The treatment of paroxysmal nocturnal hemoglobinuria (PNH) is undergoing a second revolution with the introduction of proximal complement inhibitors.¹ Indeed, recent clinical studies have shown that molecules targeting either the C3, the complement factor B, or the complement factor D (this latter in association with anti-C5 therapy) have been effective in increasing hemoglobin levels in PNH patients with residual anemia despite standard anti-C5 treatment.^2-4 The effectiveness of this class of complement inhibitors is achieved by preventing C3-mediated extravascular hemolysis, which we have identified as the major factor limiting hemoglobin normalization in PNH patients treated with anti-C5.^{1, 5} Indeed, unlike C5 inhibitors, proximal complement inhibitors are able to almost completely normalize the lifespan of PNH erythrocytes, leading to the paradox that a better hematological response is associated with a significant and large increase in both the proportion and the mass of the population of ‘crippled’ PNH erythrocytes. Eventually, the increased size of the population of PNH erythrocytes resulting from the therapeutic effectiveness of proximal complement inhibitors has raised the concern that clinically severe intravascular breakthrough hemolysis (BTH) may occur if the therapeutic blockade is lost, even temporarily. An interesting debate on the risk of BTH is currently ongoing, spurring discussions about whether proximal complement inhibitors should be used as monotherapy or in a combination with anti-C5.⁶ These discussions are based on comparisons of different clinical trials but suffer from the paucity of data. In addition, there is an almost complete lack of knowledge about the clinical course of BTH in patients with very large proportions of PNH erythrocytes (even higher than 90%) because, simply, this condition has never been observed in PNH patients before the introduction of treatments with proximal complement inhibitors. Thus, even index cases provide valuable insights that enhance the understanding of this novel condition within the PNH community.

We report the unique circumstance of three PNH patients treated with the factor D inhibitor vemircopan as monotherapy within the clinical trial NCT04170023.⁷ The trial was unexpectedly terminated by the sponsor's decision, following an interim analysis claiming that vemircopan monotherapy “Did not appropriately control IVH; significantly increased rates of BTH and LDH excursions (LDH excursions is defined by LDH values >x2ULN) compared with ravulizumab” (https://clinicaltrials.gov/study/NCT04170023). Consequently, these patients, despite achieving a very good hematological response associated with a large population of PNH erythrocytes, were compelled to discontinue this effective treatment, and to be switched to standard-of-care anti-C5 treatment (single 2700 mg loading dose, followed 2 weeks later by 3300 mg maintenance, then given every 8 weeks, all intravenously). To minimize the risk of massive intravascular hemolysis, the switch to the standard-of-care ravulizumab was planned 2 weeks before vemircopan discontinuation. Thus, vemircopan discontinuation only occurred after completing the ravulizumab loading phase, guaranteeing that ravulizumab was in its therapeutic range at time of vemircopan discontinuation.

Since the lack of an established strategy to mitigate possible extravascular hemolysis (compassionate add-on danicopan was not yet available, and commercially available pegcetacoplan was labeled only as monotherapy after failure of anti-C5 treatment), we addressed this risk by planning in advance the use of the factor B inhibitor, iptacopan, through a named-patient compassionate use study program. This study program, associated with a strict monitoring of hemolysis biomarkers, was approved by the local IRBs. The patients (once vaccine boosters were performed, if needed) could initiate iptacopan treatment at Day 42 from vemircopan discontinuation. Nevertheless, after a vemircopan washout period of at least 14 days, earlier treatment was possible in cases of severe anemia or breakthrough hemolysis. Breakthrough hemolysis qualifying for early rescue treatment was defined as meeting one or more of the following criteria: (i) Hemoglobin decrease ≥3 g/dL from baseline; (ii) requirement for red blood cell transfusion; (iii) severe signs and/or symptoms of hemolysis.

