Leandro Bugoni
email: lbugoni@yahoo.com.br

Kate Griffiths

Robert W Furness

Sex-biased incidental mortality of albatrosses and petrels in longline fisheries: differential distributions at sea or differential access to baits mediated by sexual size dimorphism?

Keywords: Adult sex ratio, seabirds, incidental capture, longline fishery, sexual dimorphism Zusammenfassung Geschlechterverschobene zufällige Mortalität von Albatrossen und Sturmvögeln bei der Langleinenfischerei: Unterschiedliche Verteilungen auf See oder unterschiedlicher Zugang zu die ASR bei Albatrossen und Sturmvögeln ermittelt haben, die auf See gefangen wurden, Wir zeigen, dass ein verschobenes ASR bei Albatrossen und Sturmvögeln in der Aggregation, die Fischerboote begleitet, häufig ist, was für Vögel im Allgemeinen gefundene Ergebnisse bestätigt. Es bestand keine Korrelation zwischen einem bei der Fischerei getötet wurden, ergab, dass das Aggregation, die wir untersucht haben, oder bei zufälligen Fängen bei der Fischerei. Eine unterschiedliche Verteilung der Geschlechter auf See scheint eine bessere Erklärung sowohl

Skewed Adult Sex Ratio (ASR) has been proposed as a common pattern in birds, frequently biased towards males and with larger biases in globally threatened species. In albatrosses and petrels, it has been suggested that differential mortality of one gender in fisheries is caused either by sexual size dimorphism giving males a competitive advantage, which allows more access of the larger sex (i.e. males) to discards and/or baits, or to at sea segregation of sexes.

Here we tested these hypotheses by determining ASRs in albatrosses and petrels trapped at sea when attending longline fishing vessels for discards in the Southwestern Atlantic Ocean, and searched in the literature for patterns in ASR in albatrosses and petrels killed by fisheries in this area and elsewhere. We show that skewed ASR is common in albatrosses and petrels in the community attending vessels for discards, confirming results found for birds in general.

There was no correlation between skewed ASR and conservation status, or between ASR and sexual size dimorphism. Our review of the sex of birds incidentally killed in fisheries found skewed ASR toward males, females or parity to be equally reported. Thus, sexual dimorphism in size does not explain skewed ASR in the community we sampled or in incidental captures in fisheries in the review. Differential at sea distribution of sexes appears to be a better explanation of the patterns found in the community sampled at sea as well as skewed ASR in seabird fatalities, particularly distant from breeding areas.

Introduction

Sex ratio in birds has received much attention from researchers, often studied at primary (at hatching) and secondary (at fledgling) levels, while tertiary level or adult sex ratio (ASR) is far less known [START_REF] Mayr | The sex ratio in wild birds[END_REF][START_REF] Donald | Adult sex ratios in wild bird populations[END_REF][START_REF] Becker | Population sex ratio shift from fledging to recruitment: consequences for demography in a philopatric seabird[END_REF]. In a recent review [START_REF] Donald | Adult sex ratios in wild bird populations[END_REF] emphasized that many questions posed by [START_REF] Mayr | The sex ratio in wild birds[END_REF] remain unanswered, and reported that skewed ASRs are common in wild bird populations (65% of studies), with males outnumbering females by around 33% on average despite offspring sex ratio not differing from parity. An emerging pattern from Donald"s review was that globally threatened species are more prone to distortion in ASR, with profound implications for conservation.

Procellariiformes (albatrosses, petrels, shearwaters) are a particularly suitable group for testing this latter hypothesis because they are monogamous, extreme k-strategist species, i.e. long-lived with low fecundity and low adult mortality, occur in multi-specific flocks, and have several threatened and non-threatened species [START_REF] Marchant | Handbook of Australian, New Zealand and Antarctic birds[END_REF][START_REF] Tickell | Albatrosses[END_REF][START_REF] Brooke | Albatrosses and petrels across the world[END_REF]).

Unbalanced ASR resulting from skewed mortality impacts on fecundity by reducing effective population size, an effect that persists long after biased mortality ceases in albatrosses [START_REF] Mills | Modelling impacts of long-line fishing: what are the effects of pair-bond disruption and sex-biased mortality on albatross fecundity?[END_REF]. In Wandering Albatross (Diomedea exulans) skewed primary and secondary sex ratios towards males were balanced at recruitment age due to higher mortality of immature males, but again biased towards males at adulthood by a slightly higher mortality of females [START_REF] Weimerskirch | Population sex ratio variation in a monogamous long-lived bird, the Wandering Albatross[END_REF]. The higher adult mortality of females is associated with different at sea distribution, males occupying Antarctic waters (50 -60°S) while females are mainly found in subtropical waters (35-45°S) and overlap more extensively with fisheries [START_REF] Weimerskirch | Population dynamics of the Wandering Albatross Diomedea exulans, of the Crozet Islands: causes and consequences of the population decline[END_REF][START_REF] Croxall | Recoveries of Wandering Albatrosses Diomedea exulans ringed at South Georgia 1958-1986[END_REF].

Several studies have reported ASRs of albatrosses and petrels incidentally captured in fisheries, with some reporting skewed ASR towards males (e.g. [START_REF] Ryan | Consistent male-biased seabird mortality in the Patagonian toothfish longline fishery[END_REF][START_REF] Nel | Seabird mortality in the Patagonian toothfish longline fishery around Prince Edward Island, 1996-2000[END_REF], some towards females (e.g. [START_REF] Bartle | Sexual segregation of foraging zones in procellariiform birds: implications of accidental capture on commercial fishery longlines of Grey Petrels (Procellaria cinerea)[END_REF][START_REF] Roma | Composição sexual e etária do Albatroz-de-sobrancelha-negra Thalassarche melanophrys [sic] e da Pardela-preta Procellaria aequinoctialis (Ordem: Procellariiformes) do Sul-Sudeste do Brasil[END_REF], variable asymmetry towards either gender, or not differing from parity (e.g. [START_REF] Gales | Seabird mortality in the Japanese tuna longline fishery around Australia, 1988-1995[END_REF][START_REF] Gandini | Spatial and temporal patterns in the bycatch of seabirds in the Argentinian longline fishery[END_REF]. Reasons for biased sex ratios in fisheries fatalities have been suggested, but not specifically tested. Hypotheses for the discrepancies include different at sea distributions of males and females, and differential access to feeding resources (baits and discards) due to sexual size dimorphism, differential aggressiveness and manoeuvrability. The access to natural food or discard from fisheries is related to body size among seabird species [START_REF] Hudson | Utilization of discarded fish by scavenging seabirds behind whitefish trawlers in Shetland[END_REF][START_REF] Ballance | Seabird community structure along a productivity gradient: importance of competition and energetic constraint[END_REF]), thus we hypothesized that if sexual dimorphism also plays a role in determining ASRs in fishery fatalities (see [START_REF] Ryan | Consistent male-biased seabird mortality in the Patagonian toothfish longline fishery[END_REF][START_REF] Nel | Seabird mortality in the Patagonian toothfish longline fishery around Prince Edward Island, 1996-2000[END_REF]) ASR will deviate more from parity in species with marked dimorphism, and be similar in closely related species.

In this study we determined the ASRs and sexual size dimorphism (from [START_REF] Bugoni | Age composition and sexual size dimorphism of albatrosses and petrels off Brazil[END_REF] in a number of albatross and petrel species associated with longline fisheries in Brazilian waters. In addition, we reviewed ASRs in Procellariiformes incidentally captured in fisheries in the study area and around the globe. The aims of the study were: a) to determine if there is a pattern in ASR towards males or females in different species attending longliners in the Southwestern (SW) Atlantic Ocean; b) to determine if ASR will deviate more from parity in species with marked dimorphism, and are similar in phylogenetically closely related species; c) to search for patterns in ASR by species and proximity with breeding grounds; d) to test the hypothesis of threatened species being more prone to asymmetry in sex ratio.

