Alvocidib (flavopiridol) for the treatment of chronic lymphocytic leukemia

Peter H. Wiernik

To cite this article: Peter H. Wiernik (2016): Alvocidib (flavopiridol) for the treatment of chronic lymphocytic leukemia, Expert Opinion on Investigational Drugs, DOI: 10.1517/13543784.2016.1169273
To link to this article: http://dx.doi.org/10.1517/13543784.2016.1169273

Accepted author version posted online: 21 Mar 2016.
Published online: 07 Apr 2016.
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Alvocidib (flavopiridol) for the treatment of chronic lymphocytic leukemia
Peter H. Wiernik
Cancer Research Foundation of New York, Bronx, NY, USA

Received 19 January 2016
Accepted 18 March 2016 Published online
5 April 2016
Alvocidib; chronic lymphocytic leukemia; cytokine release syndrome; flavopiridol; tumor lysis syndrome

1. Introduction
Chronic lymphocytic leukemia (CLL) is the most prevalent adult leukemia in the Western world. It is often an indolent disease that responds well to a variety of therapies, but, when characterized by certain adverse features, it can be quite aggressive. Most respond well to initial treatment for years. However, no treatment is curable and patients virtually always relapse and require additional treatment. Alvocidib (which now has orphan drug status in CLL) was found almost
20 years ago to have in vitro activity against CLL cells by causing apoptosis by a unique mechanism. The drug has been studied in Phase I and Phase II trials and found to have significant activity in CLL patients in general and in patients with unfavorable characteristics such as advanced age, adverse cytogenetics, and/or bulky disease. Unfortunately, several serious toxicities have been observed in these studies such as tumor lysis syndrome (TLS), cytokine release syndrome (CRS), and secretory diarrhea. Scheduling manipulations and other attempts to overcome the frequency and severity of these toxicities have only been partially successful. Therefore, the role of alvocidib in the treatment of CLL at this time is unclear.

2. Overview of the market
There are many drugs approved for the treatment of CLL but none is curative. Although allogeneic stem cell transplantation may cure an occasional patient with relapsed CLL (especially, when the procedure is complicated by graft versus host

disease [1]), most relapsed patients are not candidates for this procedure. CLL patients with a del (17) abnormality have an especially poor prognosis and only one agent (ibrutinib, a Bruton’s kinase inhibitor [2]) is approved for initial first-line treatment of that variant of CLL in the USA, having shown more favorable outcome in terms of response rate and pro- gression-free survival than all other approved drugs. In Europe, idelalisib, a PI3Kδ inhibitor, [3] is also approved for that indication as well as for patients with TP53 mutations. Ibrutinib is also approved for all CLL patients who have received one prior therapy. Several monoclonal antibodies are approved for first-line treatment of CL L (rituximab,[4] obinutuzumab,[5] and ofatumumab [6] in combination with chlorambucil). Furthermore, fludarabine (a purine nucleoside analog)[7] and the alkylating agents bendamustine [8] and chlorambucil [9] are approved for treatment of CLL. All newer agents have greater activity and more significant potential toxicity than chlorambucil, an agent that has been used for the treatment of CLL for decades. Lenalidomide,[10] an immune-modulating drug, has major activity against CLL but has not yet been approved primarily because of toxicity issues. Currently, the standard treatment for newly diagnosed patients with CLL without adverse cytogenetic findings is a combination of fludarabine, cyclophosphamide, and rituxi- mab.[11] With currently available treatments, overall response rates of 75% or greater are expected in patients with normal cytogenetics but complete response rates >15–20% are rarely observed. Newer agents have yielded progression-free survival medians of 2 years or more compared with median rates half

© 2016 Informa UK Limited, trading as Taylor & Francis Group

that for chlorambucil alone. New agents capable of inducing a complete response in the majority of patients with CLL and cure in at least a reproducible fraction are sorely needed. Venetoclax, a new investigational agent that targets BCL2, yielded a 20% complete response in previously treated, poor prognosis patients with acceptable toxicity in an early clinical trial.[12] This drug may very well ultimately become commer- cially available and play a major role in the treatment of CLL.