At baseline (last day of vemircopan treatment), hemoglobin level was remarkable in all three patients, the lowest value was 11 g/dL in patient A because of iron deficiency secondary to hypermenorrhea. All patients had normal or slightly increased LDH, consistent with adequate control of intravascular hemolysis (Figure 1 and Table 1). None of the patients showed any laboratory sign of extravascular hemolysis, with normal reticulocyte count and bilirubin, and negligible (<0.5% of PNH RBC) C3d-opsonized erythrocytes; their proportion of PNH erythrocytes was 75%, 87%, and 44% (paralleling the proportion of PNH granulocytes in all patients, Figure 1 and Table 1), highlighting the large PNH erythrocyte mass eventually at risk of complement-mediated hemolysis. This excellent clinical picture observed at baseline drastically changed once vemircopan was discontinued, despite the anti-C5 treatment with ravulizumab started 2 weeks earlier. Indeed, within 1–2 weeks, all patients developed a significant hemoglobin drop, which was very severe and symptomatic in two patients. Patient A's hemoglobin fell to around 7 g/dL within 2 weeks, with very severe fatigue and dyspnea; unfortunately, she could not be transfused because of her religious beliefs. Patient C did even worse as he experienced a complement-amplifying condition (flu-like viral infection with fever) on the same day of vemircopan discontinuation, leading to immediate symptomatic paroxysm of intravascular hemolysis (demonstrated by gross hemoglobinuria and increased LDH) and severe anemia. Indeed, the patient required red blood cell transfusions on Days 7 and 13, despite the intravascular hemolytic crisis resolved within 72 h. Notably, in these two patients, the dramatic fall in hemoglobin was not associated with massive increase of LDH, which even in patient C at time of the intravascular hemolytic crisis did not exceed two times the upper limit of normal. Interestingly, both patients quickly developed large proportions of C3d-opsonized erythrocytes (40% and 62%, respectively) and increased bilirubin levels (Table 1), suggesting that extravascular hemolysis extensively contributed to hyperacute anemia in these patients. For this reason, already at Day 14, these two patients were switched to compassionate iptacopan in monotherapy, which provided immediate clinical benefit. Indeed, signs and symptoms of hemolysis (both intravascular and extravascular) disappeared within a few hours, with hemoglobin reaching baseline values within a few weeks and C3 opsonization becoming negligible within 4–6 weeks. Patient B had a different clinical behavior; despite an early hemoglobin drop of 2 g/dL, he did not develop clinically meaningful anemia (and thus he started iptacopan later at Day 56). This was likely due to the fact that his PNH hematopoiesis was as low as 60%, with a relevant non-PNH hematopoiesis (the proportion of normal granulocytes was around 40%), which eventually prevented anemia despite a degree of hemolysis similar to that of the other patients. Indeed, we tried to estimate the extent of hemolysis by examining the absolute number of PNH erythrocyte undergoing hemolysis at time of vemircopan discontinuation, together with the loss of Hb carried by them. According to this analysis, all three patients suffered from an acute loss of 1.5–2.5 × 10⁶/μL of PNH erythrocytes, which obviously accounts for the drop in hemoglobin (Figure 1). Comparing the behaviors of the three patients, it appears that switching from vemircopan to ravulizumab led to massive, acute, extravascular hemolysis in patients A and C, while patient B experienced a low-grade residual intravascular hemolysis. One might speculate that inherited variants of complement genes could contribute to these heterogenous biological and clinical findings. This is in keeping with the highest C3 binding and hemoglobin drop seen in patient C who was heterozygous for the rare allele of the HindIII polymorphism of the complement receptor 1 (CR1) gene that has been associated with the risk of extravascular hemolysis in PNH on anti-C5 treatment.⁸