Whether ASR is more common in threatened species than non-threatened [START_REF] Donald | Adult sex ratios in wild bird populations[END_REF], we expected to find correlation between the degree of distortion in ASR and the population size, as well as a pattern when comparing closely related species (pairs within the same genus -Thalassarche spp. and Procellaria spp.) within the same Red List category, but populations with different orders of magnitude.

To better understand seabird bycatch in the study area we compared sex ratio of birds trapped at sea when attending longline vessels, with sex ratio of birds incidentally killed in the pelagic longline fisheries (same type of vessels), in the same area, years and seasons provided by [START_REF] Roma | Composição sexual e etária do Albatroz-de-sobrancelha-negra Thalassarche melanophrys [sic] e da Pardela-preta Procellaria aequinoctialis (Ordem: Procellariiformes) do Sul-Sudeste do Brasil[END_REF].

Methods

Study area and seabird sampling

Albatrosses and petrels (n = 258) were attracted close to the vessel using fishing baits and offal (shark liver) and captured non-destructively using a cast net (Bugoni et al. 2008b).

Seabirds usually attend fishing vessels in the area to scavenge on baits and waste. Vessels were targeting tuna (Thunnus spp.), sharks (mainly Blue Shark Prionace glauca) and Swordfish (Xiphias gladius) through a range of hook-and-line and pelagic longline fishing methods as described by Bugoni et al. (2008c). This fleet operates in deep waters over the continental shelf and in offshore waters in southern Brazil throughout the year [START_REF] Mayer | Swordfish (Xiphias gladius) and blue shark (Prionace glauca) fishery and the dynamics of the fleet off the southeastern Brazilian coast[END_REF] in the region under the influence of the Subtropical Convergence [START_REF] Garcia | Oceanografia física[END_REF]. We captured and sampled birds non-destructively during six cruises and 58 trapping days from February to June 2006 and August-September 2007, from 25 to 35ºS (Fig. 1).

Molecular sexing

Blood samples (1 mL) were taken by syringe and needle from the tarsal vein of every bird.

Subsamples of blood were stored in 1.5 mL vials with absolute ethanol. Sexing of birds was carried out after DNA extraction and PCR amplification of CHD genes using primers 2550F (Fridolfsson and Ellegren 1999) and 2757R (R. Griffiths, unpublished; Bugoni and Furness 2009) and genes separated in 2% agarose gel by electrophoresis. DNA extraction negative controls were included for every 23 seabird samples. Positive and negative controls were included for each PCR reaction. One third of all DNA extractions were repeated to confirm gender assignment. Furthermore, we used samples of previously sexed birds as controls: one male Atlantic Yellow-nosed Albatross (Thalassarche chlororhynchos) killed in fisheries and sex determined by necropsy, and four male and female Wandering Albatrosses of known sex from observations at the breeding colony (A. Wood and R.A. Phillips, British Antarctic Survey, in litt.).

Data analysis

ASR deviations from the expected proportion of 1:1 were tested by 2 test with Yates" correction for continuity due to only one degree of freedom [START_REF] Fowler | Chichester Fridolfsson AK, Ellegren H (1999) A simple and universal method for molecular sexing of non-ratite birds[END_REF]. Trapping methods of birds can bias estimates of ASR [START_REF] Domènech | Trap type can bias estimates of sex ratio[END_REF]. However, Procellariiformes studied here are sexually monomorphic in plumage, thus we were unable to visually detect any sex difference during captures and assumed that samples were representative of the population attending fishing vessels.

Sexual size dimorphism was calculated from biometric measurements of sexed birds in [START_REF] Bugoni | Age composition and sexual size dimorphism of albatrosses and petrels off Brazil[END_REF]. The percentage of dimorphism between sexes in each trait was calculated as:

100 / ) (  f f m x x x (1)
where m

x and f

x are mean values for males and females, respectively (Copello et al. 2006).

We calculated dimorphism percentages for culmen, wing and tarsus lengths, and pooled the three percentages in a single dimorphism index for each species by calculating the average of these values. Tail and body mass were not used because we found the former a poor descriptor of dimorphism [START_REF] Bugoni | Age composition and sexual size dimorphism of albatrosses and petrels off Brazil[END_REF] and the latter is very variable according to status of birds [START_REF] Croxall | Sexual size dimorphism in seabirds[END_REF]. Species included in the analyses attended fishing vessels for discards and most had been previously recorded to be incidentally captured in longline fisheries in the area (Bugoni et al. 2008a, c).

Values of population size of seabird species to be correlated with degree of ASR was obtained from the literature, taking into account the origin of birds found in SW Atlantic (2009), with levels of threat, in increasing order as: least concern, near-threatened, vulnerable, endangered and critically endangered.

Results

All birds (n = 258) from the six species studied here, i.e. Atlantic Yellow-nosed Albatross, Black-browed Albatross, White-chinned Petrel, Spectacled Petrel, Great Shearwater and Cape Petrel, plus 41 individuals from other eight petrel and albatross species were successfully sexed using PCR methods, with no discrepancy between controls and replicates, confirming the reliability of the PCR-based method and primers for sexing Procellariiformes.

Three species (Atlantic Yellow-nosed Albatross, White-chinned Petrel and Great Shearwater) had ASR not deviating from parity, while three species had biased ASR: Blackbrowed Albatross towards females, Cape Petrel and Spectacled Petrel towards males (Table 1).

Cape Petrel and Great Shearwaters are species not threatened by extinction, and only the former species had skewed ASR (toward males). Amongst the remaining species, two had even ASR, while one had skewed ASR towards females and one towards males. Pairs of threatened species phylogenetically closely related (Thalassarche albatrosses and Procellaria petrels), show contrasting results: one species of each pair with skewed ASR (Table 1).

Average sexual size dimorphism varied from 1.3% in Great Shearwater to 4.1% in Spectacled Petrel, with larger males in all species (Table 2). The degree of sexual dimorphism was not correlated with the level of asymmetry in ASR neither with the population size (Spearman correlation coefficient r s = 0.6, P = 0.2, n = 6, r s = -0.3, P = 0.5, n = 6, respectively) (Table 1).

Eighteen published studies reported ASR of eight albatrosses, four petrel and one shearwater species captured in bottom and pelagic longline fisheries (Table 3). Studies frequently tested for ASR in several species or carried out stratified analysis by season and area. Skewed ASR in Procellariiformes was reported more frequently than parity ( 2 Yates = 4.33, P = 0.04), but considering the three possible results, males were reported 36%, females 26% and parity 38% parity, thus no tendency was found towards any gender or parity to be reported more frequently ( 2 2 = 1.17 P = 0.56).

Discussion

In the present study we showed skewed ASR in a number of species of albatrosses and petrels associated with longliners operating in Brazilian waters and offshore. However, our results

failed to demonstrate an association between skewed ASR and conservation status, both in terms of population size or level of threat, as suggested by [START_REF] Donald | Adult sex ratios in wild bird populations[END_REF]. Determination of ASR may be subject to a number of biases [START_REF] Donald | Adult sex ratios in wild bird populations[END_REF]), but sampling and determining sex of a range of species occurring simultaneously in the same area using the same method appears to be adequate to test the hypothesis proposed by [START_REF] Donald | Adult sex ratios in wild bird populations[END_REF] and propose alternative ones, such as differential distribution and sexual size dimorphism.

Sexual size dimorphism also failed to explain why pairs of closely related species (White-chinned and Spectacled Petrels; Atlantic Yellow-nosed and Black-browed Albatrosses) have contrasting sex ratios. Furthermore, while we found unbiased sex ratio for the White-chinned Petrels captured non-destructively, [START_REF] Roma | Composição sexual e etária do Albatroz-de-sobrancelha-negra Thalassarche melanophrys [sic] e da Pardela-preta Procellaria aequinoctialis (Ordem: Procellariiformes) do Sul-Sudeste do Brasil[END_REF] found more females being killed in the pelagic longline fishery in the same area, during the same period and seasons. Whether the large males have preference in access to baits, as occurs with larger species to discards in this fishery and others [START_REF] Hudson | Utilization of discarded fish by scavenging seabirds behind whitefish trawlers in Shetland[END_REF][START_REF] Votier | Changes in fisheries discard rates and seabird communities[END_REF], and postulated to explain male-biased mortality around colonies (e.g. [START_REF] Ryan | Consistent male-biased seabird mortality in the Patagonian toothfish longline fishery[END_REF][START_REF] Nel | Seabird mortality in the Patagonian toothfish longline fishery around Prince Edward Island, 1996-2000[END_REF] one would expect biased mortality toward males in the SW Atlantic

Ocean, or at the least a result similar to the proportion of sexes in the area. For Black-browed Albatross in this area, both incidental mortality and our sampling were biased toward females.

