Real-world evidence analysis of palbociclib prescribing patterns for patients with advanced/metastatic breast cancer treated in community oncology practice in the USA one year post approval.

BACKGROUND: Rapidly evolving understanding of cancer biology has presented novel opportunities to translate that understanding into clinically relevant therapy. Palbociclib, a novel, first-in-class cyclin-dependent kinase (CDK) 4/6 inhibitor was approved in the USA in February 2015 for the treatment of advanced/metastatic breast cancer. We examined real-world evidence in the first year post approval to understand the clinical and demographic characteristics of patients treated with palbociclib in community oncology practices and the dosing, treatment, and complete blood count (CBC) monitoring patterns. METHODS: This was a retrospective observational study of structured data from a US electronic medical record (EMR) database. Female patients receiving palbociclib after 31 January 2015 were followed through 31 March 2016. Our methodological rules were constructed to aggregate drugs received according to the order in which they are given, i.e., identify the line of therapy as first, second, or third line, etc., using treatment order and course description fields from the EMR. RESULTS: There were 763 patients initiating palbociclib who met the selection criteria. Of those, 612 (80.2%) received palbociclib concomitantly with letrozole. Mean follow up was 6.4 months and mean age at palbociclib initiation was 64 years. Of patients with a known starting dose (n = 417), 79.9% started on palbociclib 125 mg. Dose reductions were observed in 20.1% of patients. Percentages of patients according to line of therapy at initiation of palbociclib were first-line, 39.5%; second-line, 15.7%; third-line, 13.1%; and fourth-line therapy or later, 31.7%. On average, two CBC tests were conducted during the first cycle of palbociclib treatment. Overall, 74.6% of patients had a neutropenic event during follow up including 47.3% and 8.0% of patients with a grade 3 or 4 occurrence, respectively. CONCLUSIONS: Real-world palbociclib use one year post US approval demonstrates a more heterogeneous patient population than that studied in the clinical trials with more than half of the patients receiving palbociclib plus letrozole in later lines of therapy. CBC testing rates suggested good provider compliance with monitoring guidelines in the USA prescribing information. The occurrence of grade 3 and 4 neutropenia (based on laboratory results) was consistent with the rates of grade 3 and 4 neutropenia in two phase-III studies (PALOMA-2, 56% and 10%; PALOMA-3, 55% and 11%, respectively). Understanding palbociclib utilization in real-world patients and how drug dosing and monitoring are performed aids in the understanding of safe and effective use of the drug.

Partitioning of electrostatic and conformational contributions in the redox reactions of modified cytochromes c.

The reduction of acetylated, fully succinylated and dicarboxymethyl horse cytochromes c by the radicals CH3CH(OH), CO2.-, O2.-, and e-aq' and the oxidation of the reduced cytochrome c derivatives by Fe(CN)3-6 were studied using the pulse radiolysis technique. Many of the reactions were also examined as a function of ionic strength. By obtaining rate constants for the reactions of differently charged small molecules redox agents with the differently charged cytochrome c derivatives at both zero ionic strength and infinite ionic strength, electrostatic and conformational contributions to the electron transfer mechanism were effectively partioned from each other in some cases. In regard to cytochrome c electron transfer mechanism, the results, especially those for which conformational influences predominate, are supportive of the electron being transferred in the heme edge region.

Cyanide reactivity of cytochrome c derivatives.

The kinetic rates and equilibrium association constants for cyanide binding have been measured for a series of cytochrome c derivatives as a probe of heme accessibility. The series included horse and yeast cytochromes iodinated at Tyr 67 and 74, horse cytochrome formylated at Trp 59 in both a low and high redox potential form, the Met 80 sulfoxide derivative of horse cytochrome and the N-acylisourea heme propionate derivative of tuna cytochrome. Native cytochromes c are well known to bind cyanide slowly in a reaction simply first order both in cytochrome and cyanide up to at least 100 mM in cyanide. The derivative demonstrate markedly different kinetics which indicate the following conclusions. (1) In spite of chemical modification at different loci, all the derivatives have highly similar reactivity, suggesting common ligation structures and mechanisms for reaction. (2) Compared to native cytochromes, reaction rates are 10-20 fold greater. This is in accord with a more accessible heme crevice, but not a completely opened crevice. For the completely opened case, rate increases are expected to be between three and five orders of magnitude. (3) Reaction rates are either independent of cyanide concentration (zero order) or show only slight variation. A mechanism which accounts for the data over four orders of magnitude in concentration postulates a protein conformation step, opening of the heme crevice, as the rate determining step. This conformation change has a limiting rate of 6 . 10(-2) s-1.