These severe hyperacute hemolytic events were not a surprise; indeed, as investigators of the NCT04170023 vemircopan monotherapy trial, we have expressed our safety concern regarding the risk of severe hemolysis (not necessarily restricted to intravascular, but possibly also extravascular) in the case of vemircopan discontinuation, asking for an appropriate risk-mitigation strategy. These concerns are not different from the broader issues emerging in the context of PNH treatment with proximal complement inhibitors when there is a significant increase in the mass of PNH erythrocytes at risk of hemolysis. Indeed, there are no guidelines for managing such risk, because of the limited knowledge of the complications that might arise when such a large mass of erythrocytes undergoes (hyper)acute hemolysis. Indirect information regarding this risk can be inferred from the PEGASUS trial. In the PEGASUS trial all patients initially underwent a 4-week ramp-up period during which they received concomitant pegcetacoplan and anti-C5 treatment. Subsequently, those patients randomized to anti-C5 monotherapy experienced an average hemoglobin level drop of nearly 4 g/dL over the subsequent 4 weeks.²

Here, we show for the first time, that rebound hemolysis following the discontinuation of effective treatment with proximal complement inhibitors is a major clinical risk that cannot be fully prevented by switching to anti-C5 therapy. Indeed, the large mass of PNH erythrocytes is susceptible not only to acute intravascular hemolysis due to incomplete blockade by anti-C5 during complement-amplifying condition, but also to massive C3-mediated extravascular hemolysis, which may develop acutely as well. In this case, terminal complement inhibitor treatment and supportive care with transfusions remain far from optimal. Therefore, whenever possible, rescue treatment with alternative proximal complement inhibitors should be strenuously pursued.

These findings provide informative guidance for treating physicians in any situations that result in a transient breach of complete complement inhibition, taking also into account that, unlike anti-C5, the currently available proximal complement inhibitors have short half-lives^{3, 4} and may also have suboptimal pharmacokinetics.^{9, 10} Similar management problems can occur in clinical conditions that prevent oral intake or drug absorption, such as gastrointestinal comorbidities or conscience impairments. Moreover, these findings highlight the importance of considering the potential risk associated with treatment discontinuation—including lack of availability, planning or incidental occurrence of pregnancy—before initiating therapy with proximal complement inhibitors.

While not the primary focus, this report highlights the intricacies of modern clinical research, where industrial considerations can sometimes hinder the development of potentially beneficial treatments or compromise patient safety. It also emphasizes the responsibilities of academic investigators in providing strong guidance in the design of clinical trials, avoiding that lack of expertise or industrial interests could compromise science and patient safety.

随着近端补体抑制剂的引入，阵发性睡眠性血红蛋白尿症 （PNH） 的治疗正在经历第二次革命。¹ 事实上，最近的临床研究表明，尽管接受了标准的抗 C5 治疗，但靶向 C3、补体因子 B 或补体因子 D（后者与抗 C5 治疗相关）的分子可有效增加残余贫血 PNH 患者的血红蛋白水平。^2-4 这类补体抑制剂的有效性是通过预防 C3 介导的血管外溶血来实现的，我们已经确定这是限制接受抗 C5 治疗的 PNH 患者血红蛋白正常化的主要因素。^1、5事实上，与 C5 抑制剂不同，近端补体抑制剂能够几乎完全使 PNH 红细胞的寿命正常化，从而导致一个悖论，即更好的血液学反应与“残疾”PNH 红细胞群的比例和质量的显着和大幅增加有关。最终，由于近端补体抑制剂的治疗效果导致 PNH 红细胞群大小的增加，这引起了人们的担忧，即如果治疗阻断消失，即使是暂时的，也可能发生临床严重的血管内突破性溶血 （BTH）。目前正在进行一场关于 BTH 风险的有趣辩论，引发了关于近端补体抑制剂是否应作为单一疗法或与抗 C5 联合使用的讨论。⁶ 这些讨论基于不同临床试验的比较，但缺乏数据。 此外，对于具有非常大比例 （甚至高于 90%） 的患者的 BTH 临床病程，几乎完全缺乏知识，因为简单地说，在引入近端补体抑制剂治疗之前，从未在 PNH 患者中观察到这种情况。因此，即使是指示病例也提供了有价值的见解，可以增强 PNH 社区对这种新病症的理解。