Explanation for this pattern of female-bias could be the predominance of females in the area.

Because Black-browed Albatross juveniles predominated in both bycatch [START_REF] Roma | Composição sexual e etária do Albatroz-de-sobrancelha-negra Thalassarche melanophrys [sic] e da Pardela-preta Procellaria aequinoctialis (Ordem: Procellariiformes) do Sul-Sudeste do Brasil[END_REF]) and our samples [START_REF] Bugoni | Age composition and sexual size dimorphism of albatrosses and petrels off Brazil[END_REF] it could be argued that there is no sex-related hierarchy due to similar aggressiveness between sexes at this life-stage. However, if this is true, and there is a similar proportion of both sexes in the area, one would expect to find unbiased sex-ratios in [START_REF] Roma | Composição sexual e etária do Albatroz-de-sobrancelha-negra Thalassarche melanophrys [sic] e da Pardela-preta Procellaria aequinoctialis (Ordem: Procellariiformes) do Sul-Sudeste do Brasil[END_REF] and the present study, again supporting the hypothesis of differential distribution of sexes at sea.

Sexual size dimorphism was not correlated with the degree in ASR, thus both sexes seem to have similar access to baits and discards, even in species with more marked dimorphism in size. The hierarchy in access to discards in seabird communities is based on body size [START_REF] Ballance | Seabird community structure along a productivity gradient: importance of competition and energetic constraint[END_REF][START_REF] Barnes | The impact of the hake Merluccius spp. longline fishery off South Africa on procellariiform seabirds[END_REF]) and this is also clear in our study area, where the preferential access to feeding discards is Diomedea > Thalassarche/Macronectes > Procellaria > Puffinus > Daption/Fulmarus (author"s, pers. observ.). However, it is probable that the magnitude of the intraspecific sexual size dimorphism in this community, and in the access to hooked baits elsewhere (see discussion on ASR review below), is not large enough to account for a male dominancy in the species studied here. On the other hand, the dimorphism could have implications in the different access to baited hooks while sinking underwater for diving species such as Puffinus shearwaters or Procellaria petrels.

There was no pattern of skewed mortality towards males in the range of ASR mortality reported in the literature, with parity, male bias and female bias being equally reported. Male biased mortality of White-chinned Petrel, Grey-headed (Thalassarche chrysostoma) and Indian Yellow-nosed Thalassarche carteri) Albatrosses reported by [START_REF] Ryan | Consistent male-biased seabird mortality in the Patagonian toothfish longline fishery[END_REF] and confirmed with a larger data set for these species plus Grey Petrel, Procellaria cinerea [START_REF] Nel | Seabird mortality in the Patagonian toothfish longline fishery around Prince Edward Island, 1996-2000[END_REF] was recorded in longline fisheries around Prince Edward Islands, where these species breed. As central-place foragers, both males and females should return to the nest for incubation or chick rearing, thus both sexes are obliged to occur around the colony. Results differed in other studies with these species (Table 3) on mortality in fisheries within the foraging range of breeding birds, such as in New Zealand and Australia [START_REF] Bartle | Sexual segregation of foraging zones in procellariiform birds: implications of accidental capture on commercial fishery longlines of Grey Petrels (Procellaria cinerea)[END_REF][START_REF] Murray | Incidental capture of seabirds by Japanese southern bluefin tuna longline vessels in New Zealand waters, 1988-1992[END_REF][START_REF] Gales | Seabird mortality in the Japanese tuna longline fishery around Australia, 1988-1995[END_REF], suggesting that the larger size of males or higher male aggressiveness do not play an important role. The most detailed study [START_REF] Gales | Seabird mortality in the Japanese tuna longline fishery around Australia, 1988-1995[END_REF]) tested for ASR in different areas and seasons and found that some species are skewed during one season or in one area but show even ASR in other areas or seasons. Those species analysed in several studies (e.g. White-chinned Petrel, Black-browed Albatross)

showed biased ASR towards males, females and no bias according to season or area.

Furthermore, the mortality in longline fisheries of the Black-browed Albatross from the Falkland/Malvinas Islands population, is unbiased within the foraging range of breeding birds [START_REF] Gandini | Spatial and temporal patterns in the bycatch of seabirds in the Argentinian longline fishery[END_REF][START_REF] Reid | Seabird mortality associated with Patagonian Toothfish (Dissostichus eleginoides) longliners in Falkland Islands waters[END_REF][START_REF] Seco-Pon | Effect of longline configuration on seabird mortality in the Argentine semi-pelagic kingclip Genypterus blacodes fishery[END_REF]), but female-biased further north off the Brazilian coast in longline catches [START_REF] Neves | Albatross mortality in fisheries off the coast of Brazil[END_REF][START_REF] Roma | Composição sexual e etária do Albatroz-de-sobrancelha-negra Thalassarche melanophrys [sic] e da Pardela-preta Procellaria aequinoctialis (Ordem: Procellariiformes) do Sul-Sudeste do Brasil[END_REF]) and attending longliners for discards (this study), indicating the role of differential distribution of sexes outside foraging range of breeding areas. The White-chinned Petrel, a species frequently killed in fisheries in several areas, also seems to have skewed male-biased mortality in fisheries around breeding colonies as mentioned above (see also [START_REF] Delord | Seabird mortality in the Patagonian toothfish longline fishery around Crozet and Kerguelen Islands, 2001-2003[END_REF] around Crozet Kerguelen Islands), but not confirmed by [START_REF] Gandini | Spatial and temporal patterns in the bycatch of seabirds in the Argentinian longline fishery[END_REF] and [START_REF] Seco-Pon | Effect of longline configuration on seabird mortality in the Argentine semi-pelagic kingclip Genypterus blacodes fishery[END_REF], while in wintering grounds it is female-biased or have even ASR [START_REF] Neves | Albatross mortality in fisheries off the coast of Brazil[END_REF][START_REF] Roma | Composição sexual e etária do Albatroz-de-sobrancelha-negra Thalassarche melanophrys [sic] e da Pardela-preta Procellaria aequinoctialis (Ordem: Procellariiformes) do Sul-Sudeste do Brasil[END_REF]current study). Further studies comparing ASR around breeding grounds and in wintering grounds are warranted.

An alternative hypothesis to explain ASR in longline fatalities is differential at sea distribution of males and females. Sexual segregation in foraging areas of seabirds during the breeding period has been clearly demonstrated in some Procellariiformes, such as Southern Giant Petrel and Northern Giant Petrel (Macronectes halli) (González-Solís et al. 2008),

Black-browed and Grey-headed Albatrosses [START_REF] Phillips | Seasonal segregation in two Thalassarche albatross species: competitive exclusion, reproductive role specialization or foraging niche divergence[END_REF]), Southern Buller"s Albatross (Thalassarche bulleri) (Stahl and Sagar 2000a), and suggested in other studies (Bartle 2000;[START_REF] Shaffer | Functional significance of sexual dimorphism in Wandering Albatrosses, Diomedea exulans[END_REF][START_REF] Weimerskirch | Population sex ratio variation in a monogamous long-lived bird, the Wandering Albatross[END_REF]. Moreover, differential at sea distribution during wintering periods has been argued as responsible for sex biases in albatrosses and petrels washed ashore [START_REF] Holmes | Unequal sex ratios among seabirds found beached-washed[END_REF] or driven inland by storm [START_REF] Bugoni | Effects of the first southern Atlantic hurricane on Atlantic Petrels (Pterodroma incerta)[END_REF]), yet not demonstrated in some species or populations (e.g. [START_REF] Anderson | Foraging ranges of Waved Albatrosses in the eastern tropical Pacific[END_REF]Stahl and Sagar 2000b).

