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Background All-trans retinoic acid (ATRA) is a biologically active isomer of retinoic acid (RA). Topical ATRA (retin-a, retin-a micro, atralin, renova, and avita) is the active pharmaceutical ingredient for FDA-approved treatments for acne and skin wrinkles. Oral formulations (Vesanoid) treat acute promyelocytic leukemia, but oral dosing can induce severe side effects. Despite benefits in various rodent models of inflammatory bowel disease (IBD), toxicity and controversial clinical observations have diminished enthusiasm for ATRA IBD clinical trials. To circumvent these issues and to use ATRA’s key role in maintaining gut tolerance, we developed a poly(lactic-co-glycolic acid) (PLGA) microsphere (MS) encapsulated ATRA formulation aimed at directing ATRA delivery to immune structures of the gut, limiting systemic exposure. Initially, ATRA MS was developed as a component of a combinatorial product (TreXTAM) that also contained encapsulated transforming growth factor (TGF)-β and ATRA in a 1:2 w/w ratio. Although the combination was optimal, benefit was also observed when ATRA MS was given alone in the CD4+ CD25-T-cell adoptive transfer (ACT) colitis model. Methods We used the ACT and DSS-induced murine models of colitis to expand on the dose-dependent effects of oral ATRA MS when given alone. The DSS model was also used to compare the efficacy of ATRA MS and soluble ATRA, while healthy animals were used to compare the pharmacokinetics of the two drugs. Results In both the ACT and DSS-induced murine models of colitis, ATRA MS was observed to be effective in ameliorating disease. ATRA MS was also observed to be more effective than soluble ATRA in these models and displayed more favorable pharmacokinetics. Conclusions We suggest ATRA MS, as a standalone product, may attenuate IBD and perhaps limit fibrosis, while limiting systemic side effects.

BACKGROUND TreXTAM® is a combination of the key regulatory cytokine transforming growth factor beta (TGFβ) and all trans retinoic acid (ATRA) microencapsulated for oral delivery to immune structures of the gut. It is in development as a novel treatment for inflammatory bowel disease (IBD). AIM To measure TGFβ levels in blood and tissue after oral administration of encapsulated TGFβ. METHODS Animals were orally administered encapsulated TGFβ by gavage. Levels of drug substance in blood and in gut tissues at various times after administration were measured by ELISA. RESULTS We made the surprising discovery that oral administration of TreXTAM dramatically (approximately 50%) and significantly (P = 0.025) reduced TGFβ levels in colon, but not small intestine or mesenteric lymph nodes. Similarly, levels in rat serum after 25 d of thrice weekly dosing with either TreXTAM, or microencapsulated TGFβ alone (denoted as TPX6001) were significantly (P < 0.01) reduced from baseline levels. When tested in the SCID mouse CD4+CD25- adoptive cell transfer (ACT) model of IBD, oral TPX6001 alone provided only a transient benefit in terms of reduced weight loss. CONCLUSION These observations suggest a negative feedback mechanism in the gut whereby local delivery of TGFβ results in reduced local and systemic levels of the active form of TGFβ. Our findings suggest potential clinical implications for use of encapsulated TGFβ, perhaps in the context of IBD and/or other instances of fibrosis and/or pathological TGFβ signaling.

It has previously been shown that genital tract infection with Neisseria gonorrhoeae in mice does not induce a state of protective immunity against reinfection but instead suppresses the development of adaptive immune responses against N. gonorrhoeae dependent on transforming growth factor beta (TGF-β) and interleukin 10 (IL-10). Intravaginal administration during gonococcal infection of IL-12 encapsulated in biodegradable microspheres (IL-12/ms) reverses the immunosuppression and promotes the production of gamma interferon (IFN-γ) and of specific antibodies in serum and genital secretions and accelerates clearance of the infection. In this study, microspheres were shown to remain largely within the genital tract lumen and to release IL-12 over the course of 4 days. Antigonococcal IgA and IgG antibodies induced by IL-12/ms treatment reacted with antigenically different strains of N. gonorrhoeae and led to resistance to reinfection with heterologous and homologous strains. Immune resistance to reinfection persisted for at least 6 months after clearance of the primary infection. Experiments performed with immunodeficient strains of mice lacking either IFN-γ or B cells demonstrated that both IFN-γ and B cells were necessary for the IL-12-induced generation of immune responses to N. gonorrhoeae and the resulting accelerated clearance of the infection. It is therefore concluded that intravaginally administered IL-12/ms achieves its effect by the sustained release of IL-12 that promotes Th1-driven adaptive immune responses, including the production of specific antigonococcal antibodies that cross-react with multiple strains of N. gonorrhoeae. IL-12-enhanced immunity to N. gonorrhoeae can be recalled against reinfection after prolonged intervals and is dependent upon both IFN-γ and antibody production by B cells. IMPORTANCE Genital infection with Neisseria gonorrhoeae (gonorrhea) is a significant cause of reproductive tract morbidity in women, leading to pelvic inflammatory disease, tubal factor infertility, and increased risk for ectopic pregnancy. WHO estimates that 78 million new infections occur annually worldwide. In the United States, >350,000 cases are reported annually, but the true incidence is probably >800,000 cases/year. Increasing resistance to currently available antibiotics raises concern that gonorrhea might become untreatable. Infection does not induce a state of immune protection against reinfection. Previous studies have shown that N. gonorrhoeae suppresses the development of adaptive immune responses by mechanisms dependent on the regulatory cytokines TGF-β and IL-10. This study shows that intravaginal treatment of gonococcal infection in female mice with microencapsulated IL-12 induces persisting anamnestic immunity against reinfection with N. gonorrhoeae, even of antigenically diverse strains, dependent on T-cell production of IFN-γ and B-cell production of antibodies.

