Geoffrey D. Miller

Basics and recent advances in peptide and protein drug delivery.

While the peptide and protein therapeutic market has developed significantly in the past decades, delivery has limited their use. Although oral delivery is preferred, most are currently delivered intravenously or subcutaneously due to degradation and limited absorption in the gastrointestinal tract. Therefore, absorption enhancers, enzyme inhibitors, carrier systems and stability enhancers are being studied to facilitate oral peptide delivery. Additionally, transdermal peptide delivery avoids the issues of the gastrointestinal tract, but also faces absorption limitations. Due to proteases, opsonization and agglutination, free peptides are not systemically stable without modifications. This review discusses oral and transdermal peptide drug delivery, focusing on barriers and solutions to absorption and stability issues. Methods to increase systemic stability and site-specific delivery are also discussed.

Resistant mutations in CML and Ph + ALL – role of ponatinib

In 2012, ponatinib (Iclusig®), an orally available pan-BCR-ABL tyrosine kinase inhibitor (TKI) developed by ARIAD Pharmaceuticals, Inc., was approved by the US Food and Drug Administration for use in resistant or intolerant chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL). Ponatinib is the only approved TKI capable of inhibiting BCR-ABL with the gatekeeper T315I kinase domain mutation, known to be the cause for 20% of resistant or relapsed CML cases. In 2013, ponatinib sales were temporarily suspended due to serious side effects seen in nearly 12% of the patient population. These side effects are thought to stem from the potent nature and pan-activity of this TKI. ARIAD Pharmaceuticals, Inc. has since been permitted to resume sales and marketing of ponatinib to a limited patient population with an expanded black box warning. In the following review, the use of ponatinib in CML and Ph+ALL will be discussed. Mechanisms of resistance in CML are discussed, which provide insight and background into the need for this third generation TKI, followed by the molecular design and pharmacology of ponatinib, which lead to its success as a therapeutic. Finally, the efficacy, safety, and tolerability of ponatinib will be highlighted, including summaries of the important clinical trials involving ponatinib as well as its current place in therapy.

Improved coiled-coil design enhances interaction with Bcr-Abl and induces apoptosis.

The oncoprotein Bcr-Abl drives aberrant downstream activity through trans-autophosphorylation of homo-oligomers in chronic myelogenous leukemia (CML).(1, 2) The formation of Bcr-Abl oligomers is achieved through the coiled-coil domain at the N-terminus of Bcr.(3, 4) We have previously reported a modified version of this coiled-coil domain, CCmut2, which exhibits disruption of Bcr-Abl oligomeric complexes and results in decreased proliferation of CML cells and induction of apoptosis.(5) A major contributing factor to these enhanced capabilities is the destabilization of the CCmut2 homodimers, increasing the availability to interact with and inhibit Bcr-Abl. Here, we included an additional mutation (K39E) that could in turn further destabilize the mutant homodimer. Incorporation of this modification into CCmut2 (C38A, S41R, L45D, E48R, Q60E) generated what we termed CCmut3, and resulted in further improvements in the binding properties with the wild-type coiled-coil domain representative of Bcr-Abl [corrected]. A separate construct containing one revert mutation, CCmut4, did not demonstrate improved oligomeric properties and indicated the importance of the L45D mutation. CCmut3 demonstrated improved oligomerization via a two-hybrid assay as well as through colocalization studies, in addition to showing similar biologic activity as CCmut2. The improved binding between CCmut3 and the Bcr-Abl coiled-coil may be used to redirect Bcr-Abl to alternative subcellular locations with interesting therapeutic implications.

Multidomain targeting of Bcr-Abl by disruption of oligomerization and tyrosine kinase inhibition: toward eradication of CML.