The role of histidine-42 in the oxidation-reduction mechanism of Chromatium vinosum high potential iron-sulfur protein.

The second order rate constants for the oxidation of high potential iron-sulfur protein (Hipip) of Chromatium vinosum by ferricyanide were determined as a function of ionic strength and pH. From the ionic strength results, calculations were done to correct the rate constant at each pH for the electrostatic interactions between Hipip and ferricyanide. The electrostatic corrections are necessary since the charge of the protein changes as a function of pH and can mask the ionization of mechanistically important amino acid residues. An apparent pKa congruent to 7 was obtained from electrostatically corrected rate-pH profile, indicating the possible participation of histidine. Perturbation difference spectroscopic studies of Hipip as a function of pH also gave apparent pKa values of 6.9 and 6.7 for the reduced and oxidized protein, respectively. That it was indeed His 42 (the only His in the polypeptide) that was responsible for the kinetic and spectroscopic pKa values was demonstrated by modification of His 42 of Hipip by the histidine selective reagent diethylpyrocarbonate. No modification of Tyr 19 could be detected. It is concluded that either deprotonation or modification of His 42 results in the destabilization of the reduced cluster and thus a faster rate of oxidation. This work provides the first experimental evidence of the 'squeeze effect' mechanism (Carter, C.W., Jr., Kraut, J., Freer, S.T. and Alden, R.A. (1974) J. Biol. Chem. 249, 6339--6346) in which the polypeptide directly modulates the stability of the iron-sulfur cluster.

Methionine-80-sulfoxide cytochrome c: preparation, purification and electron-transfer capabilities.

In order to explore the electron-transferring properties of methionine-80-sulfoxide cytochrome c, the pure, chromatographically homogeneous methionine-80-sulfoxide cytochrome c was previously published procedure (Ivanetich, K.M., Bradshaw, J.J. and Kaminsky, L.S. (1976) Biochemistry 15, 1144-1153) was found to produce a mixture of products. In the pure derivative, visible spectroscopy indicates that the 695 nm band indicative of the Met-80-Fe coordination is missing, amino acid analysis indicates that only one methionine is modified to the sulfoxide, and the E0' is found to be 240 mV vs. N.H.E. For succinate cytochrome c reductase activity, the Km for modified cytochrome was about one-ninth that of the native protein, while the maximum turnover number of the reductase with the modified protein was only about 54% of that with native protein. In contrast, the activity with cytochrome oxidase measured polarographically using ascorbate and TMPD under two different buffer/pH conditions, gave Km values that were very similar for both the native and modified cytochromes c, but the maximum turnover numbers of the oxidase with the modified protein were less than 40% of native in either buffer. It is concluded that the Met-80-sulfoxide cytochrome c in the reduced form is able to maintain substantially its heme crevice structure and thus maintain Km values similar to those of native protein. However, the low maximum turnover numbers for oxidase activity with the modified protein in the reduced state indicate that electron transfer itself has been significantly decreased, probably because the parity of acid/base and electrostatic interactions of Met-80 sulfur with the Fe in the two redox states has been disrupted.

Molecular interpretation of kinetic-ionic strength effects.

A recent and important approach to investigating electron transfer mechanisms of redox proteins has been through kinetic-ionic strength studies. There is, however, significant controversy as to whether such studies (1) yield information regarding the charge (or location) of the electron transfer site or (2) more simply reflect the influence of net or overall protein charge on the electrostatic interactions. A critical analysis using different theoretical approaches is made of our recent work and of the bulk of the published non-physiological small molecule-protein and protein-protein kinetic ionic strength studies; it is concluded that (1) the approximated Bronsted-Debye-Huckel equation can not be used at all for protein redox reactions, (2) irrespective of the theoretical approaches discussed, such studies do not provide information regarding the charge of the electron transfer site, (3) it is the net charge of the reactants that control the electrostatic interactions, (4) both the equation derived by Wherland and Gray and the full Bronsted-Debye-Huckel equation provide reasonably good approximations of net protein charge, (5) pH changes quantitatively modulate net protein charge, and (6) thus, protein redox rates need to be electrostatically corrected if relevant interpretations of kinetic-ionic strength experiments are to be made.