Background and aims: We investigated oral delivery of transforming growth factor beta 1 [TGFβ]- and all-trans retinoic acid [ATRA]-loaded microspheres as therapy for gut inflammation in murine models of inflammatory bowel disease [IBD]. Methods: ATRA and TGFβ were separately encapsulated in poly [lactic-co-glycolic] acid or polylactic acid microspheres [respectively]. TGFβ was encapsulated using proprietary phase-inversion nanoencapsulation [PIN®] technology. Results: PIN® particles provided sustained release of bioactive protein for at least 4 days and were stable for up to 52 weeks when stored at either 4C or -20C. In the SCID mouse CD4 + CD25- T cell transfer model of IBD, oral treatment starting at disease onset prevented weight loss, significantly reduced average disease score [~ 50%], serum amyloid A levels [ ~ 5-fold], colon weight-to-length ratio [~ 50%], and histological score [~ 5-fold]. Conclusions: Both agents given together outperformed either separately. Highest TGFβ doses and most frequent dose schedule were most effective. Activity was associated with a significant increase [45%] in Foxp3 expression by colonic lamina propria CD4+ CD25+ T-cells. Activity was also demonstrated in dextran sulphate sodium-induced colitis. The data support development of the combination product as a novel, targeted immune based therapy for treatment for IBD.

Among retinoic acid receptors (RARs) alpha, beta, and gamma, the messenger RNA level of RAR-gamma is the most readily detectable by Northern blotting in human and mouse skin. This observation suggests that RAR-gamma may play a critical role in the modulation of the therapeutic benefits and side effects of retinoids in skin. To test this hypothesis, 11 RAR-gamma selective retinoids were synthesized based on three related structures. Each compound was found to prefer RAR-gamma when assessed by retinoid-induced transcriptional activity (RAR-gammaRAR-betaRAR-alpha). The apparent Kd for binding to recombinant receptor protein was found to follow a similar trend. To correlate this receptor selectivity with in vivo activity, the compounds were tested topically in the Rhino mouse utriculi reduction and rabbit irritation models, two assays widely used to screen retinoids for efficacy and side effects, respectively. The results indicated that for these compounds, both efficacy in the utriculi reduction assay and irritation potential in rabbits correlated positively with the RAR-gamma transactivation activity, with r2 of 0.9 and 0.8, respectively. These data suggest that RAR-gamma is an important regulator of retinoic acid efficacy in skin and further, that the irritation associated with the use of retinoids is most likely a receptor-mediated process.

Utilizing a strategy involving domain exchange between retinoic acid receptors alpha and beta (RAR alpha and RAR beta) and monitoring the transcriptional activity of the resulting chimeric receptors with receptor-selective retinoids, we identified a 70-aa region within the N-terminal portion of the RAR alpha and -beta domain E which is important for an RAR alpha- or RAR beta-specific response. Two amino acid residues within this region, serine-232 (S232) and threonine-239 (T239) in RAR alpha and the corresponding alanine-225 (A225) and isoleucine-232 (I232) in RAR beta, were found to be essential for this effect. In addition, binding studies using the chimeric receptors expressed in Escherichia coli showed that the N-terminal portion of domain E was also important for the characteristic binding profile of t-RA and various retinoids with RAR alpha or RAR beta. Structural predictions of the primary amino acid sequence in this region indicate the presence of an amphipathic helix-turn-helix structure with five hydrophobic amino acids that resemble a leucine zipper motif. The amino acid residues identified by domain swapping, S232 and T239 in RAR alpha and A225 and I232 in RAR beta, were found within the hydrophobic face of an alpha-helix in close proximity to this zipper motif, suggesting that the ligand may interact with the receptor in the region adjacent to a surface involved in protein-protein interactions. This finding may link ligand binding to other processes important for transcriptional activation.