The oncoprotein Bcr-Abl, the causative agent of chronic myeloid leukemia (CML), requires homo-oligomerization via a coiled-coil domain to function [Bartram, C. R.; et al. Nature 1983, 306 (5940), 277-280; and Zhao, X.; et al. Nat. Struct. Biol. 2002, 9(2), 117-120]. While tyrosine kinase inhibitors (TKIs) have shown great efficacy as treatment options for CML, their use may cause an acquisition of mutations in the tyrosine kinase domain, which prevent TKI binding and lead to a loss in activity [Woessner, D. W.; et al. Cancer J. 2011, 17(6), 477-486]. Previously, we have shown that a rationally modified coiled-coil domain (CC(mut3)) can disrupt this oligomerization, inhibit proliferation, and induce apoptosis in CML cells [Dixon, A. S.; et al. Mol. Pharmaceutics 2012, 9(1), 187-195]. Here, we show that using the most recently approved TKI, ponatinib (Iclusig), in combination with CC(mut3) allows a dose reduction of ponatinib and increased therapeutic efficacy in vitro measured by reduction in kinase activity, induction of apoptosis via caspase-3/7 and 7-AAD/Annexin V assays, and reduced transformative ability measured by a colony forming assay. The combination was effective not only in cells containing wild-type Bcr-Abl (K562, Ba/F3-p210) but also cells with Bcr-Abl containing the T315I mutation (Ba/F3-p210-T315I). In addition, we report for the first time the ability of CC(mut3) alone to inhibit the T315I mutant form of Bcr-Abl. This novel combination may prove to be more potent than single agent therapies and should be further explored for clinical use.

Intranasal delivery of Natesto® testosterone gel and its effects on doping markers

The laboratory profile of intranasal testosterone gel has not been previously reported from an anti-doping perspective. Because intranasal testosterone gel is newly available as a commercial product, we sought to examine the laboratory parameters following administration of this formulation, with particular attention to anti-doping guidelines. Five healthy and active male subjects were administered testosterone intranasal gel three times daily for four weeks, using a pattern of five consecutive days on, two days off. Urine was collected after each five-day round of drug administration and analyzed using a full steroid screen and isotope ratio mass spectrometry (IRMS). Windows of detection for elevated testosterone/epitestosterone (T/E) and other steroid ratios, World Anti-Doping Agency (WADA) athlete biological passport (ABP) findings, and IRMS results were analyzed in this study. In the 0-24 h window post-administration, 70% of samples were flagged with a suspicious steroid profile and 85% were flagged as atypical passport findings according to the WADA ABP steroid module. In the 24-48 h window, 0% of samples displayed suspicious steroid profiles while 40% resulted in atypical passport findings. IRMS testing confirmed the presence of exogenous testosterone in 90% and 40% of samples in the 0-24 h and 24-48 h windows post-administration, respectively. Additionally, IRMS data were analyzed to determine commonalities in the population changes in δ13 C values of testosterone, androsterone, etiocholanolone, 5αAdiol, and 5βAdiol. Though no discernible metabolic trend of the route of administration was identified, we discovered that intranasal gel testosterone is detectable using conventional anti-doping tests. Copyright

A coiled-coil mimetic intercepts BCR-ABL1 dimerization in native and kinase-mutant chronic myeloid leukemia

Targeted therapy of chronic myeloid leukemia (CML) is currently based on small-molecule inhibitors that directly bind the tyrosine kinase domain of BCR-ABL1. This strategy has generally been successful, but is subject to drug resistance because of point mutations in the kinase domain. Kinase activity requires transactivation of BCR-ABL1 following an oligomerization event, which is mediated by the coiled-coil (CC) domain at the N terminus of the protein. Here, we describe a rationally engineered mutant version of the CC domain, called CCmut3, which interferes with BCR-ABL1 oligomerization and promotes apoptosis in BCR-ABL1-expressing cells, regardless of kinase domain mutation status. CCmut3 exhibits strong proapoptotic and antiproliferative activity in cell lines expressing native BCR-ABL1, single kinase domain mutant BCR-ABL1 (E255V and T315I) or compound-mutant BCR-ABL1 (E255V/T315I). Moreover, CCmut3 inhibits colony formation by primary CML CD34+ cells ex vivo, including a sample expressing the T315I mutant. These data suggest that targeting BCR-ABL1 with CC mutants may provide a novel alternative strategy for treating patients with resistance to current targeted therapies.

