Synovo GmbH, Tübingen, Germany.
Pharmaceutical Chemistry, Institute for Pharmaceutical Sciences, Eberhard Karls University Tübingen, Tübingen, Germany.
Front Immunol. 2023 Jun 20;14:1168252. doi: 10.3389/fimmu.2023.1168252. eCollection 2023.
TLR Agonists have promising activity in preclinical models of viral infection and cancer. However, clinical use is only in topical application. Systemic uses of TLR-ligands such as Resiquimod, have failed due to adverse effects that limited dose and thus, efficacy. This issue could be related to pharmacokinetic properties that include fast elimination leading to low AUC with simultaneously high c at relevant doses. The high c is associated with a sharp, poorly tolerated cytokine pulse, suggesting that a compound with a higher AUC/c-ratio could provide a more sustained and tolerable immune activation. Our approach was to design TLR7/8-agonist Imidazoquinolines intended to partition to endosomes acid trapping using a macrolide-carrier. This can potentially extend pharmacokinetics and simultaneously direct the compounds to the target compartment. The compounds have hTLR7/8-agonist activity (EC50 of the most active compound in cellular assays: 75-120 nM hTLR7, 2.8-3.1 µM hTLR8) and maximal hTLR7 activation between 40 and 80% of Resiquimod. The lead candidates induce secretion of IFNα from human Leukocytes in the same range as Resiquimod but induce at least 10-fold less TNFα in this system, consistent with a higher specificity for human TLR7. This pattern was reproduced in a murine system, where small molecules are thought not to activate TLR8. We found that Imidazoquinolines conjugated to a macrolide or, substances carrying an unlinked terminal secondary amine, had longer exposure compared with Resiquimod. The kinetics of pro-inflammatory cytokine release for these substances were slower and more extended (for comparable AUCs, approximately half-maximal plasma concentrations). Maximal IFNα plasma levels were reached 4 h post application. Resiquimod-treated groups had by then returned to baseline from a peak at 1 h. We propose that the characteristic cytokine profile is likely a consequence of altered pharmacokinetics and, potentially, enhanced endosomal tropism of the novel substances. In particular, our substances are designed to partition to cellular compartments where the target receptor and a distinct combination of signaling molecules relevant to IFNα-release are located. These properties could address the tolerability issues of TLR7/8 ligands and provide insight into approaches to fine-tune the outcomes of TLR7/8 activation by small molecules.
TLR 激动剂在病毒感染和癌症的临床前模型中具有良好的活性。然而，临床应用仅限于局部应用。由于不良反应限制了剂量，从而影响了疗效，因此 TLR 配体（如瑞喹莫德）的全身应用已经失败。这个问题可能与药代动力学特性有关，包括快速消除导致 AUC 较低，同时在相关剂量下 c 较高。高 c 与难以耐受的细胞因子脉冲有关，这表明具有更高 AUC/c 比值的化合物可以提供更持续和可耐受的免疫激活。我们的方法是设计 TLR7/8 激动剂咪唑并喹啉，旨在使用大环内酯载体将其分配到内涵体中进行酸捕获。这可能潜在地延长药代动力学并同时将化合物靶向到靶位。这些化合物具有 hTLR7/8-激动剂活性（细胞测定中最活跃化合物的 EC50：75-120 nM hTLR7，2.8-3.1 µM hTLR8），并且 hTLR7 最大激活率在 40%到 80%之间Resiquimod。候选药物在相同范围内诱导人类白细胞分泌 IFNα，但在该系统中诱导的 TNFα 至少少 10 倍，这与对人类 TLR7 更高的特异性一致。这种模式在鼠系统中得到了复制，在鼠系统中，小分子被认为不会激活 TLR8。我们发现，与 Resiquimod 相比，与大环内酯偶联的咪唑并喹啉或带有未连接末端仲胺的物质具有更长的暴露时间。这些物质的促炎细胞因子释放动力学较慢且更为扩展（对于可比 AUC，大约达到一半的最大血浆浓度）。最大 IFNα 血浆水平在给药后 4 小时达到。此时，瑞喹莫德处理组已经从 1 小时的峰值恢复到基线。我们提出，特征性细胞因子谱可能是药代动力学改变的结果，并且可能增强了新型物质的内涵体趋向性。特别是，我们的物质旨在分配到细胞区室，其中目标受体和与 IFNα 释放相关的独特组合的信号分子位于该细胞区室中。这些特性可以解决 TLR7/8 配体的耐受性问题，并为小分子 TLR7/8 激活的结果提供微调方法的见解。