Studies on other seabirds (including gulls, terns, penguins, auks and cormorants)

suggest that males and females share foraging areas, but show niche segregation in other dimensions such as foraging depth and prey size (e.g. Wernham et al. 2002;[START_REF] Phillips | Seasonal segregation in two Thalassarche albatross species: competitive exclusion, reproductive role specialization or foraging niche divergence[END_REF] and references therein). Albatrosses, petrels and shearwaters disperse over vast areas far from breeding grounds, which could play a role in explaining sexual and age-related at-sea segregation, both by competitive exclusion or habitat specialization. Differential distribution could be an important factor in skewed ASR in albatross and petrel communities and in samples of birds captured in longline fisheries.

Differential mortality of one gender is of serious conservation concern for threatened seabirds such as albatrosses [START_REF] Mills | Modelling impacts of long-line fishing: what are the effects of pair-bond disruption and sex-biased mortality on albatross fecundity?[END_REF]

Fig. 1

 1 Figure Captions

  Great Shearwaters Puffinus gravis, and Spectacled petrels Procellaria conspicillata from Tristan da Cunha Islands; Cape Petrel Daption capense, the global population as the origin of SW Atlantic birds is unknown and there is no estimate for the Atlantic Sector (D. c. capense). Conservation status follows categories proposed by IUCN

Ocean: Black-browed Albatross Thalassarche melanophris from Falkland Islands; Whitechinned Petrel Procellaria aequinoctialis from South Georgia; Atlantic Yellow-nosed Thalassarche chlororhynchos,

  of which 19 out of 21 species are threatened by extinction(BirdLife International 2004). Due to the effects caused by asymmetrical mortality on threatened populations by reducing effective population size, this topic deserves further study on a species-specific basis. In addition, ASR could vary if the sample of incidentally killed birds is from around breeding grounds where adults predominate, or elsewhere, where juveniles predominate or age classes are mixed. In contrast to other seabird taxa, sexual segregation of albatrosses and petrels at sea appears to be a widespread pattern, which could explain most differential captures in fisheries. It is therefore recommended that fisheries observers retain seabirds killed in fishing operations for analysis of age, sex and breeding status.

Table 2

 2 Percentage of sexual size dimorphism (SSD) in external body measurements of albatrosses and petrels sampled at sea off Brazil

	Species	n	Culmen	Tarsus	Wing	Average SSD %
	Puffinus gravis	67	2.6	1.4	-0.15	1.3
	Daption capense	31	3.3	4.0	1.8	3.0
	Procellaria aequinoctialis	30	3.6	1.2	1.2	2.0
	Procellaria conspicillata	64	6.1	3.1	3.0	4.1
	Thalassarche melanophris	33	0.5	3.0	3.5	2.3
	Thalassarche chlororhynchos	33	4.5	5.5	2.0	4.0

Table 3

 3 Review of adult sex ratios of albatrosses and petrels incidentally captured in fisheries. The number of times a given result was reported

	(not skewed or towards each gender) is given
	Species	Skewed towards	Fishery Type	Location of the study	Reference
	Wandering Albatross	Females (ring	Attributed to pelagic	Austral oceans, mainly off	Croxall and Prince (1990)
	(Diomedea exulans)	recoveries)	longline	southern Brazil and
				Uruguayan coasts
		Males (3x)	Pelagic longline	Australia	Gales et al. (1998)
		Not skewed	Pelagic longline	Australia	Gales et al. (1998)
		Females (skewed	Attributed to trawlers	Crozet Island	Weimerskirch and Jouventin
		towards males in	(colonies)		(1987) and Weimerskirch et al.
		colonies)			(2005)
		Males	Pelagic longline	New Zealand	Burg (2008)
	Black-browed Albatross	Females	Pelagic longline	Southern Brazil	Calculated from data in Neves
	(Thalassarche melanophris)				and Olmos (1998)
		Females	Pelagic longline	Southern Brazil	Roma (2009)
		Not skewed	Bottom longline	Falkland Island	Reid et al. (2004)

Acknowledgements Thanks to Captain Celso Oliveira and crew of the fishing vessels "Ana Amaral I" and "Akira V" for logistic support onboard. Authors are grateful to Richard Griffiths who provided the primer 2757R used in this study, Richard Phillips for comments on a previous draft of this manuscript and two anonymous reviewers. LB received a CAPES Scholarship. This study was carried out according to permits No. 0128931BR, No. 203/2006, No. 02001.005981/2005, No. 023/2006, No. 040/2006 and No. 1282/1, all granted by the Brazilian Environmental Agency (IBAMA), and International Animal Health Certificate No. 0975-06, issued by the Brazilian Government. The Scottish Executive -Rural Affairs Directorate also kindly provided us permit POAO 2007/91 to import samples into Scotland.


MD, PhD Johanna Hilli
email: johanna.hilli@utu.fi

MD Liisa Sailas

MD, PhD Sirkku Jyrkkiö

MD, PhD Seppo Pyrhönen

MD, PhD Kari Laine

NCT01110291: Induction of CYP3A activity and lowered exposure to docetaxel in patients with primary breast cancer

Keywords: docetaxel, CYP3A activity, [text deleted] peroral midazolam, dexamethasone, induction

published or not. The documents may come

Introduction

Docetaxel is a semisynthetic taxane shown to improve survival in metastatic breast cancer both as single therapy and in combination regimens [START_REF] Montero | Docetaxel for treatment of solid tumours: a systematic review of clinical data[END_REF]. According to data from two adjuvant trials, docetaxel is recommended for patients with early breast cancer at high risk for recurrence [START_REF] Montero | Docetaxel for treatment of solid tumours: a systematic review of clinical data[END_REF][START_REF] Martin | Breast Cancer International Research Group 001 Investigators (2005) Adjuvant docetaxel for node-positive breast cancer[END_REF][START_REF] Roché | Sequential adjuvant epirubicin-based and docetaxel chemotherapy for node-positive breast cancer patients: the FNCLCC PACS 01 Trial[END_REF]. The use of docetaxel is, however, associated with severe toxicity, especially, in adjuvant settings [START_REF] Bear | The effect on tumor response of adding sequential preoperative docetaxel to preoperative doxorubicin and cyclophosphamide: preliminary results from National Surgical Adjuvant Breast and Bowel Project Protocol B-27[END_REF]. The most common and severe adverse events are neutropenia and neutropenic fever [START_REF] Martin | Breast Cancer International Research Group 001 Investigators (2005) Adjuvant docetaxel for node-positive breast cancer[END_REF][START_REF] Bear | The effect on tumor response of adding sequential preoperative docetaxel to preoperative doxorubicin and cyclophosphamide: preliminary results from National Surgical Adjuvant Breast and Bowel Project Protocol B-27[END_REF]. Incidence of adverse events increases along with higher docetaxel exposure [START_REF] Goh | Explaining interindividual variability of docetaxel pharmacokinetics and pharmacodynamics in Asians through p henotyping and genotyping strategies[END_REF]. Docetaxel clearance is highly variable ranging about 6-fold between individuals (from 5.4 to 29.1 liters/h/m 2 ) [START_REF] Hirth | The effect of an individual's cytochrome CYP3A4 activity on docetaxel clearance[END_REF]. This interindividual variability is explained by variations in docetaxel elimination rate. Docetaxel is mainly inactivated in the liver via cytochrome P450 (CYP) 3A4 and 3A5 enzymes [START_REF] Shou | Role of human cytochrome P450 3A4 and 3A5 in the metabolism of taxotere and its derivatives: enzyme specificity, interindividual distribution and metabolic contribution in human liver[END_REF]. In addition, the intestinal P-glycoprotein (Pgp) plays a key role in the fecal elimination of docetaxel by modulating its reabsorption after hepatobiliary secretion [START_REF] Van Zuylen | Role of intestinal P-glycoprotein in the plasma and fecal disposition of docetaxel in humans[END_REF]. P-gp has been shown to have no effect, however, on the disposition of docetaxel in the systemic circulation [START_REF] Van Zuylen | Role of intestinal P-glycoprotein in the plasma and fecal disposition of docetaxel in humans[END_REF]. Also, the plas ma concentration of alpha-1-acid glycoprotein, which has been suggested to be the main determinant of docetaxel plasma binding variability, has been shown to affect docetaxel clearance [START_REF] Bruno | A population pharmacokinetic model for docetaxel (Taxotere): model building and validation[END_REF][START_REF] Urien | Docetaxel serum protein binding with high affinity to alpha 1-acid glycoprotein[END_REF].