Complications of organic solvents in synthetic drug application.

Aziridinylbenzoquinone (AZQ), Acridinyl Anisidide (m-AMSA), Tenipsode Thenvlidene-Lignan-(VM-26), and PCNU are relatively new synthetic drugs believed to have a wide spectrum of antitumor activity. Preparation of these drugs for administration requires reconstitution with an organic solvent which decomposes the thermoplastic components of I.V. infusion devices and chemo-dispensing pins. The products of this decomposition may lead to cellular damage and complicate the infusion of these agents.

Using entropies of reaction to predict changes in protein stability: tyrosine-67-phenylalanine variants of rat cytochrome c and yeast Iso-1 cytochromes c.

Using the voltammetric method of square-wave voltammetry, a direct electrochemical examination was made of the wild type and Tyr67Phe mutant of both rat cytochrome c and yeast iso-1-cytochrome c. In addition to determining the equilibrium reduction potential (E0') for each cytochrome, the entropy of reaction, deltaS0'(Rxn)(deltaS0'(Rxn) = S0'(Red) - S0'(Ox)), for the reduction process was determined via the non-isothermal method. Having determined deltaS0'(Rxn) and E0', deltaH0' was calculated. For rat cytochrome c, it was found that deltaS0'(Rxn) = -43 J mol(-1) K(-1) for the wild type and -53 J mol(-1) K(-1) for the Tyr67Phe variant, with the deltaH0' for both the wild type and variant nearly identical, indicating that the changes in reduction potential and probably stability are due to changes in deltaS0'(Rxn). In contrast the measured deltaS0'(Rxn) for yeast iso-1-cytochrome c demonstrated significant changes in both entropic and enthalpic contributions in going from wild type to mutant cytochrome c. The entropy of reaction provides information regarding the relative degree of solvation, and very likely the degree of compactness, of the oxidized state versus the reduced state of the redox protein. A thermodynamic scheme and stability derivation are presented that show how the entropies of reaction of wild type versus variant cytochromes contribute to and predict changes in stability in going from oxidized to reduced protein. For yeast iso-1-cytochrome c, the thermodynamically predicted change in stability was very close to the experimentally observed value, based on previous differential scanning calorimetric stability measurements. While such data is not available for rat cytochrome c, consideration of the enormously increased local stability of the rat oxidized cytochrome c variant predicts that the reduced rat variant will be even more stable than the already stabilized oxidized variant.

Characterization of the recombinant Clostridium pasteurianum ferredoxin and comparison of its properties with those of the native protein.

Ferredoxins (Fds) constitute an important class of nonheme iron-sulfur proteins. One of the most studied Fds is the [8Fe-8S] Fd from Clostridium pasteurianum. The gene for this Fd has previously been cloned and sequenced. We report the expression of this Fd in Escherichia coli, and the characterization and comparison of this recombinant protein to the native Fd. We have found that the purified recombinant protein has the same enzymatic, redox, magnetic and electronic properties as the native Fd isolated from C. pasteurianum, which indicates that the two [4Fe-4S] clusters present in the Fd were correctly formed in E. coli.

High-potential iron-sulfur proteins and their possible site of electron transfer.