3. Introduction to the compound
Alvocidib, originally named flavopiridol, was initially found to have significant clinical activity against CLL. More recently, it was found to have clinical activity against acute myeloid leukemia (AML).[13] In addition, laboratory studies suggest activity against hepatocellular carcinoma,[14] ovarian carci- noma,[15] breast cancer,[16] prostate cancer,[17] and uterine leiomyoma.[18] This review concentrates on the activity of alvocidib in CLL.

4. Chemistry
Alvocidib with a molecular weight of 401.1 is a semisynthetic flavone derived from the chromone alkyloid rohitukine, iso- lated from the stem bark of Dysoxylum binectariferum, a plant indigenous to India.[19] Its structure is shown in the Drug Summary Box 1.

5. Pharmacodynamics
Alvocidib is a cyclin-dependent kinase 1,2,4, 6,7, and 9 inhibi- tor that competitively binds those kinases at the ATP-binding pocket which results in apoptosis in CLL cells by activating caspase-3 and -4 without modulation of BCL2 or TP53 activity. [21–23] The drug also reduces mitochondrial oxygen con- sumption and autophagy.[24,25] Furthermore, alvocidib induces endoplasmic reticulum stress that contributes to cell death.[26] Alvocidib also exhibits groove-directed intercalation with DNA by an enthalpy-driven exothermic process.[27] Autophagy is a mechanism of resistance to the drug.[22]

6. Pharmacokinetics and metabolism
High intravenous doses of alvocidib are required in order to achieve therapeutic blood levels due to significant binding of the drug to plasma proteins, especially albumin.[28] An aver- age peak serum concentration of 271 uM/L is achieved with a 72-hour continuous infusion of 50 mg/m2. Although this result was above the predicted LC50 for alvocidib, no clinical responses were seen due to protein binding of the drug. A bolus dose plus infusion schedule was devised with a target serum concentration of 1500 uM/L sustained for 4 hours which exceeded the target area under the curve (AUC) of 4.51–31.4 μM/h,[21] and responses in CLL were observed as detailed below. Alvocidib is metabolized to a glucuronide, and both the parent compound and the glucuronide metabolite are substrates for the SLCO1B1/OATP1B1 transporter [29] as well as p-glycoprotein, breast cancer resistance protein, and multi- drug resistance-associated protein 1.[30]
The maximum tolerated dose of alvocidib given as a bolus injection is 80 mg/m2 and 78 mg/m2/day when given as a 72- hour continuous infusion. The plasma half-life for the 80 mg/ m2 bolus injection is 13.2 ±8.8 hours, and plasma concentra- tion is >1.5 uM/L.[21] Brain and testis penetration by alvocidib are limited, in part due to interaction with p-glycoprotein and breast cancer resistance protein.[31]

7. Clinical efficacy
7.1. Phase I studies
In an early Phase I study, [20] 76 patients with refractory solid tumors were treated with alvocidib 72-hour continuous infu- sions every 2 weeks. The maximum tolerated dose was deter- mined to be 50 mg/m2/day × 3 days after secretory diarrhea was observed to be dose-limiting at a dose of 62.5 mg/kg/ day × 3 days. Subsequent patients were pretreated with anti- diarrheal prophylaxis and the maximal tolerated dose for that cohort was determined to be 78 mg/m2/day × 3 days after hypotension was found to be dose-limiting at 98 mg/m2/ day × 3 days. A proinflammatory syndrome with alterations in acute phase reactants was also observed primarily in patients who received a dose >50 mg/m2/day × 3 days, which required discontinuation of therapy in some patients. A mean plasma alvocidib concentration of 271 uM was achieved with the 50 mg/m2/day × 3 days regimen and 344 nM with the 78 mg/m2. These plasma concentrations are within the 200–400 uM range previously determined to be required for cyclin-dependent kinase inhibition in preclinical models. Based on observed tolerability, a regimen of 50 mg/ m2/day × 3 days was recommended for Phase II study.
Despite significant activity of alvocidib against a variety of neoplasms including CLL, continuous infusion clinical trials were disappointing. It was thought that failure was due to major binding of the drug to plasma proteins, which resulted in insufficient available drug for therapeutic activity. Byrd et al. [32,33] on the basis of pharmacokinetic modeling designed a Phase I trial in 52 patients with refractory CLL that provided a bolus loading dose followed by a 4-hour infusion administered weekly for 4 of 6 weeks. The study involved 3 dosing cohorts:

