Rachid Ridouane

Susanne Fuchs

Phil Hoole

LARYNGEAL ADJUSTMENTS IN THE PRODUCTION OF VOICELESS OBSTRUENT CLUSTERS IN BERBER

Laryngeal adjustments in voiceless obstruent clusters in Tashlhiyt Berber were examined by means of simultaneous transillumination, fiberoptic films and acoustic recordings. This language allows a rich variety of voiceless clusters naturally. Several combinations of /s/ and /k/ clusters including singleton and geminate consonants were examined. We focused on the number of glottal opening gestures, the influence of manner of articulation and effects of word boundaries. Results of this study provide evidence that the manner of articulation of segments and their position in the clusters have a major impact both on the number and on the location of glottal abduction movements. Word boundaries did not influence laryngeal adjustment to the same extent.

INTRODUCTION

A number of studies have investigated laryngeal articulation in the production of voiceless consonant clusters in order to study coarticulation patterns at the laryngeal level. The first main objective of these works is to compare laryngeal adjustments observed in voiceless singleton obstruents with those attested in consonant clusters. A second objective is to understand the way laryngeal adjustments are coordinated with supralaryngeal events in complex sequences and the mechanisms which might determine the number, the amplitude and the location of glottal opening gestures. Most of these studies used a similar methodology -photoelectroglottography combined with fiberoptics -and a speech material consisting of different /s/-stop combinations. Germanic languages (English, German, Icelandic and Swedish) are by far the most studied languages. [START_REF] Pétursson | Timing of glottal events in the production of aspiration after /s[END_REF], [START_REF] Fukui | Laryngeal adjustments in Danish voiceless obstruent production[END_REF] and Jessen (1999), for example, observed glottal adjustment and the number of glottal abduction gestures in fricative-stop clusters in some of these languages. One general aspect that comes out from these studies is that laryngeal adjustments can be organized in one or more continuous opening and closing gestures.

In [START_REF] Pétursson | Timing of glottal events in the production of aspiration after /s[END_REF], Icelandic [s#t h ] and [s+t h ] clusters show two glottal opening peaks whereas only one peak occurs in [#st] cluster. The author concludes that there are discrete phoneme commands for glottal abduction although the intervening boundary might have influenced the pattern too. In [START_REF] Fukui | Laryngeal adjustments in Danish voiceless obstruent production[END_REF], the two-peakedness observed in Danish [s#p h ] clusters was rather speaker-dependent. One explanation of these differences could be that two different glottal abduction gestures are underlyingly present; in a fast speech rate condition they overlap, whereas no overlap would be found in a slow speech rate condition. [START_REF] Munhall | Gestural aggregation in speech: laryngeal gestures[END_REF] investigated this issue based on English [s#t h ] clusters and simulated gestural overlap as a sum of the two underlying movements. Their simulations fit the transillumination data with respect to timing characteristics, but differences were found concerning glottal opening amplitudes. The simulations showed higher amplitudes than the experimental data. They explain these differences in terms of the non-calibration possibility of the transillumination technique as well as in terms of rate effects which can decrease movement amplitudes. Jessen (1999) raised the question of whether the monomodal versus bimodal distribution of glottal opening movements would be an effect of the word boundary or whether aspiration of the stop in the fricative-stop cluster could explain the distinction. His findings drawn from German confirm the latter idea, initially based on results from Löfqvist and Yoshioka (1980a), and [START_REF] Yoshioka | Laryngeal adjustment in the production of consonant clusters and geminates in American English[END_REF] on Swedish and English respectively. These authors observed that glottal opening is characterized by one-, two or three-peaked patterns according to the nature of the voiceless obstruents and the way they are combined. Each voiceless obstruent or geminate accompanied by aspiration or frication noise tends to require a specific separate peak glottal opening. They interpret these independent glottal apertures as assuring the aerodynamic requirements for turbulent noise production during aspirated stops or fricative segments.

Laryngeal adjustments in voiceless obstruent production will be examined here in Berber voiceless clusters and compared to the organization of the laryngeal adjustment in other languages, mainly some of the Germanic languages mentioned above. We will present some general properties that appear to be commonly shared by speakers of different and unrelated languages.

Berber is an Afro-Asiatic language spoken in North Africa. Tashlhiyt Berber (Henceforth TB), which is the variety of Berber analysed in this article, is a dialect spoken in the Southern part of Morocco. Even if some variation is pervasive across the Tashlhiyt domain, it does not hinder mutual comprehension. TB uses both voicing and gemination distinctively. Both singleton and geminate velar and dental stops may be aspirated. Following a commonly accepted view, we consider a geminate as a single melodic unit associated with two prosodic positions [START_REF] Clements | CV Phonology: A Generative Theory of the Syllable[END_REF]. Such a representation accounts for the dual behaviour of the geminate which behaves in some respects as a consonant cluster and, in others, as a single consonant. Heteromorphemic geminates are a sequence of two identical segments separated by a morpheme boundary. They are generally homophonous with tautomorphemic geminates. In certain contexts, however, as is the case of /k#k/, the first velar stop may either be released or spirantized. Spirantization is an optional phonological process affecting simple non-coronal stops which become fricatives (i.e. /k/ is realized as [x]1 ). TB is of special interest in contributing to a better understanding of the organization of laryngeal adjustment, since this language allows an unusually rich combination of voiceless sounds, large series of voiceless words and different types of geminates in initial, medial and final positions [START_REF] Ridouane | Suite de consonnes en berbère : phonétique et phonologie [Consonant clusters in Berber: phonetics and phonology[END_REF].

The aim of this study is to improve our understanding of laryngeal adjustment in voiceless consonant cluster production and the mechanisms underlying the temporal coordination of laryngeal and oral articulations in sequential voiceless sounds. Our data can provide additional evidence for the interpretation that a static glottal opening position of the glottis is unlikely to occur (e.g. [START_REF] Yoshioka | Laryngeal adjustment in the production of consonant clusters and geminates in American English[END_REF][START_REF] Munhall | Gestural aggregation in speech: laryngeal gestures[END_REF]), since we included combinations of up to 6 voiceless consonants in our data (various combinations of /s/ and /k/). In this study we will focus on laryngeal adjustments during the production of different voiceless clusters and examine:

1. The influence of manner of articulation (stops and fricatives) or of gemination (tautomorphemic and heteromorphemic); 2. The effect of preceding, following or intervening word boundaries; 3. The presence or absence of aspiration in stops preceded by /s/.

METHOD

Two male native speakers of TB, RR, the first author, and RF were recorded by means of simultaneous transillumination (hereafter PGG), fiberoptic filming (hereafter FF), and acoustic recordings in the ZAS Laboratory in Berlin. A standard endoscope (Olympus ENF type P3) was inserted in the subjects' pharynx and a photosensor was glued externally on the subjects' neck. The endoscope was attached to a camera and connected to a video recorder with a monitor. The video images enabled the otorhinolaryngologist to control the position of the tip of the endoscope throughout the experiment. The video signals were taped to enable qualitative interpretation of the transillumination data. To provide the relevant amount of cold light for the tip of the endoscope, an external light source was attached to the endoscope 2 .

Acoustic and transillumination data have a sampling frequency of 24 kHz, PGG data were further downsampled to 200 Hz, and the FF data have the standard video format of 25 i/s. The velocity signal of the PGG signal was calculated as the first derivative. By analyzing the velocity signal we defined the beginning and end of glottal opening and closing using a 5% threshold criterion (i.e. the point where the signal crossed 5 per cent of the corresponding 2 Detailed discussion on the transillumination procedure and the factors affecting its reliability is given in [START_REF] Hoole | Techniques for investigating laryngeal articulation. Section A: Investigation of the devoicing gesture[END_REF]. movement segment). In the acoustic data we labeled for /k/ closure onset (clon) as the second formant offset of the preceding vowel, burst (b), aspiration offset (aspoff) as the end of high frequency energy, and for /s/ frication onset (fricon) and frication offset (fricoff) 3 . An example of our data is presented in Figure 1. The top panel shows the acoustic waveform of the cluster /s#ks/. The second panel shows the glottographic pattern, which indicates the duration, the degree and the number of glottal opening peaks. The vertical axis shows the amount of light in arbitrary unit. In this figure, we can see that two glottal opening peaks were produced, being located during the production of the two fricatives. The third panel indicates the velocity of glottal opening and closing gestures (i.e. the derivative of the transillumination data).

Insert FIGURE 1 HERE.

Both the transillumination technique and fiberoptic filming require a wide pharyngeal cavity, which was taken into consideration in selecting the linguistic material. The real word speech material consisted of several combinations of singleton and geminate /s/ and /k/ consonants. All the clusters were preceded and followed by the vowel /i/. Forms indicated in table 1 were produced by two subjects (RR and RF). RR participated in two experimental sessions4 . The results of the two sessions have been merged. The second subject (RF) participated in the second session. The number of repetitions for each subject is indicated between brackets, RR produced 160 tokens and RF 77 tokens. "#" indicates word boundary.