[text deleted]

Hepatic CYP3A4 activity measured by erythromycin breath test or clearance of intravenously administered midazolam has been shown to correlate with docetaxel clearance [START_REF] Goh | Explaining interindividual variability of docetaxel pharmacokinetics and pharmacodynamics in Asians through p henotyping and genotyping strategies[END_REF][START_REF] Hirth | The effect of an individual's cytochrome CYP3A4 activity on docetaxel clearance[END_REF]. Both of these assessments require intravenous administration and, erythromycin breath test, additionally, radioactive labelling. Thus, application of these methods into clinical setting is laborious. We wanted to study the association between CYP3A activity and docetaxel concentration using oral administration of midazolam, which could be easily applied into clinical practice. [text deleted] In addition, the possible changes in the CYP3A activity during the treatment cycles were investigated.

Furthermore, the relationship between [text deleted] docetaxel exposure and patient outcome was assessed.

Materials and methods

Patients

Altogether 20 patients aged 32-58 years with histologically verified high risk breast cancer, for which docetaxel was indicated, were included in this study conducted in the University Hospital of Turku, Finland, between years 2003-2004. High risk for recurrence was predicted by positive lymph nodes (17 patients) or, if node negative, by a histological grade 3 of a T2 tumour (3 patients; table 1). None of the patients had evidence of distant metastases. For eligibility, the patients had to have a performance status of 0 or 1 according to the World Health Organization's (WHO) performance scale. They had to have adequate bone marrow reserve defined as haemoglobin greater than 100 g/l, a leukocyte count greater than 3.0 x 10 9 /l (or a neutrophil count greater than 1.5 x 10 9 /l) and a platelet count greater than 120 x 10 9 /l. Their liver function had to be adequate, which was defined as an alanine aminotransferase level no greater than 1.5 times the normal level, an alkaline phosphatase level no greater than 2.5 times the normal level and a normal bilirubin level. Severe physical or psychiatric disease, pregnancy or lactation and substance abuse were criteria for exclusion. Also, concomitant medication with a known CYP3A substrate, inducer or inhibitor was not allowed. The patients were informed on the study both verbally and in writing, and a written informed consent was obtained. The study protocol was approved by the Ethics Committee of the Hospital District of Varsinais-Suomi, Finland, and the National Agency of Medicines, Finland.

Study plan

Chemotherapy

Docetaxel 80 mg/m 2 of body surface area (BSA) was given as an i.v. infusion during 60 minutes on day 0 repeated three times in a 21-day schedule. The docetaxel dosage was reduced by 20% in altogether 11 patients (in 9 patients during the second and in 2 patients during the third cycle) due to grade 3-4 toxicity (excluding nausea). The cycle was postponed for 2-7 days in altogether 5 patients due to low neutrophil count (2 patients) or practical reasons (3 patients). The docetaxel concentration was measured from blood samples collected on day 0 in cycles 1 and 3. Blood samples were drawn into heparinised tubes 2-4 minutes before the end of the infusion and 15 and 45 minutes, as well as 3, 6, 12, and 24 hours after the end of the infusion. The blood samples were centrifuged for 10 min at 2000 g, and the separated plasma was stored at -20 °C until analyzed.

Three weeks after the last docetaxel regimen, the patients were additionally given a standard combination treatment, composed of cyclophosphamide, epirubicin and fluorouracil (CEF), which was repeated three times.

Pre-medications

All patients were pre-medicated with dexamethasone to reduce the incidence and severity of fluid retention and hypersensitivity reactions. The patients received dexamethasone 7.5 mg p.o. altogether six times, in the evening on day -1, one hour before the docetaxel infusion and in the evening on day 0, in the morning and evening on day 1 and in the morning on day 2. In addition, the patients were given dexamethasone 0.2 mg/kg i.v. and tropisetron 5 mg i.v. just before the docetaxel infusion to reduce nausea. One patient with unstable diabetes was not given the intravenous dexamethasone, but tropisetron was given to all patients.

CYP3A phenotyping with midazolam

Midazolam phenotyping was done a day before (day -1) and a day after (day +1) the docetaxel infusion in cycles 1 and 3. On day -1 the midazolam phenotyping was completed before the first dose of dexamethasone. The patients were given midazolam 7.5 mg p.o. in the morning, and repeated blood samples for analysis of midazolam and its CYP3A-dependent metabolite 1-OH-midazolam were drawn into EDTA tubes just before as well as 1, 2, 4, 6 and 8 hours after midazolam administration. The blood samples were centrifuged for 10 min at 2000 g, and the separated plasma was stored at -20 °C until analyzed. [text deleted]

Evaluations of safety and toxicity

For safety and practical reasons the patients were hospitalized from the morning of day -1 until the evening of day +1 of each cycle. The following laboratory tests were performed no more than 7 days prior to the first docetaxel infusion: blood haemoglobin, white blood cell (WBC) count, neutrophil count, platelet count and serum creatinine, alanine aminotransferase, alkaline phosphatase, bilirubin, albumin and ionized calcium level. These tests (excluding the ionized calcium) were also repeated on day 0 before the docetaxel infusion and on days 7 and 14 of each docetaxel cycle and three weeks after the last docetaxel infusion. Blood haemoglobin, WBC, neutrophil and platelet counts were also performed prior to the start of chemotherapy of CEF cycles.

The evaluation of toxicity was done before the start of each docetaxel cycle and three weeks after the last docetaxel cycle by using a validated scoring system following the WHO's instructions (National Cancer Institute/National Institutes of Health Common Toxicity Criteria, available online at http://ctep.cancer.gov/reporting/ctc.html). Of all the recorded evaluations, the clinically most significant adverse events associated with docetaxel therapy were included in statistical analysis. These were hospitalization due to febrile neutropenia and low neutrophil count. The neutrophil counts, which were measured three times between the docetaxel cycles, were graded according to WHO and the lowest grading of cycles 1 and 3 were used in the analysis.

In addition, the lowest grading of each of the three cycles was added together and this sum was used in the analysis (table 2).

Analytical methods

Assessment of midazolam and 1-OH-midazolam concentrations

Samples for analysis of midazolam and 1-OH-midazolam were prepared for analysis with liquid-liquid extraction. After the addition of 50 l of internal standard solution (100 ng/ml of nitrazepam in 5 % methanol/water) the mixture was extracted with 5 ml of ethyl acetate. About 4.5 ml of the organic layer was evaporated to dryness in PPtubes under a gentle stream of nitrogen at about 40 o C and the residue was dissolved in 100 l of a solution containing 50 % methanol / 50 % 5 mM ammonium formate (v/v). The sample was transferred into an autosampler vial from which 22 l was injected into the high-performance liquid chromatography (HPLC) -mass spectrometer (MS) / MS system.