The three retinoic acid receptors (RARΑ, RARΒ and RARΓ) are known to modulate the transcription of target genes through interaction of the individual receptors with their naturally occurring ligand, retinoic acid (RA). Since RA has multiple effects in vivo, considerable effort has recently been devoted to finding selective compounds to elucidate the functions of individual receptors and to relate these functions to specific in vivo effects. The racemic synthetic retinoid 6-[(5,5,8,8-tetramethyl- 5,6,7,8-tetrahydro-2 – naphthyl)hydroxy-methyl]-2-naphthalene carboxylic acid has recently been identified as an RARΓ-selective agonist. A synthetic method involving lipase-mediated transformation has been developed to prepare the individual enantiomers. Discrimination between the two enantiomers is seen in both transcriptional activity and binding to recombinant receptors with the (S)-enantiomer being the more active. Differences between the two compounds are also seen in the Rhino mouse utriculi reduction assay and the rabbit irritation model. In both animal models, the (S)-enantiomer consistently gave a greater response. Taken together, these results suggest that the activity and irritation seen with RA and related compounds is receptor mediated. Further, the strong selectivity of the compounds reported here for RARΓ suggests that this receptor plays an important role in these in vivo biological activities. The discrimination between these enantiomers may be useful in the design of novel retinoids with uniquely defined biological properties.

In search for retinoic acid receptor (RAR) selective ligands, a series of 6-substituted 2-naphthoic acid retinoids were synthesized and evaluated in vitro in a transactivation assay and a competition binding assay for all RARs. These derivatives, in general, showed RAR β,γ selectivity. Among these naphthoic acids, oxime derivative 12 was identified as a potent RAR γ-selective retinoid, while olefinic derivative 11 was found to be comparable to retinoic acid and slightly RAR β,γ selective. For the bioassays, a general correlation was observed between the binding affinity of the ligand to the receptors and the potency of the compounds in the transactivation assay. The structure−activity relationship of these naphthoic acids will be discussed.

Controlled-release, injectable polymer microspheres provide a clinically feasible alternative to gene-modification for the local, sustained delivery of cytokines to tumors for cancer immunotherapy. Long-term release kinetics, bioactivity profiles, and stability of interleukin-2 (IL-2), interleukin-12 (IL-12), and granulocyte- macrophage colony-stimulating factor (GM-CSF)-encapsulated microspheres prepared by phase inversion nanoencapsulation (PIN) were evaluated. While all formulations released physiologically relevant quantities of cytokine for up to 30 days, the individual release kinetics were different. Recovery of specific activity after encapsulation was 40%, 60%, and 90%-that of pre-encapsulation levels for IL-2, GM-CSF and IL-12, respectively. Upon storage, the IL-12 microspheres rapidly lost activity, whereas IL-2 and GM-CSF microspheres remained stable for at least 9 weeks. These studies demonstrate that biochemical properties of microsphere formulations vary depending on the cytokine, and rigorous characterization of formulations is a prerequisite to in vivo testing.

The transcriptional response mediated by retinoic acid involves a complex series of events beginning with ligand recognition by a nuclear receptor. To dissect the amino acid contacts important for receptor-specific ligand recognition, a series of retinoic acid receptor (RAR) mutants were constructed. Transcriptional studies revealed that serine 232 (Ser232) in RARalpha and methionine 272 (Met272) in RARgamma are critical residues for the recognition of their respective receptor-selective analogs. The identification of these key amino acids in the ligand binding pocket is confirmed by the reported crystal structure of RARgamma. Interestingly, the serine at position 232 in RARalpha gives an explanation for the observed differences in the affinity of the naturally occurring ligand, all-trans-retinoic acid (t-RA), in this receptor compared with that for the other receptors, since hydrogen bonding would not be permitted between the hydroxyl of serine and the hydrophobic linker of t-RA. Using this model, a molecular mechanism for the transcriptional antagonism of a synthetic analog is suggested that involves an alteration in the structure of the receptor protein in the region around the AF2 domain in helix 12.

In search for retinoic acid receptor (RAR) selective ligands, a series of 6-substituted 2-naphthoic acid retinoids were synthesized and evaluated in vitro in a transactivation assay and a competition binding assay for all RARs. These derivatives, in general, showed RAR beta,gamma selectivity. Among these naphthoic acids, oxime derivative 12 was identified as a potent RAR gamma-selective retinoid, while olefinic derivative 11 was found to be comparable to retinoic acid and slightly RAR beta,gamma selective. For the bioassays, a general correlation was observed between the binding affinity of the ligand to the receptors and the potency of the compounds in the transactivation assay. The structure-activity relationship of these naphthoic acids will be discussed.