Insert Table 1 HERE.

The number of glottal opening peaks was calculated algorithmically as the number of (negative) zero-crossings in the velocity signal. To guard against counting spuriously large numbers of local maxima (e.g. due to noise during periods of low velocity) we defined a noise band as 5% of the average peak velocity over all utterances (separately for each speaker) and only counted those zero-crossings falling in movement segments that crossed this noise band above and below zero velocity. To obtain some further basic information on the shape of the glottal movements we also counted the number of positive velocity peaks during opening movements and the number of negative velocity peaks in closing movements5 . In the labeling of the figures below these velocity peak counts are referred to as "Vel. peaks (ab)" and "Vel. peaks (ad)", respectively (i.e. for abduction and adduction phases). The simplest movements are those in which each glottal opening peak is associated with exactly one positive and one negative velocity peak. While there can never of course be less velocity maxima than amplitude maxima, if either the number of positive velocity maxima or negative velocity maxima exceeds the number of amplitude maxima, then this indicates a more complex -generally more gradual -movement pattern for abduction or adduction movements respectively.

In the figures below showing the movement pattern for each linguistic category we have included the average counts of the amplitude and positive and negative velocity maxima. For the sake of clarity we have omitted any measure of variability from the figures (e.g. standard deviation). In most cases, particularly for amplitude peaks, this information is redundant as it can be derived from the average peaks counts. For example, a peak count of 1.1 with n=10 indicates 9 tokens with one peak and one token with two peaks.

To generate plots of the average movement pattern for each utterance type, the transillumination signal was normalized with respect to both time and amplitude. For the time normalization, the average duration of each utterance type was calculated over the interval from the start to the termination of glottal abduction (ON to OFF in Figure 1), and then each individual token was time-warped to this duration 6 . Amplitude normalization was designed to take account of gross changes in signal gain which may be caused for example by shifts in endoscope position over the course of the experiment. To this end, first the minimum amplitude preceding glottal abduction in each item was subtracted from the signal, then the average signal amplitude between ON and OFF labels was calculated for all items in a block of repetitions, and the transillumination signal was then normalized by this value. Ensemble averages for each utterance type were then calculated from this time-and amplitudenormalized data. On the basis of the amplitude-normalized data we believe that statistical comparison of glottal opening amplitude between utterance types is justified, if carried out cautiously, e.g. by using non-parametric tests.

RESULTS AND DISCUSSION

Clusters with two consonants

Results for two-consonant clusters are shown in Figures 2 and3. Figure 2 shows clusters with /s/ in first position, while Figure 3 shows those with /k/ in first position. As can be seen from these figures two-consonant clusters for both subjects exhibit a clear single-peaked 6 One item was excluded from this procedure as being highly untypical (one repetition of /kk#skk/ with a duration more than 50% longer than the average duration). pattern in the averaged traces regardless of the placement of word boundaries7 . The peak almost always occurs during the fricative.

Insert FIGURE 2 HERE. Insert FIGURE 3 HERE.
This is in agreement with results from Löfqvist and Yoshioka (1980a) based on data from Swedish. Two different strategies can explain the fact that the location of peak glottal opening follows the location of the fricative in the cluster. First, this is caused by aerodynamic conditions. The fricative requires a higher oral airflow than the stop and hence the peak glottal abduction occurs during the /s/. Second, this might be caused by two underlying glottal opening gestures which overlap -a larger one for /s/ and a smaller one for /k/. This latter view is advocated by [START_REF] Munhall | Gestural aggregation in speech: laryngeal gestures[END_REF] on the basis of an experimental exploration of gestural aggregation, showing that such clusters consist underlyingly of two gestures. For [START_REF] Browman | Towards an articulatory phonology[END_REF], the single-peaked glottal opening observed in word initial fricative-stop clusters is a phonological regularity of syllable-initial position in English, suggesting that the single peak is a property of the whole syllable onset. They capture the relevant timing of laryngeal-oral coordination in the following rule:

-If a fricative gesture is present, coordinate the peak glottal opening with the midpoint of the fricative. Otherwise, coordinate the peak glottal opening with the release of the stop gesture. This rule, tested over various voiceless clusters in different languages, does not appear to be completely accurate (see Hoole et al. 2004). Berber material also shows that the peak glottal opening is not systematically coordinated with the midpoint of the fricative. The generalization that can be drawn from our data is that peak glottal opening is almost always located within the fricative both for stop-fricative and for fricative-stop sequences. The timing of this opening peak tends to shift to a relatively earlier point in the fricative when it follows a stop (at 23.49 % of the fricative) and to later point in the fricative when it precedes a stop (at 66.06% of the fricative), regardless of the word boundary location. Figure 4 illustrates these timing patterns for each subject.

Insert FIGURE 4 HERE.

Contrary to what has been observed in different languages (e.g. English and Swedish), in TB /k/ can be acoustically aspirated after /s/ whether separated or not by a word boundary.

Previous work (e.g. as summarized by [START_REF] Löfqvist | Interarticulator programming in stop production[END_REF] suggests that typically each fricative and aspirated plosive requires a separate laryngeal peak. In Swedish (Löfqvist and Yoshioka 1980a) and English [START_REF] Yoshioka | Laryngeal adjustment in the production of consonant clusters and geminates in American English[END_REF]), a sequence of voiceless fricative + voiceless aspirated stop usually contain two separate peak glottal openings located during the fricative and just before stop release. In TB, this generalization has to be qualified since interestingly the language shows aspirated plosives following fricatives in initial position, but nevertheless the results, as illustrated in Figure 2, make clear that only one opening peak occurs. This pattern may well be related to the fact that voiceless geminates also show aspiration -in fact a very similar amount of aspiration to the singleton consonants. It was shown in [START_REF] Ridouane | Suite de consonnes en berbère : phonétique et phonologie [Consonant clusters in Berber: phonetics and phonology[END_REF] that aspiration duration, based on an acoustic study for 7 subjects, is not a significant criterion for distinguishing between singleton and geminate stops8 . Both are produced with virtually identical aspiration durations. For dentals measurements showed that aspiration duration varies between 45 to 65 ms for singletons and between 35 to 50 ms for geminates. For velars, aspiration duration varies between 45 to 70 ms for singletons and between 45 to 65 ms for geminates.

A photoelectroglottographic study, based on one subject, showed however important differences between singletons and geminates both in terms of timing and amplitude of glottal opening (cf. [START_REF] Ridouane | Suite de consonnes en berbère : phonétique et phonologie [Consonant clusters in Berber: phonetics and phonology[END_REF]. The maximum glottal opening, measured in arbitrary units, shows clearly that geminates are systematically produced with larger glottal opening than singletons. The interval between peak glottal opening and oral release is longer for geminates than for single stops. For dentals, this interval varies between 0 to 10 ms for singletons and between 55 to 120 ms for geminates. For velars, the interval varies between -10 to 20 ms for singletons and between 55 to 70 ms for geminates.

Laryngeal abduction for geminates is much larger in amplitude and longer in overall duration compared to singletons. However, the glottal opening at stop release appears to be similar for both. Figure 5 below, which is arranged so as to show the glottal opening of a minimal pair involving a geminate and a singleton stop, shows that the size of glottal opening at stop release is nearly identical for both singletons and geminates which may thus explain the fact that aspiration duration for both stops is virtually identical.

Insert FIGURE 5 HERE. [START_REF] Löfqvist | Interarticulator programming in stop production[END_REF] has argued rather persuasively for the importance of laryngeal-oral timing in contrasting aspirated and unaspirated plosives, but the present results for TB provides a striking example of the contrary phenomenon, namely radically different timing patterns (of peak glottal opening relative to closure release) leading to similar degrees of aspiration duration. One might speculate that the somewhat atypical aspiration in initial fricative-plosive clusters is using the same timing pattern that has emerged for the geminates, suggesting that aspiration, as was defined by [START_REF] Kim | A theory of aspiration[END_REF], is essentially a function of the degree of glottal opening at stop release.

Clusters with three consonants

Tautomorphemic vs. heteromorphemic geminates

Insert FIGURE 6 HERE.

Figure 6 contains the glottographic patterns of three-consonant-clusters produced by RR.