(1) 30 mg/m2 loading dose, then a 30 mg/m2 4-hour infusion,
(2) 40 mg/m2 loading dose and 40 mg/m2 4-hour infusion, and
(3) cohort 1 doses and schedule for treatments 1–4, then a 30 mg/m2 loading dose followed by a 50 mg/m2 4-hour infu- sion. The dose-limiting toxicity in this study was severe TLS with hyperkalemia which in some cases required hemodialysis, which could be prevented by aggressive prophylaxis and excluding patients with a white blood cell count >200,000/ μL. A partial response with a median 1-year response duration was observed in 40% of the 52 treated patients, including 39% of 12 patients with del(17p) and 74% of 18 patients with del (11q). Pharmacokinetic studies demonstrated that the alvoci- dib AUC correlated with clinical response and incidence of CRS and AUC for the glucuronide metabolite correlated with inci- dence of TLS.[33] In an effort to reduce the incidence of serious alvocidib toxicity, the same group conducted a small Phase I study of alvocidib bolus + infusion proceeded by rituximab and cyclophosphamide. Only 9 patients were trea- ted, but this regimen seemed to have prevented or reduced the severity of TLS.[34] The results of this study gave the investigators encouragement that alvocidib could ultimately play a role in the treatment of CLL. Finally, in a recently reported Phase I study, [35] a bolus + infusion regimen of alvocidib was combined with lenalidomide for heavily pre- treated patients with CLL in an effort to debulk patients prior to lenalidomide treatment, and thereby prevent or reduce the severity of toxicity. The overall response rate was 51% and no increased TLS or tumor flare was observed, sug- gesting that this regimen may deserve further development.

7.2. Phase II studies
Cancer and Leukemia Group B performed 2 sequential early Phase II studies of alvocidib in previously treated patients with CLL.[36] Patients in the first trial received alvocidib, 50 mg/ m2day as a continuous intravenous infusion for 72 hours every 2 weeks, and patients in the second trial received the same daily dose of alvocidib as a 1-hour bolus injection daily for 3 days every 3 weeks. Sixty percent of the 16 patients in the first study had high-risk disease. No responses were obtained in that study, although 27% of patients had stable disease. The bolus study included 36 patients and 64% had high-risk dis- ease. Partial responses were observed in 11% and 53% had stable disease after treatment. The median progression-free survival was 2 months in the first study and 3 months in the second study. The median overall survival was 27 months in the continuous infusion study and 24 months in the bolus dosing study. Toxicity in the 2 studies was manageable and consisted of myelosuppression, infection, secretory diarrhea, and fatigue. The investigators concluded that alvocidib had noteworthy activity in CLL and should be further studied, especially with different schedules of drug administration.
A later Phase II study enrolled 64 patients with CLL with a median age of 60 years who had received a median of 4 prior treatment regimens.[37] All had previously received a purine analog. Enrolled patients had a white blood cell count of
<200,000μL. Alvocidib, 30 mg/m2, was given intravenously as a 30 minute bolus followed by 30 mg/m2 as a 4-hour contin- uous infusion for the first dose. Patients who did not develop

