%, P less than 0.01). An anti-fibronectin monoclonal antibody directed against the heparin binding domain of fibronectin had no effect on PC attachment. Addition of the specific calcium ion chelating agent EGTA to the culture media similarly decreased attachment from 16.9 +/- 2.0% to 5.1 +/- 1.1% (P less than 0.01). Fibronectin-mediated attachment of PC to AM did not result in phagocytosis of the organisms by the AM as determined by chemiluminescence measurements. Therefore, the data indicate that PC attachment to AM is a calcium-dependent process mediated by the cell binding domain of fibronectin which does not trigger a phagocytic response by the AM.

Pneumocystis carinii attachment to cultured lung cells by pneumocystis gp 120, a fibronectin binding protein.

Pneumocystis carinii is an extracellular organism which is thought to require attachment to alveolar epithelial cells for its growth and replication in humans. Fibronectin (Fn) binding to P. carinii is essential for optimal P. carinii attachment. This study demonstrates that gp120, a 110-120-kD membrane glycoprotein on P. carinii, mediates attachment of the organism to cultured lung cells and is the site of Fn binding to P. carinii. A 51Cr-labeled P. carinii binding assay was used to quantify attachment of the organism to the alveolar epithelial cell line A549. Addition of free gp120, purified from whole P. carinii organisms, caused a significant decrease in attachment of P. carinii to A549 cells from 44.2 +/- 5.5% to 22.4 +/- 4.2% (P less than 0.01). Preincubation of the P. carinii organisms with a polyclonal antibody to gp120 also resulted in a marked decrease in P. carinii attachment to A549 cells from 46.8% +/- 5.2% to 21.3 +/- 4.8% (P less than 0.01). Furthermore, addition of free gp120 to P. carinii organisms caused a significant reduction in specific binding of 125I-Fn to P. carinii (from 83.3 +/- 8.5 ng to 47.1 +/- 5.9 ng, P less than 0.01). Similarly, anti-gp 120 antibody decreased specific Fn binding to P. carinii from 74.3 +/- 8.4 ng to 25.5 +/- 5.3 ng (P less than 0.001). Solubilized P. carinii organisms separated by gel electrophoresis and blotted with 125I-Fn demonstrated specific binding of the 125I-Fn to gp120. In addition, a specific anti-beta 1-integrin antiserum reacted with gp120 by Western blot, suggesting structural homology between gp120 and the beta-subunit of integrins. Thus, the data suggest that the P. carinii membrane glycoprotein gp120 functions as a Fn binding protein and is required for optimal P. carinii attachment to alveolar epithelial cells.

Oxidant injury of lung parenchymal cells.

Hyperoxia and paraquat ingestion are two clinical examples of lung injury thought to be mediated by oxidant mechanisms. An in vitro cytotoxicity assay using freshly explanted 51Cr-labeled lung tissue as the target was used to quantify the ability of hyperoxia and paraquat to directly injure lung parenchymal cells in an environment where indirect mechanisms such as recruitment of inflammatory cells were not possible. There are clear species differences in the susceptibility of lung parenchyma to direct injury by hyperoxia (95% O2) and paraquat (10 microM--10 mM) for 18 h at 37 degrees C, with human and rat lung being more sensitive than rabbit lung. Oxygen radical inhibitors, particularly catalase (1,100 U/ml) and alpha-tocopherol (10 micrograms/ml), reduced hyperoxia and paraquat-induced lung injury, although their ability to do so depended on the oxidant and the species. The simultaneous use of hyperoxia and paraquat accelerated the in vitro lung parenchymal cell injury in each species tested. These studies demonstrate that both oxygen and paraquat can directly injure the cells of the lower respiratory tract without enlisting the aid of additional blood-derived inflammatory cells. In addition, the 51Cr-labeled lung explant assay used for these studies allows for the quantitative assessment of direct lung cell injury and thus may prove useful as an in vitro model by which to investigate lung injury of other etiologies.

Surfactant protein A suppresses reactive nitrogen intermediates by alveolar macrophages in response to Mycobacterium tuberculosis.

Mycobacterium tuberculosis attaches to, enters, and replicates within alveolar macrophages (AMs). Our previous studies suggest that surfactant protein A (SP-A) can act as a ligand in the attachment of M. tuberculosis to AMs. Reactive nitrogen intermediates (RNIs) play a significant role in the killing of mycobacteria. We have demonstrated that RNI levels generated by AMs were significantly increased when interferon-gamma-primed AMs were incubated with M. tuberculosis. However, the RNI levels were significantly suppressed in the presence of SP-A (10 microg/ml). The specificity of SP-A's effect was demonstrated by the use of F(ab')2 fragments of anti-SP-A monoclonal antibodies and by the use of mannosyl-BSA, which blocked the suppression of RNI levels by SP-A. Furthermore, incubation of deglycosylated SP-A with M. tuberculosis failed to suppress RNI by AMs, suggesting that the oligosaccharide component of SP-A, which binds to M. tuberculosis, is necessary for this effect. These results show that SP-A-mediated binding of M. tuberculosis to AMs significantly decreased RNI levels, suggesting that this may be one mechanism by which M. tuberculosis diminishes the cytotoxic response of activated AMs.