These clusters are composed of two types of geminates, tautomorphemic and heteromorphemic ones. As was outlined above, one of the main topics of this study was to determine the possible influence of gemination on glottal adjustments. Comparisons between /kk#s/ and /k#s/ (middle column of Figure 3) on the one hand and between /ss#k/ and /s#k/ (middle column of Figure 2) on the other hand show that all these clusters are always produced with one peak glottal opening. This suggests that the presence of a geminate, instead of a singleton, is not accompanied with an additional separate laryngeal peak. There is some increase in movement complexity, however: the opening phase of /kk#s/ and the closing phase of /ss#k/ are rather gradual (indicated by velocity peak counts of 1.67 for the former and 1.33 for the latter). A major difference is nonetheless worth noting. The cluster with the velar geminate stop is systematically produced with larger glottal opening amplitude than the singleton counterpart, in fact approximately double on the basis of the ensemble averages 9 . This seems to correspond to a systematic difference, as already mentioned above, observed between singleton and geminate stops in intervocalic position. By contrast, peak amplitude is practically identical for the singleton and geminate fricatives 10 .

The comparison between tautomorphemic and heteromorphemic geminates indicates that the number and the location of peak glottal opening vary according to the phonetic nature of the voiceless obstruents and the way they are combined. The glottographic patterns for the form with the velar tautomorphemic geminate /kk#s/ show that the peak glottal opening is most often located during the geminate (towards the burst). The three realizations of the form /k#ks/ were not produced with the same number of amplitude peaks. One repetition was realized with two glottal openings, and two repetitions were realized with only one. A closer examination of the three repetitions shows that for the bimodal realization, the first velar was pronounced as a fricative [x]; the additional peak was thus located during this first velar fricative 11 . This configuration, as we shall show in the following section, is identical to the one attested in the three-phone combinations where the first segment and the third are dental fricatives. Regardless of the number of observable amplitude peaks, all repetitions of /k#ks/ had 2 velocity peaks on the opening phase, which, accordingly, is much more gradual than the closing phase. 9 A non-parametric test of this difference proved highly significant, though note that we have only 3 tokens for the geminate. 10 The absence of systematic differences in terms of laryngeal amplitude between singleton and geminate fricatives has also been observed in [START_REF] Ridouane | Suite de consonnes en berbère : phonétique et phonologie [Consonant clusters in Berber: phonetics and phonology[END_REF], where intervocalic labiodental and dental singletons were compared to their geminate counterparts. 11 The symbol indicating the burst for stops is maintained for forms realized as spirants. In this case, the symbol indicates the offset of the velar consonant.

/ss#k/ and /s#sk/ are similar to the extent that both sequences always have only one amplitude peak. Peak glottal opening for /ss#k/ is always located during the geminate /ss/. The cluster with a heteromorphemic geminate /s#sk/, produced with an uninterrupted frication noise during the two fricatives, is also produced with only one large glottal opening located during the geminate fricative. The two sequences differ in that the closing phase is longer for /s#sk/ (which has 3 velocity peaks, compared to a count of 1.33 for /ss#k/).

Overall one might say that there is some indication for greater movement complexity in the heteromorphemic compared to the tautomorphemic sequences, the former having a greater number of velocity peaks.

Clusters without geminates

Figure 7 compares two three-phone combinations /s#ks/ and /k#sk/. Looking first at /s#ks/ (right panels of Figure 7), one finds a very clear two-peaked pattern for RR. Word final /s/ in /s#ks/ is produced with a single opening peak and word initial /ks/ is produced with another one. Again, peak glottal abduction is always located during the fricatives. The same configuration was also observed for identical clusters in Swedish (Löfqvist & Yoshioka 1980a), Icelandic (Löfqvist & Yoshioka 1980b) and American English [START_REF] Yoshioka | Laryngeal adjustment in the production of consonant clusters and geminates in American English[END_REF]). The glottis is only slightly adducted without complete closure between the two opening maxima. This transitory glottal closing movement is not necessarily a consequence of a pause due to a word boundary. No specific opening gesture is observed for the velar stop /k/. This segment is rather produced within the closing phase of the glottal gesture for the preceding fricative and within the opening phase for the following /s/ segment.

Insert FIGURE 7 HERE.

The pattern for RF is at first sight more ambiguous: the amplitude peak count of 1.4 indicates that two repetitions were monomodal and three bimodal. Closer inspection indicates that the monomodal patterns had shorter durations. Taking into account that the velocity peak counts are very close to 2 for both opening and closing, this suggests that an underlyingly bimodal amplitude pattern changes into a monomodal one as the duration of the cluster decreases. This seems to correspond to what has been observed by Löfqvist and Yoshioka (1980b) in their analysis of voiceless clusters in Icelandic. For the production of the Icelandic sequence [s#sp], for example, where the duration is less than 300 ms, there is only a single glottal opening movement. For the longer productions two glottal opening movements occur during the cluster.

On average, the /k#sk/ cluster is realized with one single opening peak located during the /s/ for both subjects as is shown in Figure 7. Only two repetitions out of seven for RR were realized with two peak glottal openings, one during the fricative /s/ and the second during the following velar consonant. Closer examination of the phonetic nature of these bimodal forms shows that this velar consonant was also produced as a fricative [x] in both cases, presumably thus favouring two peaks12 . All these sequences contain two dental fricatives. They are most often produced with two glottal opening peaks located during these two fricatives.

Clusters with four to six consonants

Insert FIGURE 8 HERE.

These results are, once again, in accordance with results drawn from various languages. [START_REF] Yoshioka | Laryngeal adjustment in the production of consonant clusters and geminates in American English[END_REF], for instance, observed that American English fricative + stop + fricative + stop sequences were also produced with two peak glottal openings located during the fricatives. The same laryngeal adjustments were produced during the Icelandic sequence [st#sp] (Löfqvist & Yoshioka 1980b). Similar configurations observed in different unrelated languages provide a striking illustration of the tight temporal coordination of laryngeal and oral articulations in voiceless obstruent production: a sequence with two non-adjacent fricatives is most of the time produced with two glottal opening gestures. Another important observation is that /ks#ks/ and /k#sks/ are produced with virtually the same laryngeal adjustments for both subjects, showing once again that word boundaries are quite transparent as far as laryngeal gestures are concerned.

Insert FIGURE 9 HERE.

Figure 9 firstly compares two similar four-phone combinations with different word boundary locations: /k#kss/ (left panel) and /kk#ss/ (middle panel). These clusters are produced with only one glottal opening peak achieved during the fricative. This always held true despite the placement of the word boundary and despite some slight differences in the number of velocity peaks in the opening and closing phases. Compared to the three-consonant clusters /kk#s/ and /k#ks/ (see Figure 6, top left and right, respectively), these clusters exhibit quite similar movements but the location of the peak shifts slightly more into the fricative in the sequences with geminate fricatives. The comparison here between Figure 9 and Figure 6 also in effect compares geminate with singleton fricatives. It was already mentioned that geminate fricatives do not increase the glottal opening amplitude as much as geminate plosives do (compared to corresponding singletons). Here, too, there is not much indication that fricative gemination results in a strong increase in amplitude. /k#kss/ does not appear larger than /k#ks/ but the small number of tokens effectively precludes statistical testing13 . /k#kss/ and /kk#ss/, as realized here by RR, are homophonous, both forms being produced with a long closure duration followed by a release, i.e. in the first half of the heteromorphemic geminate the oral closure was maintained and the release follows in the second half of the geminate. Similar pronunciations are not produced in adjacent identical consonant sequences in which one segment is a geminate (e.g. /k#kk/ or /kk#k/). In such sequences, the geminate stop must be released and the singleton is either produced as a released stop or, if the optional spirantization rule operates, as a spirant. The sequence /k#kk/, for example, is phonetically distinct from /kk/, the first singleton velar being produced either as a stop or as a fricative (i.e. [x#kk]). This latter pronunciation was the one produced during the three realizations of /k#kks/ (right panel of Figure 9). These forms are always produced with two glottal opening peaks, the first being generally produced during the fricative [x] and the second either during the burst of the geminate /kk/ or at the onset of the fricative /s/.

Figure 10 contains clusters of five consonants. The sequence /ssk#kk/ (right panels) is produced either with two or three glottal abduction gestures. The first peak is always produced during the dental fricative /ss/. RR produced seven repetitions with 3 peak glottal openings and three repetitions with 2 peaks. The second peak in the bimodal productions is always located during the closure of the geminate stop /kk/. The third peak in the trimodal production is located during the singleton velar segment, generally realized as a spirant [x].

RF always produced this cluster with a bimodal gesture. The second peak is either realized during the spirantized singleton velar stop [x] (in 1 repetition) or during the occlusion of the geminate stop /kk/.

Insert FIGURE 10 HERE.

The glottographic pattern of the /kk#skk/ cluster (left panels) also varies according to the subjects. For RR, the overall pattern shows two amplitude peaks. The second peak is rather weak, and in two cases only one peak was detected based on our criteria. In one case, a very weak third peak was detected. The velocity peak counts are consistent with the idea that the basic pattern has two glottal opening movements, but with both opening and closing being somewhat more gradual than in the simplest two-peaked patterns. The first opening peak was located roughly at the boundary between the first geminate and the following fricative, the second peak during the second geminate 14 .