Separations for analysis of the content of midazolam and 1-OH-midazolam were performed with a Symmetry C 18 100 x 2.1 mm i.d. (3.5 M) column coupled with an integrated Symmetry C 18 guard column (Waters Corp). The mobile phase consisted of eluents A (methanol) and B (5 mM ammonium formate buffer). The eluent system was isocratic: 68 % of A and 32 % of B. The eluent B was filtered before use through a 0.45 m HV filter (Millipore). The flow rate was 0.2 ml/min. Mass spectrometric detection was carried out using a PE SCIEX API 365 triplequadruple instrument using positive ISI (ion spray ionisation) and MRM (multiple reaction monitoring) mode. The needle potential was set to 3.5 kV and the temperature of the heated nitrogen gas was 380 o C. The declustering potential was set to 25 V, focusing potential to 100 V, and entrance potential to 8.0 V for midazolam, to 4.0 V for 1-OH-midazolam and to 5.0 V for internal standard. The collision energy was set to 33 V for midazolam and to 29 V for 1-OH-midazolam and internal standard. Nebulizer gas (nitrogen) was set to value 12, curtain gas (nitrogen) was set to value 12 and collision gas (nitrogen) was set to value 4.0. The parent ionsplitter ion pairs detected were m/z 326.1 -291.0 (midazolam), m/z 342.1 -324.0 (1-OHmidazolam) and m/z 282.1 -236.0 (internal standard).

The quantitation of the analytes was accomplished with HPLC-MS/MS. The calculations of quantitations were based on peak areas of the analytes and the internal standard. The data from the HPLC-MS/MS analyses were collected using Applied Biosystems Analyst 1.2 software. The peak integrations, calibration curves and quantitations were generated with the same software. The standard curves (concentration range 0.20-50 ng/ml) were generated using weighted (1/x) linear regression. The lower limits of quantification were 1.0 ng/ml and 0.2 ng/ml for midazolam and 1-OH-midazolam, respectively. The interday coefficient of variation at 5 ng/ml concentration was 8.3% for midazolam and 10.7% for 1-OH-midazolam. Liquid chromatography was performed using a HP1100 system from Agilent. The analytical column (100 x 2.1 mm i.d.) was packed with a 5 m Hypersil BDS C18 stationary phase. The elution was carried out with a mobile phase made of a mixture of methanol and 0.1 % of formic acid (60/40; v/v) filtered and degassed by a 0.2 m solvent filter before use. The mobile phase was used at a flow rate of 0.25 ml/min.

Assessment of docetaxel concentrations

The HPLC system was connected to a PE Sciex API 3000 MS/MS system, operating in the Turbo Ion Spray positive mode. The MS/MS system was focused in the MRM mode to monitor the ion transitions: 808.5 -527.2 (docetaxel) and 832.4 -491.1 (internal standard). The API 3000 MS/MS system was remotely controlled by a Power Macintosh model G3 using a MAC OS 7 (or higher revision) operating system.

A plasma standard curve was daily elaborated between 1 and 500 ng/ml of docetaxel in heparinised human plasma. The concentrations of pharmacokinetic samples were calculated from calibration curves obtained by a 1/x 2 weighted linear regression analysis. A method set-up was performed before the start of study sample analysis and all method set-up results were in the predefined acceptance criteria (relative standard deviation (RSD) ≤15 %, except ≤20 % at the lower limit of quantitation (LLQ); bias ± 15 %, except ± 20 % at LLQ for calibration standards and quality control samples).

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Pharmacokinetic analysis

The ratio of the area under the concentration-time curve (AUC 0-∞ ) values of 1-OHmidazolam and midazolam (i.e. AUC ratio) was determined and used as a marker for CYP3A activity. In six patients the midazolam or the 1-OH-midazolam concentration was below the lower limit of quantification in samples taken 1, 6 or 8 hours after drug intake. For these values, half of the quantification limit value was used in the analysis.

One patient had some detectable midazolam in the baseline (0 h) sample taken on day 1 in the cycle 3. This baseline concentration was reduced from the measurements prior to pharmacokinetic analysis. The pharmacokinetics of docetaxel was described by AUC 0-∞ , the peak plasma concentration (C max ), the time from drug intake to peak concentration (t max ), the mean elimination half-life (t 1/2 ) and clearance (Cl). The pharmacokinetic analyses were performed using the WinNonlin Professional program, version 4.1 (Pharsight Corporation, Mountain View, California, USA).

Statistical analysis

The analysis of variance for repeated measures was used to study the changes in drug concentrations. The analysis of variance (ANOVA) or the Kruskall-Wallis (Mann Whitney U) test and correlation analysis (Pearson or Spearman) were used in comparisons between adverse events and drug concentrations/genetics. The Cox proportional hazard analysis was used to study survival. P values of 5 % or less were regarded as significant. The data which failed to fit the normal distribution was logtransformed prior to analysis. The non-parametric methods were used if the data failed to fit the normal distribution after log-transformation. The data was analysed using the SAS Enterprise Guide for Windows, version 3.0 (SAS Institute Inc., Cary, NC, USA).

Results

Docetaxel pharmacokinetics

The mean docetaxel pharmacokinetic parameters after a dose of 80 mg/m 2 are presented in table 3. Eleven patients had a dose reduction to 60 mg/m 2 after cycle 1 or 2. The mean docetaxel AUC was about a half of that previously reported in the literature in European populations (table 3). Docetaxel clearance was accordingly clearly greater than what has been seen in other studies with corresponding doses (table 3).

CYP3A activity before and after docetaxel infusion

The mean midazolam AUC was significantly lower on days +1 than on days -1 (mean 58.5 h*ng/ml vs. 95.6 h*ng/ml, p<0.0001, cycle 1; mean 65.0 h*ng/ml vs. 113.6 h*ng/ml, p<0.0001, cycle 3) (figure 1, table 4). There were no significant difference between the day -1 values (p=0.069) or day +1 values (p=0.188) of cycles 1 and 3 (figure 1, table 4). The AUC ratio 1-OH-midazolam/midazolam was also significantly changed during the cycles (p=0.018). The mean AUC ratio was higher on days +1 (mean 0.32 and 0.30) than on days -1 (mean 0.27 and 0.26; table 4), but statistically the difference was significant only between the day +1 of cycle 1 and the day -1 of cycle 3 (p=0.017, figure 1,table 4).

Association between docetaxel and midazolam concentrations

Docetaxel AUC or clearance did not correlate with midazolam concentrations on day -1 in cycle 1 (figure 2). The studied parameters for midazolam concentration were midazolam AUC calculated to the last measuring point (AUC last ) (p=0.22 and p=0.42) and to infinity (AUC 0-∞ ) (p=0.54 and p=0.63), midazolam weight-adjusted clearance (p=0.89 and p=0.67), AUC ratio (1-OH-midazolam/midazolam) (p=0.88 and p=0.76), midazolam concentration at 4 hours (p=0.38 and p=0.50) and at 6 hours (p=0.89 and p=0.93), and AUC ratio at 4 hours (p=0.88 and p=0.97) and 6 hours (p=0.86 and p=0.56). In cycle 3, docetaxel AUC correlated positively with midazolam AUC ratio (r=0.50, p=0.025), and midazolam AUC ratio at 4 hours (r=0.67, p=0.0012) and 6 hours (r=0.65, p=0.0017). Docetaxel clearance correlated negatively with midazolam AUC ratio (r=-0.38, p=0.094), AUC ratio at 4 hours (r=-0.53, p=0.017) and AUC ratio at 6 hours (r=-0.52, p=0.018) in cycle 3.

Docetaxel concentration and adverse events

Altogether three patients (15 %) were hospitalized due to febrile neutropenia during the docetaxel treatments, two in cycle 1 and one in cycle 2. The patient hospitalized in cycle 2 had first only fever and neutropenia developed later. She was diagnosed with influenza A. Two of the hospitalized patients received leukocyte growth factors. All the patients had grade 3 or 4 neutropenia at some point during the docetaxel treatment. The neutrophil count gradings are shown in table 2. Docetaxel AUC correlated with the given docetaxel dose (based on body surface area) in cycle 3 (r=0.53, p=0.016), but only weakly in cycle 1 (r s =0.38, p=0.097). Docetaxel clearance did not correlate with the given dose in either cycle (p=0.49 and p=0.67). There were no associations between the given docetaxel dose and adverse events ( low neutrophil count, p=0.47 and p=0.50 in cycle 1 and 3, respectively, or febrile neutropenia, p=0.95 in cycle 1 only).