HPT-Axis Effects and Urinary Detection Following Clomiphene Administration in Males

Context Clomiphene is a performance-enhancing drug commonly abused by males in sport, but the extent to which testosterone increases in healthy males following its use is unknown. In addition, evidence suggests that clomiphene, a mixture of cis- and trans-isomers zuclomiphene and enclomiphene, is detectable in urine for months following use; the isomer-specific urinary detection window has yet to be characterized in a controlled study. Objective To determine the effect of once-daily, 30-day clomiphene treatment on serum testosterone and gonadotropin levels in the subject population studied and the urinary clearance and detection window of clomiphene isomers following administration for antidoping purposes. Participants and Design Twelve healthy males aged 25 to 38 years, representing a recreational athlete population, participated in this open-label, single-arm study. Intervention Oral clomiphene citrate (50 mg) was self-administered once daily for 30 days. Serum and urine samples were collected at baseline and at days 7, 14, 21, 28, 30, 32, 35, 37, 44, 51, and 58; urine collections continued periodically up to day 261. Results Mean testosterone, LH, and FSH levels increased 146% (SEM, ±23%), 177% (±34%), and 170% (±33%), respectively, during treatment compared with baseline. Serum drug concentrations and urinary excretion were nonuniform among individuals as isomeric concentrations varied. The zuclomiphene urinary detection window ranged from 121 to >261 days. Conclusions Clomiphene significantly raised serum testosterone and gonadotropin levels in healthy men and thus can be abused as a performance-enhancing drug. Such abuse is detectable in urine for ≥4 months following short-term use.

Evaluation of serum markers for improved detection of autologous blood transfusions

Autologous blood transfusion is used by athletes to enhance performance by increasing oxygen transport to muscles. It is the only form of blood doping that is currently not detectable using a direct method. Autologous blood transfusions are detected by an indirect method which monitors the

The Anti-Doping Movement.

Historical reports of doping in sports date as far back as the ancient Greek Olympic Games. The anti‐doping community considers doping in sports to be cheating and a violation of the spirit of sport. During the past century, there has been an increasing awareness of the extent of doping in sports and the health risks of doping. In response, the anti‐doping movement has endeavored to educate athletes and others about the health risks of doping and promote a level playing field. Doping control is now undertaken in most countries around the world and at most elite sports competitions. As athletes have found new ways to dope, however, the anti‐doping community has endeavored to strengthen its educational and deterrence efforts. It is incumbent upon sports medicine professionals to understand the health risks of doping and all doping control processes.

Abstract 3852: Improved coiled-coil design enhances interaction with Bcr-Abl and induces apoptosis

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The fusion protein Bcr-Abl responsible for the oncogenic activity in chronic myelogenous leukemia (CML) depends heavily on oligomerization for function. Formation of Bcr-Abl oligomers occurs through a coiled-coil domain existing at the N-terminal portion of the Bcr segment. Previously, we have shown that disruption of this oligomerization utilizing a modified coiled-coil domain (CCmut2) both decreases the proliferation of CML cells and results in the induction of apoptosis. An important contributing factor to these results stems from the increased destabilization of CCmut2 homo-dimers. This phenomenon occurs due to the rationally mutated residues in the modified coiled-coil which allow for favorable interactions with the wild-type Bcr-Abl, but destabilizing interactions with other CCmut2 molecules, thus discouraging the formation of CCmut2 homo-dimers. In turn, this increases the availability and affinity for the modified coiled-coil to interact with and inhibit endogenous Bcr-Abl in CML cells. An additional mutation (K39E) was included that could potentially further destabilize the mutant homo-dimer. Added to the other mutations found in CCmut2 (C38A, S41R, L45N, E48R, Q60E), the new construct was termed CCmut3. To show that CCmut3 results in enhanced binding to a wild-type coiled-coil domain (representative of endogenous Bcr-Abl), 2 separate assays were performed. Colocalization studies were used to show the degree to which CCmut3 localizes intracellularly with Bcr-Abl. Additionally, a mammalian two-hybrid assay was conducted to show the relative selective dimerization between CCmut3 and the Bcr-Abl coiled-coil. Next, caspase 3/7 assays were performed and showed the ability of CCmut3 to induce apoptosis. The CCmut 3 also showed nuclear segmentation (a measurement of late-stage apoptosis) and decrease in oncogenic proliferation (via the colony forming assay). Taken together, these results provide strong evidence for using CCmut3 as a therapeutic for CML. Lastly, attempts to truncate the CCmut3 to find the smallest possible sequence needed (e.g., helix 2) to bind and kill cells effectively is currently underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3852. doi:1538-7445.AM2012-3852