All the realizations of RF, by contrast, are characterised by one opening gesture. A closer examination of this monomodal pattern reveals that this configuration is presumably related to the very short closure duration for the two velar geminates. Peak glottal opening is always located during the fricative /s/. This was thus the linguistically longest sequence in our material with a clearly monomodal pattern.

Insert FIGURE 11 HERE. 14 One of the 10 repetitions of this form by RR was not included in the average curve or the peak counts. The first repetition of the 5 recorded by RF was not considered further as it was much longer than the others (720 ms instead of 280 ms for the other realizations which are also shorter than expected probably due to degemination). The longer duration of this form is due to a long silent pause maintained during the word boundary. This silent pause was associated with clear glottal adduction (without complete closure).

The glottographic patterns for the six-consonant clusters are presented in Figure 11. For /ssk#ssk/ (left panels) the clearly predominant pattern for both subjects is for two glottal opening peaks, located during the first and second geminate fricatives respectively (note, however that both opening and closing velocity peak counts are well above two for both subjects). The movement pattern for this 6-consonant sequence is quite similar to the corresponding singleton pattern /sk#sk/ in the left panels of Figure 8. In the longer sequence, however, -with the usual caveat because of the small number of tokens -there was significantly greater opening amplitude for both subjects for the sequence containing geminate fricatives -perhaps because the double change from singleton to geminate has an amplifying effect.

The remaining sequences /ssk#skk/ and /ssk#sks/ exhibit much variability even within the same subject. Taking the simplest case first, namely /ssk#skk/ for RF, it is observed that this has a fairly clear two-peaked pattern. The most instructive sequence for comparison is once again /sk#sk/ from Figure 8 (i.e. reducing each geminate to a singleton). The overall movement pattern is very similar, supported by similar peak counts for amplitude and velocity, and there is a similar location of the opening peaks in the fricative segments. The linguistically longer sequence simply, and not surprisingly, has longer duration (464 ms for the longer sequences and 360 ms for the shorter on average) and also significantly greater opening amplitude. For RR, on the other hand, inspection of the curve as well as the high values for all the peak counts indicates substantially more movement complexity in this sequence than in either /ssk#ssk/ just discussed above, or the comparison singleton sequence /sk#sk/.

Turning finally to /ssk#sks/, this is the sequence where greatest movement complexity might be expected as it is the only sequence in the material with three separate fricative segments.

By and large, this expectation is borne out. RR had a clear preference for 3 opening peaks, quite clearly associated with each of the three fricative segments (albeit rather different in amplitude). This was the only occasion in the whole material where the mean amplitude peak count went above three (caused by one exception where 4 peaks occurred). For RF the amplitude peak count was more evenly balanced: 3 repetitions with 2 peaks, and 2 repetitions with 3. In the averaged trace, peaks are found associated with the first two fricative segments.

For both subjects the complexity of the movement pattern is made clear by the very high count for closing velocity peaks (4.29 for RR and 4.2 for RF), which is substantially higher than for any other sequence.

CONCLUSION

The main results of the present study are in general agreement with those obtained from some Germanic languages. We thus have further evidence from different unrelated languages that laryngeal activity during the production of voiceless obstruent clusters is organized in one, two or more glottal opening peaks. The manner of articulation of obstruents and their position in the clusters have a major impact on the number of glottal opening peaks and their locations. A major generalization that comes out from this study is that sequences produced with n fricative segments are most often produced with n peak glottal openings; these peak glottal openings are located during the production of these fricatives. In other words, segments produced with a high rate of oral air flow are produced with a separate laryngeal opening gesture. Geminates, mainly stops, are usually produced with larger glottal amplitude than their singleton counterparts. But the presence of a geminate in a cluster, instead of a singleton, is not accompanied with an additional separate laryngeal peak, although the geminate sequences exhibit an increase in movement complexity (i.e. the opening and closing phases during these sequences are rather gradual).

Another topic of interest in this article was to determine if there are laryngeal correlates of word boundaries. From the data analyzed here it appears that when a word boundary is marked by a long silent pause, it is associated with glottal adduction. According to Löfqvist & Yoshioka (1980a), such an adduction gesture is made to prevent air flow and waste of air during an ongoing utterance. Another possible interpretation would be that word boundaries are in themselves accompanied by glottal adduction. TB material however shows that glottal adduction is not necessarily associated with linguistic boundaries. Recall that adduction gestures are usually found in clusters without apparent word boundaries. Another piece of evidence showing that word boundaries are transparent as far as laryngeal gestures are concerned is illustrated by the numerous cases where two clusters with two different word boundary locations are often produced with similar laryngeal adjustments. Glottal adduction found in these sequences seems better ascribed to segmental properties.

In addition to TB, other languages present aspirated stops after /s/. The absence of aspiration in such segments is thus not a universal phonetic feature, contrary to what was assumed by [START_REF] Lindqvist | Laryngeal articulation studied on Swedish subjects[END_REF]. [START_REF] Pétursson | Aspiration et activité glottale[END_REF] cited cases observed in some Indian languages as well as in some Colombian Spanish dialects. This aspiration is rather exceptional, not only because it is not attested in the most widely studied languages, but also, at least in the case of TB, because it seems totally independent of word boundaries. The TB case is thus typologically interesting, making it possible to determine the two possible laryngeal mechanisms accounting of stop aspiration after /s/: on the one hand a large amplitude and a delay in peak glottal opening relative to fricative onset; on the other hand two peak glottal openings, each corresponding to one of the two obstruents. The second realization is the one produced in /s/stop heteromorphemic sequences in some Germanic languages. In sum, different combinations of interarticulatory timing and glottal opening size can result in similar amounts of aspiration. Löfqvist and Yoshioka (1980a: 800) in their analysis of long voiceless consonant clusters noted that: « There is little, if any evidence that the glottis ever opens and maintains a static open position in speech.» TB material, which included up to 6 voiceless consonant clusters, provides further evidence that the glottis does not simply remain open for voiceless utterances. These results can be combined with those obtained from an earlier study on TB voiceless words [START_REF] Ridouane | Les mots sourds en berbère chleuh : analyses fibroscopiques et photoglottographiques. 25 es[END_REF]). Here the whole utterances, which contain up to 10 obstruents, were voiceless, and one might have expected that the devoicing gesture might be regarded as superfluous and simply eliminated, leaving the glottis in respiratory position.

However, just as in the present experiment, the results showed clearly that the glottal aperture is continuously modulated in a manner that can be related quite systematically to the individual segments in the voiceless sequence. This gives a compelling demonstration of how intimately laryngeal and supralaryngeal articulations are linked.

Figure 8

 8 Figure 8 contains the glottographic patterns of the three clusters /sk#sk/, /ks#ks/ and /k#sks/.

Figure XXX, 1 .

 1 Figure XXX, 1. Example of defined time landmarks concerning acoustic and PGG data in /s#ks/.Fricon = frication onset, Fricoff = frication offset, b = burst; ON = glottal opening onset, PGO = peak glottal opening, OFF = glottal opening offset.

In our transcriptions, forms enclosed between slashed lines are intended to represent the underlying structure of these forms. When a form has a phonetic output different from its underlying structure, the phonetic form is enclosed between brackets.

The labeling of the acoustic data was done by visual and perceptual inspection of the signals and spectrograms.

The second experiment took place in Berlin about a year after the first one.

This was done analogously to the amplitude peaks by counting zero-crossings in the acceleration signal

The mean count of amplitude peaks indicates only two tokens that did not conform to the overall pattern: one token each of /k#s/ and /ks#/ for speaker RR.

For dentals and velars. TB lacks voiceless bilabial stops. The other voiceless stops (the uvular /q/ and the emphatic /T/ and their geminate counterparts) are not aspirated.

This displays the same pattern observed in some repetitions of /k#ks/ discussed above. Note also that all /k#sk/ repetitions for RR are characterized by a very gradual closing phase, with a high number of velocity peaks.

If /k#kss/ and /kk#ss/ are merged to a geminate group, and /k#ks/ and /kk#s/ to a singleton group (with 6 tokens in each group) no significant difference in amplitude is found.

ACKNOWLEDGEMENTS

We would like to thank Dr. Klaus Dahlmeier for his expertise during the transillumination recording, Jörg Dreyer for technical support, and Christine Mooshammer for help with postprocessing of the data. This work was supported by a grant from the German Research Council (DFG) GWZ 4/8-1, P.1.