severe TLS received 30 mf/M2 bolus doses and continuous infusion doses of 50 mg/m2 for subsequent treatments. The treatment was given weekly for 4 doses every 6 weeks for up to 6 cycles. During the study, modifications were made to reduce toxicity: The cycle length was reduced from 42 to 28 days, the number of treatments per cycle was reduced from 4 to 3, and dexamethasone, 20 mg intravenously was added to each treatment day to reduce interleukin-6 production in an effort to favorably modify CRS and TLS severity.[38] Rasburicase was added to the regimen 2 hours before the first and second alvocidib doses to prevent a rapid rise in serum uric acid, and thereby reduce the likelihood of renal failure requiring dialysis. Pegfilgrastim was also added on Day 16 of each cycle to protect against neutropenia. Patients who needed hemodialysis for TLS continued treatment with a 10 mg/m2 reduction in both the bolus and continuous infu- sion doses. The first 27 patients enrolled on study were treated with the original regimen, 32 received the amended regimen, and another 5 early patients transitioned from the original to the modified regimen while on study. The median age of all treated patients was 60 years. More than three quarters of the patients had advanced stage disease and 73% had bulky lymphadenopathy. The majority of patients (59%) had purine analog-refractory disease. Many patients had high-risk cytoge- netics: 33% had del(17p), 44% had del(11q), and 42% had a complex karyotype. Post-amendment patients were somewhat better risk in that as a group they were younger, had fewer prior treatment regimens, and had a lower rate of bulky lymphadenopathy than patients treated earlier on the study.
Toxicities observed in the study were generally transient and included cytopenias, electrolyte and live function abnorm- alities, fatigue, diarrhea, and infection. However, Grades 3–4 infections occurred in 25% of patients. Planned dosage escala- tion was accomplished in 58 of 62 patients who received at least 2 doses of alvocidib. Four patients who experienced severe TLS were not dose escalated. Three of those 4 patients required hemodialysis, recovered and continued treatment with a 10 mg/m2 dose reduction in both the bolus and con- tinuous infusion segments of the regimen. In total, the median number of delivered treatment cycles was 4 and only 17 patients completed planned therapy. Toxicity was the cause of discontinuing therapy in 8 patients. Only 1 of 27 patients on the original regimen completed planned treatment, whereas 47% post-amendment patients did so. The incidence of CRS was reduced from 56% to 22% with the amendment. Overall, 53% of patients responded to therapy, 41% with the pre- amendment regimen and 63% with the amended regimen. Overall, 52% had a partial response and 1 additional patient had a complete response. There was no significant difference in response rate between patients with adverse cytogenetic or bulky disease and others. A surprising result of the study is that women had a response rate almost double that of men (77% vs. 40%). The median progression-free survival of evalu- able patients was 12 months and did not differ between high- risk and other patients.
A Phase II multicenter international study employing the same dosing and scheduling of alvocidib as in the above study was recently reported.[39] In order to continue on study beyond Cycle 2, patients must have achieved at least a 35%

reduction in lymphocytosis or lymph node volume. Again, TLS prophylaxis consisted of allopurinol and rasburicase adminis- tration, and the first 2 cycles of treatment were administered in hospital. The study enrolled 165 patients with a white blood cell count <150,000/μL and a median age of 61 years. A total of 78% of the patients were male, 81% were Stage III or IV, and 70% had bulky lymphadenopathy. High-risk cytogenetics (del (17p), 35%; del(11q), 31%) were frequent. All patients were previously treated with fludarabine and 90% were refractory to it. Almost all patients had been previously treated with alkylating agents and 87% had received rituximab previously. Of the 165 patients, 159 actually received alvocidib, 35 patients completed the planned 6 cycles of treatment and 79 patients received 3 or more therapy cycles Thirty percent of patients were taken off study due to disease progression and 25% discontinued treatment due to toxicity. Dose escala- tion did not occur due to the prevalence of TLS. Median follow-up for all treated patients was 19.8 months. Responses included 6 complete responses (4%), 44 (27%) partial responses, and stable disease was noted in 66 patients (40%). Progressive disease on therapy was observed in 26 patients (16%). Response was not determined in 23 patients (14%). Stage and number of prior treatment regimens were associated with response, but adverse cytogenetics was not. Median progression-free survival for all responders was 7.6 month and the median duration of response for complete and partial responders was 13.7 months. Median overall survi- val for all patients was 14.6 months.
Toxicity in this study was significant. An adverse event of Grade 3 or 4 occurred in 87% of patients including diarrhea (18%), TLS including 1 death (21%), infections (30%), and
cytopenias (61%).
These studies clearly demonstrate that alvocidib is highly active in previously treated patients with CLL including those with poor-risk factors. However, serious toxicity, irrespective of drug scheduling, continues to plague the development of alvocidib in CLL. Methods to eliminate the need to administer alvocidib in hospital by eliminating CRS and TLS as major toxicities of this agent need to be developed in order to make alvocidib competitive with recently approved new drugs for CLL. Combinations of alvocidib with other agents active in CLL may lead to regimens with less alvocidib toxicity. Recent combination studies in AML have yielded such results.[40]

7.3. Phase III studies
There are no reported Phase III studies of alvocidib in CLL and no such studies are listed on www.clinicaltrials.gov.