The docetaxel AUC (p=0.87) and clearance (p=0.37) of the three patients with neutropenic fever were not significantly different from the other patients' values, although the patients with febrile neutropenia did have somewhat lower mean docetaxel clearance than the other patients (mean 76 l/h vs. 88 l/h). The patients with febrile neutropenia also tended to have higher docetaxel C max values than the others (mean 2029 ng/ml vs. 1566 ng/ml, p=0.064, figure 3). There were no associations between the docetaxel AUC (p=0.16, Kruskall-Wallis test or r s =-0.24, p=0.30, cycle 1; p=0.32, ANOVA or r=-0.35, p=0.13, cycle 3), clearance (p=0.91, Kruskall-Wallis test or r s =0.18, p=0.45, cycle 1; p=0.74, ANOVA or r=0.29, p=0.21, cycle 3) or C max (p=0.14, Kruskall-Wallis test or r s =-0.17, p=0.47, cycle 1; p=0.32, ANOVA or r=-0.32, p=0.17, cycle 3) and the low neutrophil count (nadir or sum of nadirs).

CYP3A activity and adverse events

CYP3A activity defined as the AUC ratio of 1-OH-midazolam and midazolam showed no association with neutrophil count (data not shown). There was a trend towards lower AUC ratio among the three patients with neutropenic fever compared with the other patients (mean 0.19 vs. 0.29, p=0.088, figure 3).

[text deleted]

Survival analysis

The mean follow-up time of the patients after the initiation of the study treatments was 4.4 years (range 0.75 -5.8 years). Four patients died during the follow-up period, 9 (two patients), 12 or 23 months after initiation of therapy, respectively. Midazolam AUC or AUC ratio (assessed before docetaxel infusion), docetaxel AUC or clearance, low neutrophil count or febrile neutropenia was not associated with survival after treatment. Also, known prognostic factors lymph node metastases, Her2, Ki-67, estrogen and progesterone receptor status were not predictors of survival (data not shown).

Discussion

Although docetaxel clearance has been shown to be highly variable, the docetaxel concentrations in this study were generally clearly lower than what has been reported in the literature. The mean docetaxel AUC was about a half of and the mean clearance more than double to that reported with corresponding doses of docetaxel in European populations (table 3). Only Caucasians were included in this comparison to avoid the possible influence of race on docetaxel metabolism. It has been suspected, for example, that Asians may have lower CYP3A activity than Caucasians [START_REF] Lin | Differences in the urinary excretion of 6-beta-hydroxycortisol/cortisol between Asian and Caucasian women[END_REF]. Docetaxel metabolism has been shown to be inducible by CYP3A inducers [START_REF] Marre | Hepatic biotransformation of docetaxel (Taxotere) in vitro: involvement of the CYP3A subfamily in humans[END_REF] and, therefore, other drugs and herbal products affecting CYP3A activity were forbidden during the study. We found that the CYP3A activity was markedly and rapidly induced when assessed a day after docetaxel infusion as evidenced by significantly lower midazolam AUC values and higher 1-OH-midazolam/midazolam AUC ratios on day +1 and by low docetaxel exposure. By the point of assessment of CYP3A activity, the patients had received dexamethasone 7.5 mg perorally altogether four times (30 mg) and all except one patient also 0.2 mg/kg intravenously (11-19 mg).

Dexamethasone is a known inducer of CYP3A activity [START_REF] Shou | Modeling, prediction, and in vitro in vivo correlation o f CYP3A4 induction[END_REF][START_REF] Roberts | The influence of CYP3A5 genotype on dexamethasone induction of CYP3A activity in African Americans[END_REF]. However, Goh et al.

have shown that peroral dexamethasone 8 mg x 6 administered every 12 hours, beginning either 24 hours before or after docetaxel treatment, did not significantly alter docetaxel pharmacokinetics in Asian patients with solid tumours [START_REF] Goh | Explaining interindividual variability of docetaxel pharmacokinetics and pharmacodynamics in Asians through p henotyping and genotyping strategies[END_REF]. Docetaxel has been shown to induce CYP3A4 gene expression in vitro in peripheral mononuclear cells of 16 previously untreated lung cancer patients [START_REF] Fujitaka | Induction of cytochrome P450 3A4 by docetaxel in peripheral mononuclear cells and its expression in lung cancer[END_REF]. Also, in another in vitro study, another taxane, paclitaxel, has been shown to increase CYP3A4 mRNA in human hepatocytes [START_REF] Kostrubsky | Induction of cytochrome P4503A by taxol in primary cultures of human hepatocytes[END_REF]. Hence, taxanes seem to autoinduce their own metabolism at least in in vitro conditions. The CYP3A induction we found here in patients could have been caused by either dexamethasone or docetaxel itself or both. However, the inductive effect of docetaxel has been present in earlier studies as well, so that dexamethasone re mains the most likely candidate for the induction. As a comparison to the before mentioned study by Goh et al. [START_REF] Goh | Explaining interindividual variability of docetaxel pharmacokinetics and pharmacodynamics in Asians through p henotyping and genotyping strategies[END_REF], we used a different dosing schedule and route of administration in a genetically different population. In table 3 there are three studies where dexamethasone has been used as premedication in European populations [START_REF] Harvey | Phase III trial comparing three doses of docetaxel for second-line treatment of advanced breast cancer[END_REF]18,19]. As a comparison to these studies, we used a larger total dose of dexamethasone of which about a half was administered intravenously. In addition, in our study the dexamethasone premedication was administered during a shorter time period before docetaxel infusion than in the other three studies (in 12 hours vs. in [START_REF] Puisset | Data are given as arithmetic mean ± arithmetic SD *p-values between docetaxel pharmacokinetic parameters of cycle 1 and 3 after 80 mg/m 2 of docetaxel Premed.: dexamethasone 7.5 mg p.o. 12 h and 1 h before infusion plus 11-19 mg i.v. just before infusion 100 5000 43[END_REF]

hours). [text deleted]

Although the docetaxel exposure in this study was smaller than expected, grade 3 or 4 neutropenia occurred in all patients at some point of docetaxel treatment. As a comparison, in a large adjuvant setting with primary breast cancer patients in comparable performance status, and with peroral dexamethasone premedication 8mg x 6 every 12 hours beginning 24 hours before chemotherapy, (n=744) the incidence of grade 3 or 4 neutropenia was 65.5% [START_REF] Martin | Breast Cancer International Research Group 001 Investigators (2005) Adjuvant docetaxel for node-positive breast cancer[END_REF]. On the other hand, febrile neutropenia occurred in 25-29% of those patients, whereas its incidence was only 15% in our study [START_REF] Martin | Breast Cancer International Research Group 001 Investigators (2005) Adjuvant docetaxel for node-positive breast cancer[END_REF]. In another study in Asian patients receiving docetaxel alone, grade 3 or 4 neutropenia occurred in 17 of 23 (74%) and febrile ne utropenia in 6 of 23 (26%) of patients after 75 mg/m 2 of docetaxel [START_REF] Goh | Explaining interindividual variability of docetaxel pharmacokinetics and pharmacodynamics in Asians through p henotyping and genotyping strategies[END_REF]. These patients received peroral dexamethasone 8mg x 6 every 12 hours beginning 24 hours after docetaxel treatment in the first treatment cycle and 24 hours before docetaxel treatment in the second treatment cycle [START_REF] Goh | Explaining interindividual variability of docetaxel pharmacokinetics and pharmacodynamics in Asians through p henotyping and genotyping strategies[END_REF]. Interestingly, after an approximate follow-up period of 5 years, 80% of the patients in our study were alive and free of disease. In the study by Martin et al. [START_REF] Martin | Breast Cancer International Research Group 001 Investigators (2005) Adjuvant docetaxel for node-positive breast cancer[END_REF] estimated rates of disease-free survival at five years was 75 % in patients who received adjuvant docetaxel. Thus, although the docetaxel exposure was smaller than expected, it did not seem to affect survival. Naturally, firm conclusions of the incidence of adverse events or survival cannot be made based on the quite small sample size of our study. For the same reason, known prognostic factors of the primary tumour failed to associate with survival in this material.