Julie Bertrand
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Influence of pharmacogenetics on indinavir disposition and short-term response in

Keywords: Pharmacokinetics, Nonlinear Mixed Effects Modeling, Protease inhibitors, CYP3A4, Safety, Efficacy Céron, X. Duval, statistics: F. Mentré;

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Introduction

Indinavir has been one of the preferred protease inhibitor (PI) included in highly active antiretroviral therapy (HAART). Nowadays, even if not recommended as initial therapy, indinavir is still used in patients who initiated their therapy with this PI and kept a viral load below the limit of quantification with an acceptable safety profile. Further compared to others PI, indinavir exhibits a high penetration into viral reservoirs such as genital compartments and CNS [START_REF] Solas | Discrepancies between protease inhibitor concentrations and viral load in reservoirs and sanctuary sites in human immunodeficiency virus-infected patients[END_REF]. It was found that the indinavir better distribution is leading to better outcomes in neurological complications related to HIV [START_REF] Letendre | Validation of the CNS penetration-effectiveness rank for quantifying antiretroviral penetration into the central nervous system[END_REF]. The pharmacokinetics (PK) of indinavir is characterised by high maximal concentrations leading to potential toxicity, notably nephrolithiasis [START_REF] Collin | Indinavir trough concentration as a determinant of early nephrolithiasis in HIV-1-infected adults[END_REF] and low minimum concentrations with respect to the 95% inhibitory concentration of the virus. These low residual concentrations result from an extended oxidative metabolism by the cytochrome P450 (CYP3A) isoenzyme [START_REF] Flexner | HIV-protease inhibitors[END_REF].The co-administration of ritonavir, whose molecular structure leads to CYP3A inhibition, therefore enhances exposure to indinavir [START_REF] Moltke | Protease inhibitors as inhibitors of human cytochromes P450: high risk associated with ritonavir[END_REF][START_REF] Hsu | Ritonavir. Clinical pharmacokinetics and interactions with other anti-HIV agents[END_REF]. As a booster, ritonavir is given at lower dose than for therapeutic use, but was shown nevertheless to influence metabolic profiles especially with respect to lipids disorders [START_REF] Soriano | Efficacy and safety of replacing lopinavir with atazanavir in HIV-infected patients with undetectable plasma viraemia: final results of the SLOAT trial[END_REF][START_REF] Collot-Teixeira | Impact of different low-dose ritonavir regimens on lipids, CD36, and adipophilin expression[END_REF].

The large inter-patient and intra-patient variability of indinavir pharmacokinetics is well referenced [START_REF] Csajka | Population pharmacokinetics of indinavir in patients infected with human immunodeficiency virus[END_REF][START_REF] Brendel | Population pharmacokinetic analysis of indinavir in hivinfected patient treated with a stable antiretroviral therapy[END_REF][11]. Genetic polymorphisms could partly explain this variability, as far as proteins involved in the metabolism and transport of PI are concerned. Few studies have investigated the impact on indinavir pharmacokinetics of ABCB1 polymorphisms, a gene coding for P-glycoprotein, and of CYP3A5 and CYP3A4*1B polymorphisms. Solas et al. [START_REF] Solas | Minimal effect of MDR1 and CYP3A5 genetic polymorphisms on the pharmacokinetics of indinavir in HIV-infected patients[END_REF] have shown that the ABCB1 C3435T genotype affects the absorption constant of indinavir whereas Verstuyft et al. [START_REF] Verstuyft | Absence of association between MDR1 genetic polymorphisms, indinavir pharmacokinetics and response to highly active antiretroviral therapy[END_REF] have reported an absence of association. Anderson et al. [START_REF] Anderson | Pharmacogenetic characteristics of indinavir, zidovudine, and lamivudine therapy in HIV-infected adults: a pilot study[END_REF] have observed that CYP3A5 expressors (CYP3A5*1 carriers) have significantly faster oral clearance than non-expressors. To date no relationship was found between CYP3A4*1B polymorphism and alterations in CYP3A substrate metabolism, but clinical data have shown an association between CYP3A*1B polymorphism and disease risk/treatment toxicity [START_REF] Lamba | Genetic contribution to variable human CYP3A-mediated metabolism[END_REF].

Efficacy [START_REF] Lichterfeld | Long-term efficacy and safety of ritonavir/indinavir at 400/400 mg twice a day in combination with two nucleoside reverse transcriptase inhibitors as first line antiretroviral therapy[END_REF][START_REF] Duval | Indinavir plasma concentration and adherence score are codeterminant of early virologic response in HIV-infected patients of the APROCO cohort[END_REF] as well as adverse events [START_REF] Dieleman | Urological complaints in relation to indinavir plasma concentrations in HIV-infected patients[END_REF][START_REF] Collin | Indinavir trough concentration as a determinant of early nephrolithiasis in HIV-1-infected adults[END_REF] has been related to indinavir plasma concentrations. Thus, therapeutic drug-monitoring appears to be a potent tool to achieve undetectable HIV-RNA and prevent toxicity for this drug. The COPHAR 2 -ANRS 111 trial is a multi-centre non-comparative pilot trial of early therapeutic drug-monitoring in HIVpositive patients naive of protease inhibitor containing highly active antiretroviral treatment (HAART) [START_REF] Duval | Benefit of therapeutic drug monitoring of protease inhibitors in HIV-infected patients depends on PI used in HAART regimen -anrs 111 trial[END_REF]. We focused on the PK sub-study from the group of patients receiving indinavir boosted with ritonavir. The aims of this paper were to estimate the population PK parameters and variability of indinavir and ritonavir in HIV patients, to evaluate the impact of genetic polymorphisms on indinavir PK and to study the link between indinavir concentrations and short-term efficacy and metabolic safety.

Methods

Study

The COPHAR 2 -ANRS 111 study is a multi-centre non-comparative prospective pilot trial of early-dose adaptation in HIV-positive patients naive of protease inhibitors (PI) starting a PI containing HAART treatment. The trial started on July 2002 and was completed by the end of March 2005. The objective was to assess the benefit of pharmacological advice based on trough plasma concentrations of PI. The study involved three groups treated with indinavir, nelfinavir or lopinavir respectively. In the present study we analysed the data obtained during the first month of treatment in the indinavir group. A similar analysis of the data in the nelfinavir group was performed by Hirt et al [START_REF] Hirt | Effect of CYP2C19 polymorphism on nelfinavir to M8 biotransformation in HIV patients[END_REF][START_REF] Duval | Benefit of therapeutic drug monitoring of protease inhibitors in HIV-infected patients depends on PI used in HAART regimen -anrs 111 trial[END_REF], details of the study can be found in that paper.

Patients were required to have a baseline plasma viral load value > 1000 copies/mL, and to be naïve of treatment with protease inhibitor. Patients initiated a HAART treatment containing 400, 600 or 800 mg of indinavir b.i.d. associated with ritonavir booster (100 mg b.i.d.) and two nucleoside analogues. The first dose was left to physicians' discretion and no dose adaptation was performed from week 0 (W0) to W4. A detailed pharmacokinetic study was performed at W2. Adherence was evaluated at W2 by means of a validated autoquestionnaire [START_REF] Carrieri | The dynamic of adherence to highly active antiretroviral therapy: results from the french national APROCO Cohort[END_REF] and patients were classified as adherent when reporting no shift in their treatment schedule during the last 4 days and non-adherent otherwise.

Viral load and CD4 count were collected at baseline (D0) and at W2. Biochemical profiles of total cholesterol, high density lipoprotein cholesterol, triglyceride and glycaemia as well as creatinine clearance and clinical events (diarrhoea grade of 2) were collected 4 weeks before treatment initiation (W-4) and at W4.

The study was performed in accordance with the Declaration of Helsinki and its amendments. All subjects gave written informed consent and the protocol was approved by the Ethic Committee of the Bicêtre Hospital (France) as well as the amendment for the pharmacogenetic study.

Indinavir and ritonavir concentration measurements

During a visit to the hospital at W2, the patients were sampled on arrival to measure trough concentrations. Patients were asked to record the time at which the dose was taken on the previous evening. Then they were given their medications, and sampled again 1 h, 3 h and 6 h after drug administration. Plasma concentrations were assumed at steady state with trough concentrations considered as following the drug intake using the delay reported by the patient from previous dosing. Plasma concentrations were determined in the laboratories of the hospitals by specific high-performance liquid chromatography. The participant laboratories were cross-validated before starting the study. Results of the blind inter-laboratory quality control at three concentrations for indinavir and for ritonavir were within 15% of the target values for medium and high values and within 20% for low values. Lower limits of quantification (LOQ) were 0.02 mg/L for indinavir and 0.025 mg/L for ritonavir.