The electron-transfer mechanism of the Fe4S4 high-potential iron-sulfur proteins (HiPIP's) was explored via a stopped-flow spectrophotometric kinetic study of the reduction of Chromatium vinosum and Rhodopseudomonas gelatinosa HiPIP's by both native and trinitrophenyllysine-13 horse cytochrome c. The influence of electrostatic effects was also effectively partitioned from the redox process per se. The corrected rates were 12.3 X 10(4) and 3.8 X 10(4) M-1 s-1 for native with C. vinosum and R. gelatinosa HiPIP, respectively, and 17.5 X 10(4) and 5.46 X 10(4) M-1 s-1 for TNP-cytochrome c with the two HiPIP's, respectively. The faster rates of TNP-cytochrome c with the HiPIP's are unexpected in terms of possible steric interaction since lysine-13 is at the top of the heme crevice. In understanding the somewhat faster rates of the TNP-cytochrome c over native cytochrome c it is possible that (1) TNP-cytochrome c reacts more quickly since modification of the lysine-13 residue destabilizes somewhat the heme crevice or (2) in light of the hydrophobic nature of the trinitrophenyl group and the X-ray crystallographic structure of HiPIP, the TNP group facilitates electron transfer by interacting with a hydrophobic region on the HiPIP molecular surface. The region about the S4 sulfur atom is the most exposed and accessible hydrophobic region on the HiPIP surface, in addition to being the point of closest approach of the S4 to the external environment.

Redox properties of several bacterial ferredoxins using square wave voltammetry.

The equilibrium reduction potential of the 2[4Fe-4S] ferredoxin (Fd) isolated from four different bacterial strains was determined at a methyl viologen-modified gold electrode using square wave voltammetry. The observed reduction potential at pH 8 for Clostridium thermoaceticum Fd was -385 mV; Clostridium pasteurianum, -393 mV; Clostridium thermosaccharolyticum, -408 mV; and Chromatium vinosum, -460 mV versus normal hydrogen electrode at 25 degrees C. The reduction potential of the C. pasteurianum Fd was found to be pH independent from pH 6.4 to 8.7, indicating that the electron transfer mechanism does not involve proton exchange. In contrast, the reduction potential of the C. thermosaccharolyticum Fd was found to be pH dependent from pH 6.4 to 8.7, with pKox approximately 7 and pKred approximately 7.5. The +30 mV change in reduction potential from pH 8.7 to 6.4 was attributed to an electrostatic interaction between the iron-sulfur cluster II and the protonated histidine 2 residue located about 6 A away. The Ch. vinosum Fd interacted reversibly at the methyl viologen-modified gold electrode, and its reduction potential was verified using visible spectroelectrochemistry. The reduction potential of Ch. vinosum Fd was found to be 30 mV more positive than previously reported. The similarities of the bacterial Fd reduction potentials are discussed in terms of the homology of their primary structure as reflected by the similarities in the visible and circular dichroic spectra.

Direct square-wave voltammetry of superoxidized [4Fe-4S]3+ aconitase and associated 3Fe/4Fe cluster interconversions.

We report a direct square-wave voltammetric study of the iron-sulfur enzyme, aconitase, at the pyrolytic graphite edge electrode. New and established redox driven reactions were observed and the equilibrium reduction potential for each couple was determined: E0'[3Fe-4S]1+/0 = -268 mV, E0'[4Fe-4S]2+/1+ = -450 mV, E0'[4Fe-4S]3+/2+ = +100 mV, E0'Linear Form = -281 mV, and putatively, E0'[3Fe-4S]0/2- congruent to -1000 mV, all versus normal hydrogen electrode. Most importantly we have directly observed the superoxidized [4Fe-4S]3+ form of aconitase (originally proposed by Emptage, M. H., Dreyer, J.-L., Kennedy, M. C., and Beinert, H. (1983) J. Biol. Chem. 258, 11106-11111) and directly followed its conversion to the [3Fe-4S]1+ form; this intermediate is required for the deactivation of aconitase. Without exogenous ferrous iron, [3Fe-4S]0 aconitase is apparently super-reduced at very negative potentials to the [3Fe-4S]2- form and the concomitant formation of [4Fe-4S]2+ aconitase was followed over time. It is the apparent decomposition of super-reduced [3Fe-4S]2- aconitase that provides the source of ferrous iron for the interconversion of [3Fe-4S]0 aconitase to the [4Fe-4S]2+ form. Voltammetry of free and substrate bound [4Fe-4S]2+ aconitase showed that the latter is less susceptible to oxidation but, surprisingly, has the same E0'[4Fe-4S]3+/2+.

A spectroelectrochemical cell designed for low temperature electron paramagnetic resonance titration of oxygen-sensitive proteins.