我们在临床试验NCT04170023中报告了 3 名 PNH 患者接受因子 D 抑制剂 vemircopan 作为单一疗法治疗的独特情况。⁷ 该试验因申办者的决定而意外终止，此前中期分析声称 vemircopan 单一疗法“没有适当控制 IVH;与 ravulizumab 相比，BTH 和 LDH 偏移率显着增加 （LDH 偏移由 LDH 值 >x2ULN 定义） （https://clinicaltrials.gov/study/NCT04170023）。因此，这些患者尽管取得了与大量 PNH 红细胞相关的非常好的血液学反应，但被迫停止这种有效的治疗，并转为标准护理抗 C5 治疗（单次 2700 mg 负荷剂量，随后 2 周后维持 3300 mg，然后每 8 周给药一次，全部静脉注射）。为了最大限度地降低大量血管内溶血的风险，计划在 vemircopan 停药前 2 周改用标准护理 ravulizumab。因此，vemircopan 停药仅在完成 ravulizumab 负荷阶段后发生，保证了 ravulizumab 在 vemircopan 停药时处于其治疗范围内。

由于缺乏减轻可能的血管外溶血的既定策略（同情附加药物 danicopan 尚不可用，并且市售的 pegcetacoplan 在抗 C5 治疗失败后仅被标记为单药治疗），我们通过命名患者同情使用研究计划提前计划使用因子 B 抑制剂 iptacopan 来应对这一风险。该研究计划与溶血生物标志物的严格监测相关，已获得当地 IRB 的批准。患者（如果需要，一旦进行了疫苗加强剂）可以在停用 vemircopan 后第 42 天开始 iptacopan 治疗。然而，在至少 14 天的 vemircopan 清除期后，对于严重贫血或突破性溶血病例，可以进行早期治疗。符合早期救援治疗的突破性溶血定义为满足以下一项或多项标准： （i） 血红蛋白从基线减少 ≥3 g/dL;（ii） 需要输注红细胞;（iii） 溶血的严重体征和/或症状。

在基线 （vemircopan 治疗的最后一天），所有 3 例患者的血红蛋白水平均显著，患者 A 的最低值为 11 g/dL，因为继发于痛经的缺铁。所有患者的 LDH 正常或略有升高，与血管内溶血的充分控制一致 （图1 和 表1）。所有患者均未表现出任何血管外溶血的实验室体征，网织红细胞计数和胆红素正常，C3d 调理红细胞可忽略不计 （<0.5% of PNH RBC）;他们的 PNH 红细胞比例为 75% 、 87% 和 44% （与所有患者中 PNH 粒细胞的比例平行，图 1 和 表 1），突出了最终有补体介导溶血风险的大 PNH 红细胞块。尽管 2 周前开始使用 ravulizumab 进行抗 C5 治疗，但一旦停用 vemircopan，基线时观察到的这种出色的临床表现发生了巨大变化。事实上，在 1-2 周内，所有患者都出现了明显的血红蛋白下降，其中两名患者非常严重且有症状。患者 A 的血红蛋白在 2 周内降至 7 g/dL 左右，伴有非常严重的疲劳和呼吸困难;不幸的是，由于她的宗教信仰，她无法输血。患者 C 的情况更糟，因为他在停用 vemircopan 的同一天经历了补体扩增情况（流感样病毒感染伴发热），导致血管内溶血的即时症状发作（表现为肉眼血红蛋白尿和 LDH 升高）和严重贫血。事实上，患者需要在第 7 天和第 13 天输注红细胞，尽管血管内溶血危象在 72 小时内消退。 值得注意的是，在这两名患者中，血红蛋白的急剧下降与 LDH 的大幅增加无关，即使在患者 C 的血管内溶血危象时，LDH 也没有超过正常上限的两倍。有趣的是，两名患者都很快出现了大比例的 C3d 调理红细胞 （分别为 40% 和 62%） 和胆红素水平升高 （表1），表明血管外溶血广泛导致了这些患者的高急性贫血。出于这个原因，早在第 14 天，这两名患者就被转为富有同情心的 iptacopan 单药治疗，这提供了立竿见影的临床益处。事实上，溶血的体征和症状（血管内和血管外）在几个小时内消失，血红蛋白在几周内达到基线值，C3 调理素作用在 4-6 周内变得可以忽略不计。患者 B 的临床行为不同;尽管早期血红蛋白下降了 2 g/dL，但他没有出现具有临床意义的贫血（因此他在第 56 天晚些时候开始使用 ipatacpan）。这可能是由于他的 PNH 造血率低至 60%，具有相关的非 PNH 造血功能（正常粒细胞的比例约为 40%），尽管溶血程度与其他患者相似，但最终预防了贫血。事实上，我们试图通过检查停用 vemircopan 时发生溶血的 PNH 红细胞的绝对数量以及它们携带的 Hb 损失来估计溶血的程度。根据这项分析，所有 3 名患者的 PNH 红细胞急性损失为 1.5-2.5 × 10⁶/μL，这显然是血红蛋白下降的原因（图 1）。 比较三名患者的行为，似乎从 vemircopan 转换为 ravulizumab 导致患者 A 和 C 出现大量急性血管外溶血，而患者 B 出现低度残余血管内溶血。人们可能会推测，补体基因的遗传变异可能导致这些异质性的生物学和临床发现。这与患者 C 中观察到的最高 C3 结合和血红蛋白下降一致，患者 C 是补体受体 1 （CR1） 基因 HindIII 多态性罕见等位基因的杂合子，该等位基因与抗 C5 治疗中 PNH 血管外溶血的风险有关。⁸