Genetic polymorphisms

All the genotyping analyses were performed in the same laboratory. Total deoxyribonucleic acid (DNA) was extracted from plasma samples by use of the QIAamp DNA Blood Mini Kit (Qiagen, Courtaboeuf, France). ABCB1 polymorphisms in exons 21 (GG, GT, TT) and 26 (CC, CT, TT) were determined by use of previously-published methods [START_REF] Cascorbi | Frequency of single nucleotide polymorphisms in the Pglycoprotein drug transporter MDR1 gene in white subjects[END_REF]. Genotyping of CYP3A5 (*1*1, *1*3, *3*3, *1*6, *6*6) was performed by real-time polymerase chain reaction by use of TaqMan MGB probe technology (Applied Biosystems). Genotyping for CYP3A4 (*1B*1B, *1B*1A, *1A*1A) was determined by polymerase chain reaction (PCR) followed by direct sequencing. PCR was performed by use of a GenAmp PCR System 9700 (Applied Biosystems, Courtaboeuf, France) according to a previously-published method [START_REF] Dally | The CYP3A4*1B allele increases risk for small cell lung cancer: effect of gender and smoking dose[END_REF]. Amplified DNA was purified by use of the QiaQuick DNA Purification System (Qiagen) and sequenced by use of BigDye Terminator chemistry and an ABI PRISM 3100 genetic analyser (Applied Biosystems). For each genotyping analysis, at least 2 positive controls were used: 1 homozygous for the wild-type allele and 1 heterozygous and, when available, 1 homozygous for the mutated allele. These controls were DNA that had already been sequenced.

Allele frequencies (p for the wild allele and q = 1 -p for the mutant allele) were estimated by gene counting. Departure from Hardy-Weinberg proportions (p 2 , 2pq, q 2 ) was tested by a χ 2 test with 1 degree of freedom within each ethnic group [24]. We used two approaches to define patients belonging to an ethnic group: i. classification on the patient town, birth area and nationality and ii. classification by means of their genotype information using the Structure software [START_REF] Pritchard | Inference of population structure using multilocus genotype data[END_REF]. This software is based on a Bayesian approach and computes a posteriori probabilities for each individual of belonging to a given ethnic group. We assumed each locus to be at the Hardy-Weinberg equilibrium and patients to originate in one ethnic group (with its own characteristic set of allele frequencies).

Population pharmacokinetic analysis

We used a population approach to analyse the concentration-time data at W2 for indinavir and for ritonavir separately. Model fitting and estimation of the population model parameters were performed by use of the Stochastic Approximation Expectation Maximization algorithm (SAEM) for nonlinear mixed-effects models implemented in the MONOLIX software version 2.1 [START_REF] Kuhn | Maximum likelihood estimation in nonlinear mixed effects models[END_REF][START_REF] Lavielle | MONOLIX (MOdèles NOn LInéaires à effets miXtes)[END_REF][START_REF] Lavielle | Estimation of population pharmacokinetic parameters of saquinavir in HIV patients with the MONOLIX software[END_REF]. Both indinavir and ritonavir concentrations were fitted by a onecompartment model with first-order absorption and first-order elimination parameterised in absorption rate constant (k a ), oral clearance (Cl/F) and oral volume of distribution (V /F). Each model was assumed at steady state with trough concentrations considered as following the drug intake.

An exponential model was used for inter-individual variability where random effects were assumed to follow a normal distribution with zero mean and diagonal variance matrix. Additive, proportional and combined error models were tested and model choice was based on the likelihood ratio test (LRT) and goodness-of-fit plots (observed vs. predicted population and individual concentrations, population and individual weighted residuals vs. predicted concentrations and vs. time). We performed a visual predictive check (VPC) with 1000 simulated data sets to evaluate the basic model [START_REF] Gelman | Bayesian data analysis[END_REF].

Interaction between ritonavir and indinavir pharmacokinetics was evaluated with the individual parameters estimated from the basic model for each drug. All the different correlations were tested with the Spearman non-parametric correlation test.

Assessment of the effect of covariates

The effects of the following covariates were evaluated from the basic model: dose, concomitant use of the zidovudine lamivudine combination (AZT/3TC), co-infection by hepatitis C or B (VHC/VHB), adherence as previously defined, sex, ethnic group, the four studied genetic polymorphisms (ABCB1 exon 26, ABCB1 exon 21, CYP3A5 and CYP3A4) and the CDC classification for HIV infection as categorical variables, age, body mass index (BMI), body weight, creatinine clearance, albumin and orosomucoid levels as continous variables. The latter were centered to the median and log-transformed for model interpretation convenience.

Each of the four genetic polymorphisms was analysed by means of two binary categorisations: first, wild homozygotes versus heterozygotes or mutant homozygotes, second, heterozygotes or wild homozygotes versus mutant homozygotes. Categorisation in three classes was also tested: wild homozygotes versus heterozygotes versus mutant homozygotes. Missing continuous covariates were replaced with the median and patients with missing dicrete covariates were discarded for the corresponding analysis. The effects of covariates on the empirical Bayes estimates (EBE) of each individual pharmacokinetic parameters from the basic model were tested with the Wilcoxon non-parametric test for categorical variables and the Spearman non-parametric correlation test for continuous variables. The population covariate model was built with the covariates which were found to have an effect in this first step with a P-value < 0.1. When a genetic covariate was found to have an effect whatever the categorisation, the same categorisation as other genetic covariates also found to have an effect was chosen in model selection for consistency.

A forward selection of these covariates for the population model was performed by use of the likelihood ratio test (LRT) with a significance threshold at P < 0.05. From this ascending method, a backward elimination procedure was performed. In order to correct the inflation of the LRT type I error on small sample size [30], the backward selection was realized using permutation [START_REF] Manly | Randomization, bootstrap and monte carlo methods in biology[END_REF]. More specifically, 1000 data sets are generated by permuting the rows of the covariates matrix from the original data set. For each covariate, one likelihood ratio statistic LRT obs is estimated from the original data and one likelihood ratio statistic LRT perm is estimated from each of the 1000 data sets. Thus, we obtain j=1,...,1000 LRT perm j . The permutation P-value is the proportion : card(LRT perm j > LRT obs )/1000.

Short-term efficacy and safety and link with concentrations

As there was no change of dose before W4, we studied the link between concentration at W2 and efficacy or safety during the first two or four weeks of treatment. For short-term efficacy, the difference of log viral load between the day of treatment initiation and W2 (∆ logV L) was studied. The significance of the viral load decrease was tested by a Wilcoxon non-parametric paired test.

Individual area under the concentration-time curve (AUC), maximal plasma concentrations (C max ) and trough concentrations (C trough ) of indinavir at steady-state were derived for each patient by use of the EBE of the individual parameters from the basic model and their corresponding dose of indinavir. The relationship between indinavir dose, indinavir AUC, C max , C trough and ∆ logV L was evaluated by use of the Spearman correlation test. A Wilcoxon non-parametric test was performed to compare ∆ logV L between patients with or without a C trough below the lower limit of the therapeutic range used in the COPHAR 2 -ANRS 111 trial: 150 ng/mL. Safety was analysed through the difference between 4 weeks before and 4 weeks after treatment initiation in total cholesterol (∆ TC), high density lipoprotein cholesterol (∆ HDL), triglyceride (∆trig) and glycaemia (∆ gly) and also through appearance of diarrhoea (grade 2) between treatment initiation and W4. To our knowledge, no precocious biological markers exist for nephrolithiasis, however creatinine clearance has been found to relate to occurence of severe adverse events (including nephrolithiasis) in a multivariate analysis [START_REF] Collin | Indinavir trough concentration as a determinant of early nephrolithiasis in HIV-1-infected adults[END_REF]. Thus, we also analysed the difference in creatinine clearance (∆ClCr), computed with the Cockcroft-Gault formula using body weigth and serum creatinine 4 weeks before and 4 weeks after treatment initiation. The significance of these differences was tested by use of a Wilcoxon non-parametric paired test.

We performed Spearman correlation tests between indinavir dose, indinavir AUC, C max , C trough and ∆ TC, ∆ HDL, ∆trig, ∆ gly and ∆ClCr. We used Wilcoxon non-parametric tests to compare these differences between patients with or without an indinavir C trough over the upper limit defined in the therapeutic index (550 ng/mL). We studied the link between the appearance of grade 2 diarrhoea (yes/no) between treatment initiation and W4 and indinavir dose, indinavir AUC, C max , C trough using a Wilcoxon non-parametric test and we studied the association with or without an indinavir C trough > 550 ng/mL using a Fisher exact test.

We assessed the relation between the genetic polymorphisms remaining in the final population model and indinavir dose, indinavir AUC, C max , C trough and the relation between these genetic polymorphisms and the short-term efficacy and safety outcomes using Wilcoxon non-parametric tests.

We also derived AUC, C max , C trough for ritonavir and performed Spearman correlation tests with ∆ TC, ∆ HDL, ∆trig, ∆ gly and ∆ClCr as well as Wilcoxon non-parametric tests on appearance of grade 2 diarrhoea.

Results

Patients

Forty-two patients were included in this treatment group of the COPHAR 2 ANRS -111 trial, one patient withdrew from the study and one switched to another protease inhibitor during the first week of treatment. We therefore obtained pharmacokinetic data from 40 patients (27 men, 13 women) with a median age of 36.5 years (20.0 -59.0). Table 1 describes the main characteristics of the 40 studied patients.