In this paper we describe an anaerobic titrator made virtually from glass with a small amount of high vacuum epoxy mounted directly to a quartz EPR tube. A complete titration may be carried out with as little as 600 microliters of sample. This cell features the anaerobic manipulation of an electrochemically poised solution from an electrochemical pouch to an EPR tube. The cell uses a gold foil working electrode and Ag/AgCl reference and counter electrodes. The reference and counter electrodes are isolated from the sample by leached Vycor glass. In the work reported here, we used this cell to determine the equilibrium redox potential of methyl viologen in an EPR titration. With methyl viologen as an indicator we found that the cell has a residual oxygen level of 1.5 microM with a leak rate of 0.005 nmol/min. After moving the solution into the EPR tube, freezing, performing EPR, and thawing, the potential of the methyl viologen solution drifted only 2 mV. During the titration, the poised potentials were stable, drifting only 1 mV/min. Formal potentials as low as -630 mV in a vitamin B12-type protein have been determined with this cell (S. R. Harder, W.-P. Lu, B. A. Feinberg, and S. W. Ragsdale (1989) Biochemistry, in press).

Electrostatic effect upon association of reduced nicotinamide adenine dinucleotide and equine liver alcohol dehydrogenase.

The rate of association of equine liver alcohol dehydrogenase and its coenzymes exhibits a large pH dependence with slower rates at basic pH and an observed kinetic pKa value of approximately 9-9.5. This pH dependence has been explained by invoking local active site electrostatic effects which result in repulsion of the negatively charged coenzyme and the ionized hydroxyl anion form of the zinc-bound water molecule. We have examined a simpler hypothesis, namely, that the pH dependence results from the electrostatic interaction of the coenzyme and the enzyme which changes from an attractive interaction of the negatively charged coenzyme and the positively charged enzyme to a repulsive interaction between the two negatively charged species at the isoelectric point for the enzyme (pH 8.7). We have tested this proposal by examining the ionic strength dependence of the association rate constant at various pH values. These data have been interpreted by using the Wherland-Gray equation, which we have shown can be applied to the kinetics of enzyme-coenzyme association. Our results indicate that the shielding of the buffer electrolyte changes from a negative to a positive value as the charge on the protein changes at the isoelectric point. This result is exactly that which is predicted for electrostatic effects that depend on the charge of the protein molecule and is not consistent with predictions based upon the local active site effects. At low ionic strength values of 10 mM or less, approximately 75% of the observed pH dependence results from the enzyme electrostatic effects; the remaining pH dependence may result from active site effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Are reduction potentials of antifungal agents relevant to activity?

Cyclic voltammetry data were obtained for several categories of fungicidal agents including quinones (akrobomycin, podosporin A), iminium ions and precursors (pyridazines, 15-azahomosterol, griseofulvin-4'-oxime), and metal derivatives of chelators (pyridine-2-aldehyde thiosemicarbazones). The reductions usually occurred in the range of -0.7 to +0.3 V. Reduction potentials provide information on the feasibility of electron transfer in vivo. Catalytic production of oxidative stress from redox cycling is a possible mode of action. Alternatively, there may be interference with normal electron transport chains.

Energetics of beta-oxidation. Reduction potentials of general fatty acyl-CoA dehydrogenase, electron transfer flavoprotein, and fatty acyl-CoA substrates.

We have determined reduction potentials for porcine mitochondrial general fatty acyl-CoA dehydrogenase (GAD) and electron transfer flavoprotein (ETF) using an anaerobic spectroelectrochemical titration method. Computer simulation techniques were used to analyze the absorbance data. Nernst plots of the simulated data gave E'0, 7.1, quinone/semiquinone = -0.014 V and E'0, 7.1, semiquinone/hydroquinone = -0.036 V for ETF and E'0, 7.1, quinone/semiquinone = -0.155 V and E'0, 7.1, semiquinone/hydroquinone = -0.122 V for GAD. Using these techniques we have also determined a conditional reduction potential of -0.156 V for the chromophore producing fatty acyl-CoA substrate beta-2-furylpropionyl-CoA. From this value and our previous determination of the equilibrium constant for the transhydrogenation reaction between beta-2-furylpropionyl-CoA and the oxidized substrate crotonyl-CoA (Keq = 10.4), we have determined a reduction potential of -0.126 V for the butyryl-CoA/crotonyl-CoA couple. In light of the structural similarity between butyryl-CoA and octanoyl-CoA, the optimal substrate for GAD, the reduction potential for octanoyl-CoA should be similar to that for butyryl-CoA; i.e. fatty acyl-CoA substrates and GAD are essentially isopotential. The ability of octanoyl-CoA to reduce GAD quantitatively (Keq = 9.0) poses a dilemma in light of the nearly equal reduction potentials. We postulate that the stable charge-transfer complex formed between enzyme and optimal product is significantly lower in energy than enzyme and product and thus is responsible for pulling the reaction toward completion.