There were no associations between docetaxel and midazolam concentrations in cycle 1. An association was found in cycle 3, but it was inverse. Dexamethasone is also a known inducer of P-gp [START_REF] Kageyama | Relationship between excretion clearance of rhodamine 123 and P-glycoprotein (Pgp) expression induced by representative Pgp inducers[END_REF]. A P-gp inhibitor has been shown not to alter docetaxel clearance. However, the fecal excretion of docetaxel was markedly reduced by a P-gp inhibitor, but levels of the major CYP3A4-mediated metabolites of docetaxel in feces were significantly increased [START_REF] Van Zuylen | Role of intestinal P-glycoprotein in the plasma and fecal disposition of docetaxel in humans[END_REF]. Thus, docetaxel pharmacokinetics may have been modified by multiple mechanisms which have led to a random correlation between docetaxel and midazolam in cycle 3. However, at baseline with no prior inducers, no correlations were found. While oral midazolam is a well-established and sensitive marke r for CYP3A activity, the difference in the mode of administration vs. docetaxel (i.v.) may at least partly explain the lack of correlation between midazolam and docetaxel exposure. Also, there were no clear associations between CYP3A activity or docetaxel exposure and adverse events. Thus, it is concluded that oral midazolam cannot be used as a predictor of docetaxel exposure at least when dexamethasone is given. [text deleted] The weak associations of occurrence of febrile neutropenia and higher docetaxel C max values and lower midazolam AUC ratio suggest that slow docetaxel metabolism may predispose to adverse events.

Conclusions

In conclusion, CYP3A activity was clearly and rapidly induced after administration of dexamethasone 30-49 mg and docetaxel 60-80 mg/m 2 . This induction was disappeared after a 21-day wash-out period. The docetaxel exposure was markedly smaller than expected based on literature, but this did not have influence on survival or occurrence of neutropenia. Incidence of febrile neutropenia was, however, smaller than reported in other studies with comparable docetaxel dosage. Moreover, peroral midazolam did not seem to have value as a predictor of docetaxel pharmacokinetics in this setting. According to results from this and other studies, slow CYP3A-mediated metabolism might predispose patients to adverse events of docetaxel. P=0.017 day -1 of cycle 3 Data are given as arithmetic mean ± arithmetic SD except for t max data, which are given as mean and range

  Docetaxel concentrations were determined by a sensitive and specific assay transferred from sanofi-aventis and partially validated at Parexel International Bioanalytical Laboratories. Docetaxel and the internal standard were separated from human plasma by a solid-phase extraction (SPE) using an Empore TM 96 well extraction disk plate C18 system. The combined extracts were transferred into HPLC injection vials and placed in the pre-cooler injector. 10 l of sample extracts were injected into the LC-MS/MS system.

  , we are the first to report CYP3A induction followed by dexamethasone premedication and docetaxel infusion by investigating CYP3A activity using midazolam as a probe drug. However, since we did not have a control group in which the docetaxel pharmacokinetics would have been measured without prior dexamethasone administration, we cannot definitely conclude dexamethasone to be responsible for the induction. Also, we did not investigate the role of alpha-1-acid glycoprotein in docetaxel metabolism. The sample size in this study was too s mall to draw any firm conclusions, especially on survival. However, the low docetaxel exposure in this study may have contributed to the low incidence of febrile neutropenia and should have affected survival, as well. Studies with larger patient populations and with direct assessment of docetaxel pharmacokinetics both before and after dexamethasone are needed to confirm these results. Until that it is recomme nded to give dexamethasone as a supplemental drug according to regimens found not to influence docetaxel clearance, i.e. by keeping the pre-infusion dose as little as possible, avoiding intravenous administration of dexa methasone and managing the adverse effects of docetaxel by continuing dexamethasone administration after docetaxel infusion.

Figure 1 .

 1 Figure 1. Midazolam AUC (A) and AUC ratio of 1-OH-midazolam/midazolam (B)

Figure 2 .

 2 Figure 2. At baseline, there were no correlations between docetaxel clearance and

Figure 3 .

 3 Figure 3. Docetaxel C max values after docetaxel 80 mg/m 2 and midazolam AUC ratios

Figure 1

 1 Figure 1.

  Figure 2. A

Table 1 . Patient and tumour characteristics (n=20) CharacteristicTable 3 .

 13 Pharmacokinetics of docetaxel after 1-hour infusion in high risk breast cancer patients (current study) and in European patients with various malignancies.

	Docetaxel	AUC	Cl	C max	t 1/2	N	Reference
	dose
	(mg/m 2 )	(h*ng/ml)	(l/h)	(ng/ml)	(h)
	CYCLE 1						Current
	80	1737 ±	87 ± 27	1635 ±	17 ± 5.8	20	study
	(range 76.5-81.3)	371		512
	CYCLE 3	p=0.36* p=0.45* p=0.31* p=0.10*
	80	1905 ±	79 ± 23	1852 ±	15 ± 3.2	9
	(range 78.5-81.3)	468		538
	60	1251 ±	100 ±	1135 ±	16 ± 4.1	11
	(range 58.8-65)	379	41	453
	All patients	1545 ±	90 ± 35	1458 ±	15 ± 3.7	20
		529		604

Table 4 .

 4 Pharmacokinetics of midazolam and its main metabolite 1-OH-midazolam after 7.5 mg oral midazolam in patients with high risk breast cancer. These pharmacokinetic parameters were measured a day before and a da y after docetaxel infusion in two treatment cycles.

	Variables	Cycle 1	Cycle 3
		Day -1	Day +1	Day -1	Day +1
	Midazolam
	AUC (h*ng/ml)	96 ± 35	59 ± 19	114 ± 42	65 ± 24
	C max (ng/ml)	24 ± 6.9	22 ± 6.6	29 ± 9.0	23 ± 9.7
	t max (h)	1.7 (1-8)	1.4 (1-2)	1.5 (1-4)	1.4 (1-2)
	t 1/2 (h)	3.7 ± 1.0	2.7 ± 0.8	3.6 ± 1.0	3.4 ± 1.3
	Difference between midazolam AUC values (overall, p<0.0001)
	day -1 vs. day +1	P<0.0001	P<0.0001
	day -1 vs. day -1		P=0.069
	day +1 vs. day +1			P=0.188
	1-OH-midazolam
	AUC (h*ng/ml)	24 ± 7.4	17 ± 5.9	27 ± 10	18 ± 4.8
	C max (ng/ml)	8.1 ± 3.0	7.7 ± 4.2	9.2 ± 4.6	7.5 ± 2.6
	t max (h)	1.7 (1-8)	1.4 (1-2)	1.5 (1-4)	1.3 (1-2)
	t 1/2 (h)	3.1 ± 0.9	2.9 ± 1.1	2.9 ± 1.0	3.3 ± 1.0
	AUC ratio (1-OH-	0.27 ± 0.09	0.32 ± 0.11	0.26 ± 0.11	0.30 ± 0.09
	midazolam/ midazolam)
	Difference between midazolam AUC ratios (overall, p=0.018)
	day -1 vs. day +1	P=0.067	P=0.165
	day -1 vs. day -1		P=1.00
	day +1 vs. day +1			P=1.00
	day +1 of cycle 1 vs.

Acknowledgme nts

The authors thank Mrs Kirsi Penttilä for the skilful technical assistance. This study was financially supported by sanofi-aventis.

Data

Competing interests

This study has received financial support from sanofi-aventis.

Authors' contributions

JH gathered and interpreted the results, performed the statistical analysis and drafted the manuscript. LS participated in the design of the study and carried out the clinical phase of the study. SJ participated in the design, coordination and clinical phase of the study. SP participated in the study design and coordination. KL participated in the design and conduction of the study, in interpretation of the results and acquired funding for the study. All authors read and approved the final manuscript.