Both approaches to allocate the ethnic group provided corroborating results. Using the civic information we allocated 20 patients to the African group and 20 to the Caucasian group. Given that one patient had missing information for all genotypes, the Structure software allocated 19 patients to the Caucasian group and 20 to the African group. In the resulting two ethnic groups, Hardy-Weinberg proportions were respected for all polymorphisms under study as shown in Table 2.

Indinavir pharmacokinetics

For two patients, two samples were missing, the trough and the 6 h concentrations and only the trough concentration was available for another. Among the 155 samples, two indinavir plasma concentrations in one patient were below the limit of quantification (at 1 h and at trough) and were discarded from the analysis. Figure 1a displays indinavir plasma concentrations at W2 versus time. It shows that there is a high inter-individual variability.

The best error model was a proportional error model. The population estimates are displayed in Table 3. All the relative standard errors (RSE) were below 25% with the exception of k a and ω V /F (around 30 and 60% respectively). The inter-individual variance of k a in this study was rather important (above 100%). The simulated median and the 90 th interval are displayed in Figure 2a together with all the observed concentrations of indinavir. This graph shows good evidence of the adequacy of the model.

From that basic model, we first tested the effects of the covariates on the individual parameters estimates. Effects of age (P = 0.03) and ABCB1 exon 26 polymorphism (P=0.09) on Cl/F and of CDC classification (P = 0.09) and CYP3A4*1B polymorphism (P = 0.09) on k a were found. Both ABCB1 exon 26 and CYP3A4*1B polymorphism variables were dichotomised in mutant homozygotes versus other genotypes. After the forward selection based on LRT, the population model had CYP3A4 effect on k a (P = 0.02) and age effect on Cl/F (P = 0.03). The age effect on clearance was withdrawn from the model after the backward selection based on permutation test. In the final model, Absorption rate constant was decreased by 70% (P = 0.04, LRT by permutation) in patients *1B*1B for the CYP3A4 allele:

k a = 2.1×e -1.3×CYP3A4 with CYP3A4=0 for patients CYP3A4*1A*1A or CYP3A4*1A*1B CYP3A4=1 for patients CYP3A4*1B*1B
The population parameters of this final model and their RSE are given in Table 3 for the thirty-eight patients with available genotyping for CYP3A4*1B polymorphism. The interindividual variability for k a decreased by 27% from the basic model with the incorporation of the covariate and residual variability was 44.7%.

Ritonavir pharmacokinetics

One patient who had indinavir concentrations had no concentration for ritonavir, so we only analysed ritonavir data for 39 patients. The same 5 samples for indinavir mentioned in the preceding paragraph were also missing. Among the 151 samples, two ritonavir plasma concentrations at 1 h and at trough in one patient and one concentration at 12 h in another patient were below the limit of quantification and were discarded. Observed plasma concentrations are displayed in Figure 1b, and it should be noticed that some patients exhibited high plasma concentrations (above 2000 ng/mL) for a dose of 100 mg b.i.d. A proportional error model was selected. The population estimates are displayed in Table 4. All the RSE were below 25% with the exception of k a , partly attributable to the sparse design, and ω Cl/F as observed for the indinavir data. The VPC obtained with the basic model parameters estimates is displayed in Figure 2b, together with the observed concentrations. The results of the basic model evaluation were very satisfactory.

Effects of orosomucoid (P=0.03), albumin levels (P=0.04) and CYP3A5 polymorphism (patients with 2 wild alleles at most vs other genotypes, P = 0.04) on Cl/F were found on the individual parameters by use of non-parametric tests as well as effects of HIV disease status

Cl/F = 8.3 × Orosomucoid -0.8
The population parameters of this model and their RSE are given in Table 4.

Link between indinavir and ritonavir PK parameters

Four positive correlations between individual parameters of ritonavir and indinavir were found significant. There was a relationship between indinavir and ritonavir absorption rate constant (r = 0.4, P = 0.005). Indinavir clearance was strongly correlated to ritonavir clearance (r = 0.6, P < 0.0001) and to a smaller degree to ritonavir volume of distribution (r = 0.4, P < 0.01), while indinavir volume of distribution was highly correlated to ritonavir volume of distribution (r = 0.5, P < 0.002).

Indinavir AUC (ng.h/mL) ∆logVL (log copies/mL) -3.0

Concentrations link with short-term efficacy and safety

There was a significant decrease in viral load in the first two weeks of treatment and a significant increase in total cholesterol, glycaemia and triglycerides in the first four weeks of treatment, as shown in Table 5.

The decrease in log viral load was significantly associated with higher indinavir AUC (r = -0.4, P = 0.03) and C trough (r = -0.4, P = 0.03), as shown in Figure 3. No significant difference in viral load decrease was found between the five patients with a C trough below the lower limit of the therapeutic range and the thirty five patients with a C trough above this value.

Further, no significant relationship was found between indinavir nor ritonavir concentrations and safety measurements or grade 2 diarrhoea. No nephrolithiasis has been reported in the COPHAR 2 -ANRS 111 trial, which has prevented us to analyze the link between concentrations and this adverse event associated with indinavir.

The genetic covariate kept in the final population pharmacokinetic model was the CYP3A4*1B polymorphism categorised in 2 classes: *1B*1B versus other genotypes. Both C max and triglycerides increase were found to be significantly associated with the CYP3A4*1B polymorphism, althought not significantly correlated. In patients' homozygotes for the *1B allele, C max was significantly lower (median 3. The various doses of indinavir were not found to be associated with the CYP3A4*1B polymorphism, short-term efficacy or safety, which dismissed its potential confounding effect.

Discussion

The pharmacokinetics of indinavir was analysed by use of a one compartment model with first-order absorption and elimination at steady-state. The estimated clearance and volume of distribution were 22.2 L/h and 97.3 L respectively, both in the range of those obtained in previous studies [START_REF] Csajka | Population pharmacokinetics of indinavir in patients infected with human immunodeficiency virus[END_REF]11,32]. In this study, the ABCB1 exon 26 and 21 and the CYP3A5*3 and *6 polymorphisms were not found to significantly influence the pharmacokinetics of indinavir. Whereas, the absorption rate was 0.6 h -1 for CYP3A4*1B*1B patients and 2.1 h -1 for CYP3A4*1A*1A or CYP3A4*1A*1B patients. The CYP3A enzymes are distributed in both hepatocytes and enterocytes [START_REF] Fuhr | Appropriate phenotyping procedures for drug metabolizing enzymes and transporters in humans and their simultaneous use in the "cocktail" approach[END_REF] and their inhibition by ritonavir is well-documented [START_REF] Ikezoe | HIV-1 protease inhibitor, ritonavir: a potent inhibitor of CYP3A4, enhanced the anticancer effects of docetaxel in androgen-independent prostate cancer cells in vitro and in vivo[END_REF][START_REF] Zhou | Mechanism-based inhibition of cytochrome P450 3A4 by therapeutic drugs[END_REF][START_REF] Rittweger | Clinical pharmacokinetics of darunavir[END_REF]. In vivo, the genotype-phenotype correlation for CYP3A4*1B remains a subject of debate [START_REF] Spurdle | The CYP3A4*1B polymorphism has no functional significance and is not associated with risk of breast or ovarian cancer[END_REF][START_REF] He | Genotype-phenotype associations of cytochrome P450 3A4 and 3A5 polymorphism with midazolam clearance in vivo[END_REF][START_REF] Hesselink | Genetic polymorphisms of the CYP3A4, CYP3A5, and MDR-1 genes and pharmacokinetics of the calcineurin inhibitors cyclosporine and tacrolimus[END_REF][START_REF] Tran | Pharmacokinetics and toxicity of docetaxel: role of CYP3A, MDR1, and GST polymorphisms[END_REF], however CYP3A4*1B has been related to increased transcription [START_REF] Amirimani | Increased transcriptional activity of the CYP3A4*1B promoter variant[END_REF] in vitro. We hypothesised that in CYP3A4*1B*1B patients the ritonavir inhibition potency is lowered, which leads to higher first pass effect of indinavir although it does not impact on its clearance. The potential confounding effect of the ethnic group has been discarded, as this covariate was not significantly related to indinavir individual parameters in the sample. Still, this finding is more relevant clinically in an african population given the extremely low frequency of the CYP3A4*1B*1B genotype among caucasian. The primary objective of the COPHAR2 study was not to assess the influence of genetic polymorphisms on indinavir pharmacokinetics, and the use of modelling has helped to circumvent the limited sample size of forty patients. In addition, most of the tests in this study were performed at an exploratory step and final inclusion in the model was based on permutation to cope with departure from the asymptotic assumption [30]. No evidence for a gender effect was found as reported in some other studies on indinavir PK [START_REF] Csajka | Population pharmacokinetics of indinavir in patients infected with human immunodeficiency virus[END_REF][START_REF] Brendel | Population pharmacokinetic analysis of indinavir in hivinfected patient treated with a stable antiretroviral therapy[END_REF][11] but there were only thirteen women in the present study. The dose was not found to influence the pharmacokinetics of indinavir and using ritonavir as a boost hides dose non linearity of indinavir [START_REF] Yeh | Simultaneous investigation of indinavir nonlinear pharmacokinetics and bioavailability in healthy volunteers using stable isotope labeling technique: study design and model-independent data analysis[END_REF]. We did not assess the impact of the diet, as these data were not available, but patients were recommended to ingest the pills with food containing a sufficient amount of fats.