Partial purification and characterization of a new intracellular beta-glucosidase of Trichoderma reesei.

A new intracellular beta-glucosidase was isolated from Trichoderma reesei. It was sequentially purified by (NH4)2SO4 precipitation and chromatography and rechromatography on Sephadex G-150. The enzyme has a mol.wt. of 98 000, optimal activity at pH 6.5, pI 4.4 and Km values of 6.7 mM and 3.3 mM for sophorose and cellobiose respectively. Possible functions of the enzyme may be regulation of cellulase induction and/or to serve as a proenzyme.

Lactoperoxidase-catalyzed iodination of horse cytochrome c:monoiodotyrosyl 74 cytochrome c.

Iodination of horse cytochrome c with the lactoperoxidase-hydrogen peroxide-iodide system results initially in the formation of the monoiodotyrosyl 74 derivative. This singly modified protein was obtained in pure form by ion exchange chromatography and preparative column electrophoresis. It shows an intact 695 nm absorption band, the midpoint potential of the native protein, a nuclear magnetic resonance spectrum which indicates an undisturbed heme crevice structure, a normal reaction with antibodies directed against native horse cytochrome c, and circular dichroic spectra in which the only changes from those of the native protein can be ascribed to the spectral properties of iodotyrosine itself. This conformationally intact derivative reacts with the succinate-cytochrome c reductase and the cytochrome c oxidase systems of beef mitochondrial particle preparations indistinguishably from the unmodified protein, showing that the region including tyrosine 74 is not involved in these enzymic electron transfer functions of the protein. The circular dichroic spectra of this derivative indicate that the minima observed at 288 and 282 nm in the spectrum of native ferricytochrome c originate from tyrosyl residue 74.

Spectroelectrochemical studies of the corrinoid/iron-sulfur protein involved in acetyl coenzyme A synthesis by Clostridium thermoaceticum.

An 88-kDa corrinoid/iron-sulfur protein (C/Fe-SP) is the methyl carrier protein in the acetyl-CoA pathway of Clostridium thermoaceticum. In previous studies, it was found that this C/Fe-SP contains (5-methoxybenzimidazolyl)cobamide and a [4Fe-4S]2+/1+ center, both of which undergo redox cycling during catalysis, and that the benzimidazole base is uncoordinated to the cobalt (base off) in all three redox states, 3+, 2+, and 1+ [Ragsdale, S.W., Lindahl, P.A., & Münck, E. (1987) J. Biol. Chem. 262, 14289-14297]. In this paper, we have determined the midpoint reduction potentials for the metal centers in this C/Fe-SP by electron paramagnetic resonance and UV-visible spectroelectrochemical methods. The midpoint reduction potentials for the Co3+/2+ and the Co2+/1 couples of the corrinoid were found to be 300-350 and -504 mV (+/- 3 mV) in Tris-HCl at pH 7.6, respectively. We also removed the (5-methoxybenzimidazolyl)cobamide cofactor from the C/Fe-SP and determined that its Co3+/2+ reduction potential is 207 mV at pH 7.6. The midpoint potential for the [4Fe-4S]2+/1+ couple in the C/Fe-SP was determined to be -523 mV (+/- 5 mV). Removal of this cluster totally inactivates the protein; however, there is little effect of cluster removal on the midpoint potential of the Co2+/1+ couple. In addition, removal of the cobamide has an insignificant effect on the midpoint reduction potential of the [4Fe-4S] cluster. A 27-kDa corrinoid protein (CP) also was studied since it contains (5-methoxybenzimidazolyl)cobamide in the base-on form.(ABSTRACT TRUNCATED AT 250 WORDS)