这些严重的超急性溶血事件并不令人惊讶;事实上，作为 NCT04170023 Vemircopan 单药治疗试验的研究者，我们已经表达了我们对停用 Vemircopan 的情况下严重溶血风险（不一定局限于血管内，但也可能局限于血管外）的安全担忧，要求采取适当的风险缓解策略。这些担忧与使用近端补体抑制剂治疗 PNH 时出现的更广泛的问题没有什么不同，当时有溶血风险的 PNH 红细胞质量显着增加。事实上，没有管理此类风险的指南，因为对如此大量的红细胞经历（高度）急性溶血时可能出现的并发症的了解有限。有关 PEGASUS 试验的间接信息可以推断出来。在 PEGASUS 试验中，所有患者最初都经历了 4 周的加速期，在此期间他们同时接受了 pegcetacoplan 和抗 C5 治疗。随后，那些随机接受抗 C5 单药治疗的患者在随后的 4 周内平均血红蛋白水平下降了近 4 g/dL。^{阿拉伯数字}

在这里，我们首次表明，停止近端补体抑制剂有效治疗后反弹性溶血是一种主要的临床风险，无法通过改用抗 C5 治疗来完全预防。事实上，大量 PNH 红细胞不仅易受补体扩增条件下抗 C5 不完全阻断而引起的急性血管内溶血，而且易受 C3 介导的大量血管外溶血的影响，其也可能急性发展。在这种情况下，终末补体抑制剂治疗和输血支持性治疗仍远非最佳。因此，应尽可能努力使用替代性近端补体抑制剂进行挽救治疗。

这些发现为在任何导致短暂破坏完全补体抑制的情况下治疗医生提供了信息指导，同时还考虑到与抗 C5 不同，目前可用的近端补体抑制剂具有较短的半衰期^3、4，并且也可能具有次优的药代动力学。^9、10类似的管理问题可能发生在阻止经口摄入或药物吸收的临床情况下，例如胃肠道合并症或良心障碍。此外，这些发现强调了在开始使用近端补体抑制剂治疗之前考虑与治疗中断相关的潜在风险的重要性，包括缺乏可用性、计划或偶然怀孕。

虽然不是主要关注点，但本报告强调了现代临床研究的复杂性，其中工业因素有时会阻碍可能有益的治疗方法的开发或危及患者安全。它还强调了学术研究人员在临床试验设计中提供强有力指导的责任，避免缺乏专业知识或行业利益可能会损害科学和患者安全。