We also performed a population pharmacokinetic analysis of ritonavir concentrations. Ritonavir profiles were adequately described by a one compartment model with first order absorption and elimination processes, with estimates of the parameters in good agreement with previous studies [START_REF] Hsu | Ritonavir. Clinical pharmacokinetics and interactions with other anti-HIV agents[END_REF][START_REF] Lu | Model-based analysis of the pharmacokinetic interactions between ritonavir, nelfinavir and saquinavir after simultaneous and staggered oral administration[END_REF][START_REF] Kappelhoff | Development and validation of a population pharmacokinetic model for ritonavir used as a booster or as an antiviral agent in hiv-1-infected patients[END_REF]. The estimated inter-individual variance for the absorption constant was singularly large. We found a negative relationship between ritonavir clearance and the orosomucoid level in plasma. The affinity of ritonavir for orosomucoid protein as well as its impact on PI intracellular concentrations and efficacy has been described in both in vitro and in vivo studies [START_REF] Schön | The binding of HIV-1 protease inhibitors to human serum proteins[END_REF][START_REF] Jones | Effect of alpha1-acid glycoprotein on the intracellular accumulation of the HIV protease inhibitors saquinavir, ritonavir and indinavir in vitro[END_REF][START_REF] Zhang | The effect of increasing alpha1-acid glycoprotein concentration on the antiviral efficacy of human immunodeficiency virus protease inhibitors[END_REF]. In patients with high orosomucoid plasma level, the decrease in unbound fraction of ritonavir led to a lower clearance.

In the analysis of both protease inhibitors, the few concentrations (1.3% and 2% for indinavir and ritonavir, respectively) below the limit of quantification were discarded. This approach has been shown to provide using SAEM a less important bias than to set those to LOQ/2 [START_REF] Samson | Extension of the SAEM algorithm to left-censored data in nonlinear mixed-effects model: Application to HIV dynamics model[END_REF] and in MONOLIX 2.1, no proper method to handle LOQ was implemented.

In the analysis of the link between indinavir and ritonavir concentrations, we chose not to include ritonavir as a covariate in the indinavir model, as performed in previous studies [START_REF] Brendel | Population pharmacokinetic analysis of indinavir in hivinfected patient treated with a stable antiretroviral therapy[END_REF]11]. Indeed, such parameterisation assumes unidirectional influence of ritonavir on indinavir which is not true. Ritonavir concentrations when ritonavir is given with lopinavir are lower than when ritonavir is given with indinavir [START_REF] Kappelhoff | Development and validation of a population pharmacokinetic model for ritonavir used as a booster or as an antiviral agent in hiv-1-infected patients[END_REF]. We have instead underlined the different levels of interaction between indinavir and ritonavir PK, especially at the absorption step, with the strong correlation between their absorption constant, but also in terms of bioavailability as the oral clearances and volumes of distribution were highly correlated. In order to properly model such an interaction between PI, a joint population analysis of concentrations of indinavir and ritonavir should be considered with correlated absorption constants and bioavailabilities.

We observed significant changes in viral load after two weeks of treatment and we confirmed the association between high indinavir trough and mean concentrations and a greater decrease of viral load, which has already been described in PI naive patients [START_REF] Fletcher | Pharmacologic considerations for therapeutic success with antiretroviral agents[END_REF][START_REF] Burger | A retrospective, cohort-based survey of patients using twice-daily indinavir + ritonavir combinations: pharmacokinetics, safety and efficacy[END_REF][START_REF] Rayner | A critical evaluation of the therapeutic range of indinavir[END_REF]. We did not find any relationship between CYP3A4*1B polymorphism and viral load decrease. We also observed a significant increase, after four weeks of treatment, of total cholesterol, glycaemia and triglycerides, as already reported [START_REF] Behrens | Impaired glucose tolerance, beta cell function, and lipid metabolism: HIV patients under treatment with protease inhibitors[END_REF], which was, however, not significantly related to indinavir concentrations at week 2. Ritonavir was found in singularly high levels in the study and is known to affect metabolic profiles, yet we have found no evidence of an association between ritonavir levels and safety measurements. In patients' homozygotes for the CYP3A4*1B allele, the ritonavir-decreased inhibition on indinavir metabolism led to significantly lower indinavir C max and appeared to impact at a metabolic level trough a significantly lower increase in triglycerides in these patients.

Conclusion

We developed and validated models for indinavir and ritonavir PK with reduced sampling in indinavir HAART patients. Both the average and through concentrations were found to be predictor of the viral load decline. Only the CYP3A4*1B allele was found to influence indinavir absorption and biochemical safety, but no evidence was found of an impact of the five genetic polymorphisms under study on indinavir efficacy.

Fig. 1

 1 Fig. 1 Observed plasma indinavir concentration (a) and plasma ritonavir concentration (b) versus time collected two weeks after treatment initiation, in 40 HIV naive-patients receiving indinavir plus 100 mg of ritonavir b.i.d. In the indinavir plot the plain lines correspond to an indinavir dose of 400 mg b.i.d., the dashed lines to 600 mg b.i.d. and the dotted lines to 800 mg b.i.d. Sampling times following drug administration were measured by the nurse. Concentrations were assumed at steady state, trough concentrations are displayed as following the drug intake at sampling times deduced from the patient record.

Fig. 2

 2 Fig. 2 Visual predictive check of the basic population PK model: comparison between the median (continued line) and the 90 th interval (colored area) predicted for 1000 simulated datasets and the observed concentrations of indinavir (a) and of ritonavir (b). In the indinavir plot the open circles correspond to an indinavir dose of 400 mg, the open triangles to 600 mg and the crosses to 800 mg

Fig. 3

 3 Fig. 3 Differences in log viral load (∆ logVL) observed between treatment initiation and week 2 versus area under the concentration-time curve (a) and trough plasma concentration of indinavir (b) predicted by the model

Fig. 4

 4 Fig. 4 Peak indinavir concentrations predicted by the model (a) and differences in triglycerides (∆ triglyceride) 4 weeks before and after treatment initiation (b) versus CYP3A4 genotype. The solid-line represents the median in each group

a

  Baseline = D0 for log viral load and W-4 for safety b W2 for log viral load and W4 for safety

Table 4

 4 Population pharmacokinetic parameters of ritonavir for the basic and the final model: estimates and relative standard error (RSE)

		Basic model (N = 39)	Covariate model (N = 39)
	Parameters	Estimates	RSE (%)	Estimates	RSE (%)
	k a (h -1 )	2.4	98.7	2.2	93.5
	Cl/F (L/h) β Orosomucoid Cl V /F (L) ω ka (%) ω Cl/F (%) ω V /F (%) σ (%)	8.7 -61.6 357.7 55.9 22.8 30.4	9.4 -8.6 21.9 12.2 53.9 8.4	8.3 -0.8 60.7 346.8 52.4 23.3 30.3	9.0 46.5 8.7 21.5 12.4 51.5 8.4
	RSE = relative standard error

Table 5

 5 Median [range] of the studied short-term efficacy and safety measurements and of the change from baseline

		Baseline a	W2 or W4 b	Difference from	P-value
				baseline
	Efficacy
	Log viral load (log copies/mL)	4.9 [3.4 -6.3]	2.9 [1.8 -4.1]	-1.8 [-2.8 --0.5]	<0.001
	Safety
	Total cholesterol (mmol/L)	4.3 [1.9 -7.4]	5.0 [2.9 -7.5]	0.8 [0.8 -4.7]	<0.001
	HDL cholesterol (mmol/L)	1.1 [0.5 -1.8]	1.1 [0.4 -2.1]	0.1 [-0.7 -1.0]	0.09
	Glycaemia (mmol/L)	4.7 [3.4 -6.0]	4.9 [2.8 -7.1]	0.2 [-1.0 -2.

Acknowledgements Steering committee of COPHAR2 ANRS-111 trial: principal investigators: D. Salmon-

precious advices they provided along the analysis. During this analysis J. Bertrand was supported by a grant from Servier Research Group, France.