8. Safety and tolerability
As noted above, TLS is a major, occasionally fatal, complication of the treatment of CLL with alvocidib. In a retrospective review of 116 treated patients the incidence of TLS was 46%.
[41] Patient characteristics that significantly correlated with the development of TLS were female gender, lymphadenopa- thy ≥ 10 cm, elevated white blood cell count, elevated β2 microglobulin and decreased serum albumin.[41] The

occurrence of TLS did not influence overall or progression- free survival. The same group determined that levels of the glucuronide metabolite of alvocidib were positively associated with the incidence of TLS and females had higher glucuronide exposure than males, explaining the higher incidence of TLS in females.[42] Perhaps leukapheresis prior to alvocidib treat- ment might prevent or modify TLS severity. Leukapheresis can easily reduce the white blood cell count to normal levels in patients with CLL, and, in some cases, reduce bulky dis- ease.[43]
In a recent survey, the incidence of TLS with alvocidib was found to be higher than with any recently approved drug for leukemia.[44]
Initial treatment with low-dose alvocidib to debulk patients might allow for the safe combined treatment with drugs such as lenalidomide and avoid tumor flare which can occur with either drug.[34] Only a small number of patients have been treated in this fashion to date.
CRS has also been noted with some frequency during treatment with alvocidib and has been attributed to IL-6 release, which may respond to dexamethasone.[38] Perhaps other IL-6 inhibitors, such as cyclosporine A [45] or tocilizumab
[46] might be more effective.
A liposomal formulation of alvocidib has been developed [47], and, in mice, showed an increased elimination phase half- life, decreased clearance, and increased AUC compared with free drug administration. Therefore, the liposomal formulation may improve alvocidib pharmacokinetics and potentially reduce toxicity, but it has never been studied in the clinic.

9. Regulatory affairs
Alvocidib received orphan drug designation for treatment of CLL from the U.S. Food and Drug Administration on 13 April 2007 and for AML on 21 April 2014. EMA granted orphan drug status for CLL and AML on 23 October 2007 and 25 February 2015, respectively.

10. Conclusion
Alvocidib, a cyclin dependent kinase inhibitor, has significant activity in patients with relapsed or refractory CLL regardless of karyotype [48] or age.[49] Its clinical development as ther- apy for CLL has been hampered by a relatively high incidence of serious toxicity necessitating inpatient treatment initially, and by the recent development of agents with similar efficacy but significantly less toxicity.

11. Expert opinion
Alvocidib was the first in class cyclin dependent kinase inhibitor to come to clinical trial. The drug is highly active in relapsed and refractory CLL, but a high frequency of serious toxicity has ham- pered its development as a treatment for CLL. Studies of alter- native methods to manage or prevent TLS and CRS, or newer formulations of the drug (liposomal encapsulation) may improve the therapeutic index. Combination of alvocidib with other agents active in CLL may ultimately identify a role for alvocidib

in the treatment of newly diagnosed patients. Such combina- tions have proven to be of benefit recently in AML.
Unfortunately, with an abundance of new drugs for the treatment of CLL that have major activity, including activity in patients with poor prognostic factors and less toxicity than alvocidib, alvocidib is unlikely to emerge as a single-drug treatment for CLL. Data are insufficient for approval in the USA or Europe at present. If the drug was approved, physi- cians would be reluctant to prescribe it due to its cumbersome administration requiring hospitalization, and its serious toxi- city. No Phase III studies of alvocidib have been reported. Unless drug combinations employing much lower doses of alvocidib that show a clear advantage for addition of the drug can be devised, development of alvocidib for treatment of CLL will be discontinued.

Declaration of interest
P Wiernik is Director of the Cancer Research Foundation of New York. He has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

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